PLACE ll RETURN BOX to roman this chockout from your mood. TO AVOID FINES Mom on or baton dd. duo. DATE DUE DATE DUE DATE DUE MSU In An Affirmative Action/Equal Opponfirmy Inflation mm: SCREENING FOR MUTATIONS IN PAX3 AND MITF IN WAARDENBURG SYNDROME AND WAARDENBURG SYNDROME-LIKE INDIVIDUALS By Melisa Lynn Carey A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for a degree of MASTER OF SCIENCE Department of Zoology 1 996 ABSTRACT SCREENING FOR MUTATIONS IN PAX3 AND MITF IN WAARDENBURG SYNDROME AND WAARDENBURG SYNDROME-LIKE INDIVIDUALS BY Melisa L. Carey Waardenburg Syndrome (WS) is an autosomal dominant disorder characterized by pigmentary and facial anomalies and congenital deafness. Mutations causing WS have been reported in PAX3 and MITF. The goal of this study was to characterize the molecular defects in 33 unrelated WS individuals. Mutation detection was performed using Single Strand Conformational Polymorphism (SSCP) analysis and sequencing methods. Among the 33 WS individuals, a total of eight mutations were identified, seven in PAX3 and one in MITF. In this study, one of the eight mutations was identified and characterized in PAX3 exon seven in a WSI family (UoM1). The proband of UoM1 also has Septo-Optic Dysplasia. In a large family (MSU22) with WS-Iike dysmorphology and additional craniofacial anomalies, linkage was excluded to PAX3 and no mutations were identified in MITF. Herein I review the status of mutation detection in our proband screening set and add to the understanding of the role of PAX3 and MITF in development by exploring new phenotypic characteristics associated with WS. ACKNOWLEDGMENTS I would like to thank Drs. Tom Friedman and Jim H. Asher Jr. for their encouragement, support, patience and guidance. A special thanks to Dr. Asher for all of his efforts in collecting families for this study. His dedication and conviction were admirable and he will be missed. I would also like to thank Tom Barber, Aihui Wang, Yong Liang, Rob Morell, Lori Swenson and Maki Saitoh for their enlightening discussions both scientific and social. A special thanks to Tim Cloutier for his assistance with the project. I would like to thank my committee members, Dr. John Fyfe, Dr. Emanuel Hackel and Dr. Jeff Innis for their helpful suggestions. A thanks to all of our collaborators and the families represented in this project; whose cooperation is greatly appreciated. I would like to to take a moment to thank my parents, Barbara and Patrick Carey and the rest of my family, Mike, Donna, Courtney, Kyle and Brandon; who gave me both the encouragement and the necessary outlet to get through the trials of graduate school. This work was supported by Grant DC 01160-04 from the National Institute on Deafness and Communication Disorders, National Institute of Health. TABLE OF CONTENTS LIST OF TABLES ...................................................................... vii LIST OF FIGURES ................................................................................. ix BACKGROUND AND SIGNIFICANCE ................................................... 1 Waardenburg Syndrome Phenotype ................................. 3 Mouse Models for Waardenburg Syndrome ...................... 2 The PAX family ................................................................. 6 PAX genes responsible for several disorders ................... 8 PAX3 expression pattern .................................................. 9 Mutations in human PAX3 .................................................. 10 Micropthalmia ..................................................................... 15 Other genes causing Waardenburg Syndrome ................. 20 Understanding function ...................................................... 20 CHAPTER ONE: Screening for mutations in PAX3 and MITF in families with classical Waardenburg Syndrome (WS) and Waardenburg Syndrome-like phenotypes. INTRODUCTION ........................................................................... 22 The goals of this study ............................................... 22 Description of the proband screening set ....................... 26 RESULTS ...................................................................................... 28 SSCP analysis of PAX3 .................................................... 29 SSCP analysis of MITF ...................................................... 29 Cycle sequencing analysis of PAX3 and MITF 31 DISCUSSION ................................................................................ 32 SSCP analysis .................................................................... 33 Cycle sequencing ............................................................... 34 Mutation detection .............................................................. . 36 Evaluation of clinical data .................................................. . 38 Mutations due to deletions ................................................... 40 Mutations in regulatory regions ..................................... 41 Mutations of alternative transcripts ................................... 41 Technical obstacles with SSCP ........................................... 42 Mutations in other genes ..................................................... 45 CONCLUSION .............................................................................. 46 MATERIALS AND METHODS ..................................................... 48 Family identification and DNA isolation ............................. 48 Polymerase chain reaction .................................................. 49 Single strand conformational polymorphism ....................... 50 Allele specific amplification .................................................. 51 Cloning fragments ................................................................ 52 Sequencing .......................................................................... . 53 CHAPTER TWO: Waardenburg Syndrome in conjunction with Septo—Optic Dysplasia. INTRODUCTION ........................................................................... 55 Septo-Optic Dysplasia ....................................................... 55 Ascertainment of an individual with WSI and SOD .............. 59 RESULTS ...................................................................................... 61 SSCP analysis of PAX3 ...................................................... 61 Mutation indentification in MITF ........................................... 69 Other individuals with SOD ................................................. 70 DISCUSSION ................................................................................. 71 Description of UoM1 ............................................................. 71 Mutational analysis of PAX3 and MITF ............................. 72 Other individuals with SOD .................................................. 74 CONCLUSION ................................................................................ 76 CHAPTER THREE: Waardenburg Syndrome co-segregating with other severe craniofacial anomalies. INTRODUCTION ........................................................................... 77 Genes causing craniofacial anomalies .............................. 77 Description of MSU22 .......................................................... 78 Craniofacial syndromes ...................................................... . 78 RESULTS ........................................................................................ 82 Mutational analysis ........................................................... . 82 Linkage analysis to PAX3 .................................................... 82 DISCUSSION ................................................................................. 88 Description of MSU22 .......................................................... 88 SSCP and sequence analysis .............................................. 91 Linkage analysis ................................................................... 91 CONCLUSIONS ........................................................................... . 93 APPENDIX A: Additional Tables .............................................................. 94 APPENDIX 8: Additional lnforrnation ....................................................... 109 Blank foms .......................................................................... 109 Reprints ............................................................................... 1 15 LIST OF REFERENCES .......................................................................... 134 vi Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 LIST OF TABLES Waardenburg Syndrome Diagnostic Criteria 2 PAX3/Splotch Mutations .................................................... 4 Mi Mouse Mutations ........................................................... 5 PAX Genes ......................................................................... 6 PAX3 Mutations ................................................................... 12 MITF Mutations ................................................................... 16 Proband Screening Set ....................................................... 24 PAX3 PCR Primers .............................................................. 94 MITF PCR Primers .............................................................. 95 Cycle Sequencing Primers .................................................. 98 Collaborators for Each WS and WS-like Family ............... 99 PAX3 Linked Markers ......................................................... 100 W-lndex for members of MSU22 ........................................ 90 vii Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Table 21 Phenotypes for MSU1-MSU7 ............................................. 101 Phenotypes for MSU8-MSU14 ............................................ 102 Phenotypes for MSU15-MSU21 .......................................... 103 Phenotypes for MSU22-MSU28 ........................................... 104 Phenotypes for MSU29-MSU32; UoM1, UoM3, UoM4 ........................................................... 105 Phenotypes for UGM families ............................................. 106 Phenotypes for SOD families ............................................. 107 Phenotype Description for members of MSU22 ................ 108 viii Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 LIST OF FIGURES PAX Gene Family ................................................................ 7 PAX3 Mutations ................................................................... 13 MITF Mutations .................................................................... 17 bHLH-Zip Family .................................................................. 19 PAX3 Gene Structure with Primers .................................... 96 MITF Gene Structure with Primers .................................... . 97 Medial Surface of a 4 Month Embryo Brain ........................ 56 Normal Frontal View of the Brain ....................................... 58 Frontal View of a Brain with Septo-Optic Dysplasia .......................................................... 58 The Pedigree of UoM1 .......................................................... 60 PAX3 Exon 7 Normal and Mutant DNA and Protein Sequence .......................................................... 63 ASA of PAX3 Exon 7 ........................................................... 65 SSCP Variants Identified in UoM1 ..................................... 67 Figure 14 MSU22 WS and Craniofacial anomalies .............................. 81 Figure 15 MSU22 Pedigree: Linkage Analysis with WS phenotype 84 Figure 16 MSU22 Pedigree: Linkage Analysis with CA phenotype 86 BACKGROUND and SIGNIFICANCE Waardenburg Syndrome Phenotype There are at least four clinical sub-types of Waardenburg Syndrome as defined by McKusick in Mendelian Inheritance of Man (MIM): WSI (MIM 193500), WSII (MIM 193510), WSIII (MIM 148820), and WSIV (MIM 277580). The presence of dystopia canthorum in 98% of WSI individuals distinguish WSI from WSII“! 6'10) Waardenburg Syndrome (WS) types I, II and III are autosomal dominant disorders. WSI is characterized by congenital deafness, dystopia canthorum, heterochromia irides, poliosis, broad nasal root, synophrys, hypo- and hyperpigmentation of the skin and hair!” Other less common clinical anomalies include aganglionic megacolon, cardiac defects, cleft lip and palate and spinal bifida. WSIII has the clinical characteristics of WSI with the addition of limb abnormalities.“ ‘2) The classic example of WSIII was described by Kleinm' 13' 14) and hence WSIII is often referred to as Klein-Waardenburg Syndrome. WSIII is rare, with approximately a dozen published cases.<12- 1523) WSIV (Shah-Waardenburg Syndrome) exhibits ws along with aganglionic megacolon.(24'29) WSIV is also called Hirschprung disease (HSCR) with pigmentary anomaly. Approximately 25% of WS individuals exhibit unilateral or bilateral hearing loss“) It is estimated that 2% of all individuals with profound deafness have WS.“ 4) Classification of WS is determined by major and minor criteria established by the WS consortium as listed in TABLE 1.(2' 3) The penetrance and expressivity for all WS clinical features vary both within and between families. Thus, WS is both clinically pleiotropic and genetically heterogeneous.(25' 30' 31) Table 1: Waardenburg Syndrome Diagnostic Criteria Major Characteristics: Sensorineural deafness Iris pigmentary abnormalities Heterochromia irides Characteristic brilliant blue iris Hypopigmented iris Hair pigmentation White forelock Body hair (eyelashes; eyebrows) Dystopia canthorum (only in WSI individuals) First-degree relative previously diagnosed with WS Minor Characteristics: Congenital leukoderrna (severe areas hypopigmented skin) Synophrys Broad high nasal root Hypoplasia of alae nasi Premature graying Rare Characteristics: Hirschprung disease (classified as WSIV) Sprengel anomaly Spina bifida Cleft lip and/or palate Limb defects (characteristic of WSIII) Congenital heart abnormalities Abnormalities of vesitbular function Broad square jaw Low anterior hair line Table 1: The clinical diagnostic criteria for Waardenburg Syndrome according to the guidelines established by the Waardenburg Syndrome Consortium.(5) In order to be characterized as having WS, an individual must possess either two major characteristics, one major and a first degree relative diagnosed as affected with WS or 1 major and 2 minor characteristics. Mouse models for Waardenburg Syndrome Pigmentary anomalies associated with deafness were documented in domestic animals by Darwin,(32) and othersm' 34) may have been examples of Waardenburg Syndrome. Waardenburg Syndrome was one of the first reported examples of pigmentary anomalies with deafness in humans.“ 34) The hypothesis of a single gene being responsible for the combined clinical phenotype gained acceptance, after the observations that all of the tissues affected in WS patients were derivatives of neural crest cells.(25' 3537) Several mouse mutations including the Splofch (Sp)(38), microphthalmia (M099), piebald-Iethal (S’)(4°) and Patch (Ph) loci affect neural crest cell development, migration and/or differentiation.(“) The Splofch mice when homozygous have severe neural tube defects, pigmentary defects, muscle defects, craniofacial anomalies and usually embryonic or neonatal death‘41'52- 321) (TABLE 2). Mi mice when homozygous exhibit a white coat, eye abnormalities and ear defects (TABLE 3). Piebald homozygotes are completely white, they have megacolon and structural defects of the iris. Likely candidates for Waardenburg Syndrome were predicted on the basis of conserved syntenic relationships between mouse and human,(53) at chromosomal locations, of 2q, 39. 3q or 4p, near the proto-oncogene KIT.(54) In 1989, lshikiriyama et ai.<55-57> reported a child with WSI that had a de novo inversion of 2q35-q37.3. This was the region predicted for WS on the basis of the Sp mutant locus. demonstrated.‘57‘6°) Genetic linkage of WSI to 2q35 was then Table 2: Pax3lSpIotch mutations (modified from Chalepakis et al. 1993)(90) ALLELE PHENOTYPE HETEROZYGOTE PHENOTYPE Homozveore Sp (sd) white spotting embryonic death E14p.c. curly tail pigmentation deficiency spina bifida exencephaly meningocele Spd (sd) similar to Sp Sp2H/3P1H similar to Sp Sp4H analogous to Sp retarded growth neural overgrowth dorsal root ganglia deficiency schwann cell deficiency truncus arteriosus deficiency thyroid deficiency muscle deficiency anterior structures not affected no exencephaly . late embryonic or neonatal death most severe phenotype postimplantation lethal Table 3: Mi mouse mutations (modified from Steingrimsson et al. 1994)(167) ALLELE Mior (oak ridge) MIWh (white) mIWS (white spot) miOW (eyeless-white) mice (cloudy-eyed) mi'W (red-eyed white) mrw't (vitiligo) miSP (spotted) mibw (black-eyed white) PHENOTYPE HETEROZYGOTE slight dilution coat color pale ears and tail belly streak or heat spot dilution of coat color reduced eye pigmentation spots on toes, tail and belly inner ear defects melanocytes absent from dermis white spot on belly toes and tail often white normal appearance normal appearance normal appearance normal appearance normal appearance normal appearance PHENOTYPE HOMOZYGOTE white coat eyes small/absent incisors fail to erupt osteopetrosis white coat eyes small; inner iris pigmented spinal ganglia, adrenal medullae small inner ear defects mast cell deficiency white coat eyes pink but near normal size white coat eyes absent/eyelids never Open white coat eyes pale (cloudy white) and small inner ear defects white coat with pigmented spots head/tail eyes small and red spots on thorax and abdomen gradual depigmentation of pigmented areas old mice are nearly white retinal degeneration normal appearance reduced tyrosinase activity in skin white coat color inner ear defect The PAX Family of Genes There are several conserved DNA-binding motifs identified among the genomes of Drosophila, mouse, nematode, zebra fish, frog, turtle, chick and humanist 62) One evolutionarily conserved DNA-binding domain is encoded by the paired box, first discovered in three Drosophila segmentation genes.(63' 65) There are nine known human PAX genes that have a paired domain of 128 amino acids‘66- 67) (TABLE 4). PAX gene members may also contain an octapeptide domain and/or a paired-homeobox. Table 4: PAX Genes GENE HUMAN MOUSEa REFERENCES PAX1: 20p11.2 2 Stapleton et al. 1993 PAX2: 10q22.1-q24.3 19 Tsukamoto et al. 1992 PAX3: 2q35 1 Stapleton et al. 1993 PAX4: 7q32 6 Stapleton et al. 1993, Tamara et al. 1994 PAX5: 9p13 4 Stapleton et al. 1993 PAX6: 1p13 2 Ton et al. 1991 PAX7: 1p36.2-p36.12 4 Stapleton et al. 1993 PAX8: 2q12-q14 2 Stapleton et al. 1993 PAXQ: 14q12-q13 nd Stapleton et al. 1993 Nine PAX genes with the human and mouse chromosomal locations. (modified from Stapleton et al. 1993)(66) a = Walther et al. 1991, nd = not determined. The members are grouped according to their DNA-binding motifs (FIGURE 1). PAX1<68> and PAX9<69> contain a paired domain and an octapeptide domain. PAX2‘6" 7°72), PAX5i73' 74) and PAX8‘7577) contain a paired domain, an octapeptide domain and a small: portion of the homeodomain. PAX4I7i” and PAX6‘79' 80’ contain the paired domain and the homeodomain. These two genes do not have an octapeptide domain. PAX3 is most closely related to PAX7(8“84), containing a paired domain, an octapeptide domain and a paired-type homeodomain. The Sp phenotype was demonstrated to be due to a mutation in Pax3.(42“‘3' 85) Pax3 and its human homologue, PAX3 are members of the paired box (PAX) gene family of transcription factors. A PAX Gene Family NH2 —— COOH PD OCT PD OCT HD C . PD OCT HD D , NHZ N COOH PD HD Figure 1 PAX gene family. (A) PAX1 and PAX9. (B) PAX3 and PAX7. (C) PAX2, PAX5 and PAX8. (D) PAX4 and PAX6. (modified from Baker et al. 1995)(44) PAX genes responsible for several disorders The PAX genes have distinct functions throughout development.(62- 86' 89) There are slight overlaps in expression patterns as well as specific organ and tissue development.(86- 87' 90' 9‘) The PAX genes are expressed in the developing nervous system with the exception of Pax1i92' 93) and Pax9. Many of the Pax genes were identified through the use of syntenic relationships between mouse and human (TABLE 4). Several PAX genes have been implicated in human syndromes and disorders. Pax6 in the mouse is involved in eye development‘aot 94) and some Pax6 mutations are responsible for the small eye phenotype.(79v 9597) Mutations in human PAX6 have been identified that cause a number of disorders including: aniridia,(98'1°2) Peter's anomaly,(1°3'1°5) cataracts,(‘°1' 106) WAGR (Wllms' tumor, aniridia, genitourinary abnormalities and mental retardation)(1°7- 103) and keratitis.(1°9) A PAX2 mutation has been implicated in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux.(“°) No human disorder has been identified that is associated with a defect in PAX1. Although the undulated mouse mutant is caused by Pax1 and homozygous mice exhibit vertebral malformations along the entire cranio- caudal axis.("1'"3) Several of the PAX genes play a role in cancer development.("4'"6) PAX2m7) and PAX8‘1‘8' 119’ are implicated in the development of VWms' tumor,(12°‘122) an embryonic tumor of the kidney. Medulloblastomas express many of the PAX genes. PAX5 expression is upregulated in the tumors compared to slight increase in PAX2, PAX3 and PAX1.(123) PAX5 is a B-cell transcription factor (BSAP) that controls expression of 0019““) and may also play a role in the development of astrocytoma.(125) Fusion gene products between PAX3 and forked head (FKHR) and between PAX 7 and FKHR are responsible for alveolar rhabdomyosarcoma.(126'136) PAX3 and PAX7 gene translocations result in the 5'-end of either PAX3, t(2;13)(q35;14)<126- 127' 131. 132' 136) or PAX7, t(1;13)(q35;14)(128) adjacent to the 3'-end of FKHR. The 5'- end of both PAX3 and PAX 7 contains the DNA binding domains and the 3'-end of the FKHR gene contains the activation domains. PAX3 Expression Pattern Pax3 encodes a 479 amino acid, 56 kDa protein that is expressed during embryonic development‘90' 91' 137442) and in the adult.‘55 38) Around embryonic day 8.5 to 9, murine Pax3 expression is limited to mitotic cells in the ventricular zone of the developing spinal cord and to distinct regions of the hindbrain, midbrain and diencephalon.(137) Pax3 is expressed in neural crest derivatives, particularly the spinal ganglia and cephalic neural crest cells, including the nasal process and structures derived from the first and second brachial 1O arches.(143) Pax3 is expressed in the migrating neural crest cells and the derrnomyotome cells.(144) Pax3 expression during development is observed in the craniofacial mesectoderrn and in the limb mesenchyme.(14“43) Pax3 is also expressed in the Bergmann glia and the basket cells of the Purkinje cell layer of the cerebellar cortex.(88) Mutations in human PAX3 Mutations have been identified in PAX3 in ws individuals.(59' 60' 149-155) Hundreds of WS families have been identified by the WS consortium; approximately 80% of the mutations in PAX3 in WSI individuals have been identified.(155) To date more than 50 mutations have been identified in WSI individuals within PAX3 (TABLE 5). Until recently no common mutations in WSI individuals from unrelated families were identified. However, three identical mutations have since been identified in unrelated families with WSI (TABLE 5). The majority of PAX3 mutations that cause WSI are within the DNA- binding domains (FIGURE 2). Many of the codons with mutations are highly conserved among species. On the basis of crystal structure studies, these codons have been identified as important for DNA-binding or phosphate backbone contacts.(156' 157) 10 arches.“43) Pax3 is expressed in the migrating neural crest cells and the derrnomyotome cells.(144) Pax3 expression during development is observed in the craniofacial mesectoderrn and in the limb mesenchyme.(144‘143) Pax3 is also expressed in the Bergmann glia and the basket cells of the Purkinje cell layer of the cerebellar cortex.(88) Mutations in human PAX3 Mutations have been identified in PAX3 in ws individuals.(59' 6°~ 149-155) Hundreds of WS families have been identified by the WS consortium; approximately 80% of the mutations in PAX3 in WSI individuals have been identified.(155) To date more than 50 mutations have been identified in WSI individuals within PAX3 (TABLE 5). Until recently no common mutations in WSI individuals from unrelated families were identified. However, three identical mutations have since been identified in unrelated families with WSI (TABLE 5). The majority of PAX3 mutations that cause WSI are within the DNA- binding domains (FIGURE 2). Many of the codons with mutations are highly conserved among species. On the basis of crystal structure studies, these codons have been identified as important for DNA-binding or phosphate backbone contacts.(156' 157) 11 Table 5: PAX3 mutations. The listing includes the family name, the mutation, and the exon of the mutation. Large deletions and inversions are not included in this table. 12 Table 5: PAX3 Mutations PANEL? vwaoss 8047 MSU17 BU26 BU35 8048 vwsoz4 vm315 2kno1995 805 vwsooe ’Msua \NSps vmstoo UGM2 vvsoe BU53 vwsoeo vmsoes vmsos4 \N8003 \NSJ1 \NSJ38 HoL1995 BU7 804 BU9 BU8 BU52 vwsoso vw3001 iuHa vwsoee \NStne vwsto M805 luHs vmsooe MSU7 vvsnza 8014 8022 M309 vwsn19 vM5105 BU3O 0dM1 8025 MUTATION exon F45L N47H N47L PSOL R56L V60M V78M 681 A $84F K85E 6990 1001ns1 185del18 191 del 1 7 266del 1 4 288del1 297del28 364de15 358del1 451 ins1 452de12 556del2 A1 961‘ 598del5 OZOOX 8201 X R223X E235X F2388 E251 X 0254X V265F W266C R2700 R271 C R271 C R271 G R271 H W274X W274X 0313X 874ins1 874ins1 874ins1 916del1 954del1 0391 H 1 185ins3 mummommmmmmmmmmmmmmmmmmwm:-orAwwwMNMMNNMMNMMMNNMMM REFERENCES Tassabehji et al. 1994 ” Hoth et al. 1993 Sommer et al. 1983; Asher et al. 1995 da Silva 1991; Hoth et al. 1993, Baldwin et al. 1993 Carezani-Gavin et al. 1992; Hoth et al. 1993 Baldwin unpublished Tassabehji etal. 1995 Foy et al. 1990; Tassabehji et al. 1993 Zlotogora et al. 1995 Baldwin et al. 1995 Tassabehji et al. 1994 Morell et al. 1993 Foy etal. 1990; Tassabehji et al. 1992 Tassabehji et al. 1995 Morell et al. 1992 Foy et al. 1990; Tassabehji et al. 1992 Baldwin et al. 1994 Tassabehji et al. 1995 Tassabehji et al. 1995 Tassabehji et al. 1994 Tassabehji et al. 1994 Foy et al. 1990; Tassabehji et al. 1993 Tassabehji et al. 1995 Hol et al. 1995 Baldwin et al. 1995 Baldwln et al. 1995 Baldwin et al. 1994 Baldwin et al. 1995 Baldwin et al. 1995 Tassabehji et al. 1995 Tassabehji et al. 1995 Lalwani et al. 1995 Tassabehji et al. 1995 Tassabehji et al. 1995 Foy et al. 1990, Tassabehji et al. 1995 Asher et al.1991; Morell et al. in press Lalwani et al.1995 Tassabehji et al. 1995 Morell et al. in press Tassabehji et al. 1995 Baldwin et al. 1995 Baldwin et al. 1995 Kapur and Karam 1991; Morell et al. in press Tassabehji et al. 1995 Tassabehji et al. 1995 Baldwin et al. 1995 Carey et al. 1996 (in preparation) Baldwin et al. 1995 13 Figure 2: PAX3 mutations. A diagram of the mutations characterized in the literature. The top panel displays the paired domain, the octapeptide domain and the homeodomain in relation to the mutations. The lower panel displays the mutations in relation to the eight exons. 14 PAX3 Mutations 358del(1) 874ins(1) . /" 7 - -‘:~\ ., {(21 _. seeded I l 451del(1) 039m 452del(2) R271C R271H R271G F45L 358del(1) 288del(1) 874ins(1) 1:?2239)‘ 364del(5) £251x wz74x 1 | 2 l 3 I 4 I 5 | 6 | 7 l l I 681A 451del(1) A196T R2700 039111 V78M 452del(2) R2710 V60M 100ins(1) 566del(2) R271H R56L R2716 P50L G990 N47L V265F N47H wzsec F45L 15 In addition to alveolar rhabdomyosarcoma and WS, a mutation in PAX3 also causes Craniofacial Deafness Hand Syndrome (CDHS) (MIM 122880). CDHS was first identified in a single small family.(158) CDHS is characterized by the absence or hypoplasia of the nasal bones, profound sensorineural deafness, small and short nose with a slit like nare, hypertelorism, short palpebral fissures and limited movement at the wrist and ulnar deviation of the fingers.(158) A missense mutation, Asn47Lys, in PAX3 exon two in a highly conserved codon of the paired domain was identified.(159) There is a mutation in the same codon, Asn47His, in a WSIII family.(15) This discovery is an example of a syndrome other than WS being caused by a mutant allele in PAX3. Microphthalmia Extensive linkage studies suggested that WSII was not linked to PAX3.(7'16°) At least one additional gene was responsible for W811 mutations. After the mouse Mi gene was cloned,(161) its human homologue MITF (Microphthalmia-associated Transcription Factor) was cloned and assigned to 3p12.3-p14.1 by fluorescent in situ hybridizaiton (FISH).‘162) Analyses in WSII families established linkage to 3p12.3-p14.1. Tassabehji et al.(163) were the first to describe mutations in MITF responsible for the W811 phenotype. Approximately 10 MITF mutations have been reported to datei163) (TABLE 6). The majority of MITF mutations fall within the DNA binding domains (FIGURE 3). However, WSII appears to be genetically heterogeneous since the W811 16 phenotype in some families is unlinked to MITF. Therefore, there must be at least one more gene which when mutant, causes WSII. MITF Mutations FAMILY MUTATION EXON RBERENCES WS.002 G153+1A IN1 Tassabehji et al. 1994 WS.140 G153+1A IN1 Tassabehji et al. 1994 WS.026 A562-1C lN4 Tassabehji et al. 1994 WS.082 del3 7 Tassabehji et al. 1994 WS.115- 8250F 8 Tassabehji et al. 1994 WS.078 N278F 8 Tassabehji et al. 1994 WS.022 S298F 9 Tassabehji et al. 1994 MSU11 944del1 8 Morell et al. submitted Table 6 MITF mutations. The listing includes the family name, the mutation, the exon the mutation. Tassabehji et al.(155)discuss non-pathologic mutations which are not included in this table. MITF/Mi are members of the basic helix-loop-helix-leucine zipper (bHLH-Zip) family of transcription factors (FIGURE 4). In bHLH-Zip proteins, DNA binding is mediated through the basic domain. Dimerization occurs by the .helix-loop-helix domain and is stabilized by the zipper.“54' 165) This family of transcription factors bind as dimers and can form stable heterodimers with other members of the bHLH-Zip family.(‘66) Mi is expressed in the murine developing ear, eye, skin and in the adult heart.(157) Melanocytes are not essential for viability, however, Mi is essential for melanocyte differentiation, function and survival.(165) Figure 3: 17 MITF mutations. A diagram of the mutations characterized in the literature. The top panel displays the paired domain, the octapeptide domain and the homeodomain in relation to the mutations. The lower panel displays the mutations in relation to the nine exons. S = splice site mutations. 18 MITF Mutations i i 3293p 944del(1) del(R217) N2780 i i 1| 2 3 4 | 5 6 7 8 9 R203K 8250'” $298P 944del(1) del(R217) N27BD 19 Figure 4: bHLH Protein Family Class A and B bHLH Proteins variable loop __NH2 ——l\j//lf\‘lZZl—- COOH basic H1 residues Class C bHLH-zip Proteins variable loop NH2 COOH ilI 1. a; basic 1 H2 0 residues zip Dominant Negative (ld) HLH Proteins variable loop NH2 —lzz/\EZZ— com H1 H2 (modified from Baker et al. 1995)(44) 20 Other genes causing Waardenburg Syndrome Mutations in several genes have been identified that are associated with WSIV. Mutations have been characterized in Endothelin-3 (EDN3),(168) Endothelin Receptor B (EDNRB)(169' 170’ and the proto-oncogene RE T (Rearranged during Transfection)(171‘179) in individuals with Hirschprung disease and Waardenburg Syndrome. The endothelin family of 21 amino acid peptides act on G protein-coupled heptahelical receptorsfwo) EDNs are produced from a large prepropolypeptide precursor that is cleaved to the active 21-residue mature form.(18°) There are three endothelins (EDN1, EDN2 and EDN3) known in mammals. Each EDN gene product is encoded by a separate gene and expressed in vascular and nonvascular tissues. There are two subtypes of endothelin receptors (EDNRA and EDNRB) that are expressed in various cells. The receptors initiate several intracellular signal transduction events through heterotrimeric G proteins.(13°) EDNRB plays an essential role in the normal development of epidermal melanocytes and enteric ganglion neurons in mice and humans.(17°'18°'181) EDNRB in the mouse is called the piebald locus, which was predicted to be a candidate for WS.(54'173'181-182) Understanding function There are now at least five genes, PAX3 , MITF, EDNRB, EDN3 and RET, known to be involved in the Waardenburg Syndrome phenotypes. The expression, function, interaction with one another, if any, and the role of these 21 genes in development are now of great interest. One experimental approach to begin to elucidate the normal function of these genes is to examine the range of mutant phenotypes. Therefore, identifying additional mutations in all of these genes may help in further understand their function. CHAPTER ONE Screening for mutations in PAX3 and MITF in families with classical Waardenburg Syndrome and Waardenburg Syndrome-like phenotypes. INTRODUCTION The Goals of this study The main purpose of this study was to determine the molecular defects in WS genes segregating in WS and WS-like probands. The proband screening set consisted of 42 individuals (TABLE 7). The entire set of probands was screened for the eight exons of PAX3‘151' 183) and the nine exons of MITF‘163) by SSCP analysis, and sequencing analysis. The proband screening set included 33 WS individuals representing 33 families (in APPENDIX A TABLES 14-21) of which thirteen were WSI, nine were WSII, three were WSIV and seven were unclassified WS families. In addition, six SOD individuals and two families with WS-like clinical traits not usually considered a part of the WS phenotype were examined. The additional phenotypes of interest in the WS-like families were deafness, other neural tube defects and facial anomalies. The intent of including individuals with WS with other phenotypes and WS-like phenotypes, was to determine if these traits were caused by mutations in PAX3 or MITF. The analysis of two families, designated UoM1 and MSU22, with WS probands exhibiting WS and additional 22 23 traits will be discussed in detail in later chapters. There have been many observations of WS associated with various other phenotypes that may or may not be classified as traits of WS.("" 184490) The identification of mutations causing related phenotypes or disorders may help in further understanding the role of PAX3 and MITF in normal development. Table 7: 24 Proband screening set. The table is organized by the WS phenotypic type. The approximate number of individuals in the family are listed A = affected family members and U = unaffected family members. Any other clinical traits associated with the WS phenotype are listed including SOD = Septo-Optic Dysplasia, 18q = 18q Syndrome, CDHS = Craniofacial Deafness Hand Syndrome, AN = Anencephaly, DEAF = Deafness, CA = Craniofacial anomalies and OA = Ocular Albinism. The mutations identified in the set are included. 25 l |PROBAND scaeenme SET l i ; I 5 FAMILY l A T 0 TYPE gsscp ANALYSIS‘ MUTATION :REFERENCE M301 : 17 l 30 . WSI ' PAX3__MIIFH‘ _ ,. L.,.............. 2..---- WM302 6 ¥ 9 E ws1___W 94x3 MITF. .. _ _. ; ._ ,. M303 : 10 14 i ws1 PAX3 MITF ”100mg” WMorellet al. 1993 _____ _ MSUs : 11 , 26 ‘ WSI "PAX3 Mir?_..._-82219”386616911299“, MSU7 : 4 3 6 WSI . PAX3 Mifr"*__M74x_iMorell_etal1996 . M309 : 3 l 2 WSI PAX3 MITF.“ 874 19E1-.‘M9'9l etal._1996 _:' M3029 1 . 10 l ws1 gaxa MITF _--_.--_ “W ; UGM1-1 , 15 7 67 i ws1 PAX3 MITF __ . UGM1-2 i 10 l 20 l WSI , i PAX3 MITF 266del14 TMorelletal.1992 UGM1-3 l 4 . 11 l WSI PAX3 MITF _ ' UGM1-4 i 2 l 28 WSI PAX3 MITF ._-_-__- __ ; UofM1 7 i 9 WSI-3007 PAX3 MITF ~;‘__939_1_l_1___«lca_rey_et_a_l._1_9§6 M3025 1 ' 23 WSI-18q i PAX3 MITF L" _-...____ l l UoM4 2 5 wsn I PAX3 MITF WT" "‘"“"‘"““—'_‘ MSU15 ? 1 7 : WSI? PAX3 MITF fl " "" M3020 W? '~4 ; 14D. WSI? PAX3 MW?" ‘ "T "—7 M3030 : 1 ; 7 WSI? PAYSMMTTTW‘iT "T“T” M3022 15 . 39 WS-CA PAX3 MITF ’ ’T ”W: ‘_ MSU11 T 9 T 18 WSll-OA : PAX3 NEE... 94403211 TjMorell submitted ‘ M304 T 6 E 16 wsu j PAX3_MIT_F- l- ' “"“TT M306 ' 7 f 14 l wsil‘“ "P’AX’3 “Mm?" " ” ' J M3010 . 7 10 i wsn FAXE’MIT‘F“ “ " ' M3023 l 3 16 wsu PAX3 MITF " ’ "”"‘ ’ . M3024 2 2 wsu ; PAX3 MITF A ‘ —"” ” ; M3027 5 1o wsn ; PAX3 MITF j r UGM2-1 1 30 wsu 3 PAX3 MITF . i . UGM2-2 12 26 wsu l PAX3 MITF s WW , M3014 l 1 l 10 WSIII? , PAX3:MITP_::' " , ’ W ”"T‘ M3015 i 1 5 10 WSW? 3__WWPA)_ and M3017<159> in PAX3 exon two within the paired domain. Mutations were also identified in the homeodomain of PAX3 in MSU5, MSU7, MSU9 (Morell et al. 1996,(320) see manuscript in APPENDIX B). RESULTS The proband screening set included the probands from Waardenburg Syndrome families and Waardenburg Syndrome-like families. These individuals were screened for mutations in PAX3 and MITF. Methods for detecting mutations or sequence variants were SSCP analysis, cycle sequencing and direct sequencing techniques. A total of 34 primer pairs (TABLES 8 and 9 in Appendix A) were used for SSCP analysis. A diagram of the eight exons of PAX3 and the nine exons of MITF, with the approximate locations of each of the primers, is displayed on FIGURES 5 and 6 in APPENDIX A. Details of PCR and SSCP analysis are described in the Materials and Methods. The PCR fragments labeled with a33P- dCTP or a33P—dATP were electrophoresed on 0.5x hydrolink MDE gels. The length of electrophoresis was sequence dependent and determined empirically. Variant SSCP patterns were identified in several families. Many of the subtle SSCP pattern differences were not reproducible. All DNA fragments that displayed aberrant and reproducible SSCP patterns were subcloned and sequenced. Some of the PCR fragments were cycle sequenced without first sub-cloning. None of the PCR products was gel purified prior to SSCP analysis which could contribute to the complexity of the SSCP patterns. Only the families with reproducible SSCP variants are discussed below. 28 29 SSCP Analysis of PAX3 Among the 33 W81 and WSI-like probands in this study several SSCP variants were identified in PAX3. However not all of the variants were consistently identified in independent PCR amplification followed by MDE gel electrophoresis. For example, there were two different SSCP variants identified in exon one, in MSU1 and MSU2. Duplicate PCR amplifications were done on the genomic DNA for the probands of both of these families and the variants were not reproduced. In this WS proband screening set, mutations were identified in exon two in MSU3 and MSU17 prior to this study yet, no other SSCP variants were identified within exon two for the screening set. Prior to this study three SSCP variants were identified in exon six in MSU5, MSU7 and MSU9 , and the mutations have been characterized in these families (see APPENDIX B for reprint). In this study a SSCP variant was identified in UGM4 in exon six, no mutation was identified. The SSCP variant identified in UoM1 was reproducible and will be further discussed in chapter two. There were no SSCP variant patterns identified for any of the WS and WS-Iike probands for exons three, four or eight. SSCP analysis of MITF SSCP variants were identified in MITF. There were three different SSCP variants identified in exon one in MSU23, MSU26 and UoM1. The variant in 3O MSU26 in MITF exon one was a subtle pattern difference not present in either parent. This variant was also not reproducible in multiple PCR amplifications. The PCR product from MSU26 was included in the sequencing evaluation. The SSCP pattern identified in MSU23 was subtle. The DNA fragments from both the probands from MSU23 and MSU26 were subcloned into the TA-cloning kit pCRWII vector® (lnvitrogen) and the clones were analyzed by SSCP analysis. For each proband fifteen clones were screened by SSCP analysis. No SSCP variants were identified in any of the clones for either MSU23 or MSU26. In MSU26 along with the MITF exon one variant, there were two other possible MITF variants detected, one in exon three and another in exon eight. The parent’s genomic DNA was isolated and analyzed for the SSCP variants identified in the proband. The parents were both profoundly deaf. Neither parent had any of the three mentioned SSCP variations. Also after multiple PCR amplification of the proband’s genomic DNA, the exon three and exon eight variants were not reproduced. There were two other SSCP variants identified, one in MSU23 in exon two and one in UoM1 in exon nine. Neither of these subtle pattern variants were identified after consecutive PCR amplifications. No SSCP variants were identified for any of the other probands in the screening set in exons four, five, six or seven of MITF. 31 Cycle Sequencing Analysis of PAX3 and MITF Cycle sequencing was optimized for PAX3 exons two through seven and MITF exons one, two, six, seven and eight. The primers designed for the remaining exons of PAX3 and MITF were not suitable for cycle sequencing and were therefore omitted from the analysis. All the PCR products were gel purified on low melt agarose gels after PCR amplification. Several of the PCR primers were optimized for cycle sequencing (TABLE 10 in APPENDIX A) and were used to screen a number of the probands from the screening set. However, no sequence variants were identified in the probands screened by cycle sequencing methods. There were a few primers that were optimal for many of the DNA samples including PAX3 exon two, six and seven and MITF exon one, two and eight. However, the sequencing results, even using these primers, did not produce reliable data. PCR amplified DNA fragments were cycle sequenced from both MSU23 and MSU26. The sequence was of high quality with very little background. There were no sequence changes detected within the coding region of MITF exon one. However, not all of the 5'-untranslated region (UTR) sequence was readable by this method. This problem was addressed by subcloning these PCR fragments using the TA cloning kit (lnvitrogen). No sequence variants were detected in the region sequenced after cloning the fragments from MSU23 or MSU26. DISCUSSION The main goal of this study was to identify and characterize mutations in PAX3 and MITF in WS and WS-like individuals. The proband set included 33 WS individuals and eight WS-like individuals. There have been reports in the literature of various neurocristopathies associated with WS including meningocele,(193) meningomyelocelefm‘v 194' 195) spina bifida,(187' 196) cleft lip/palate,(186' 197) neuropathy,(198' 199) piebaldism,(188- 20°) vitiligo‘zm) and albinism.(2°2) The rationale of including WS-like individuals in this study, and many others,(155' 203) was to determine if mutations in PAX3 or MITF caused a WS-like phenotype. Several methods were employed to screen the exons of both PAX3 and MITF including SSCP analysis, direct cycle sequencing and sequencing of plasmid clones. The 42 probands (TABLE 7) were screened by SSCP analysis for all known coding exons of PAX3 and MITF (in APPENDIX A TABLES 8 and 9). All probands were included in the screen of both genes to establish controls for the normal SSCP patterns. Mutations were not expected in MITF in WSI individuals.“ 155' 160' 136- 197) PAX3 mutations were not expected in individuals with WSII. The inclusion of all samples in the screen also increased the number of chromosomes screened which could be used to demonstrate that any given variant was not a polymorphism. Once a variant was identified the genomic DNA was PCR amplified several times to assure the variant was 32 33 reproducible, not an artifact of PCR or a concentration dependent variant. Fragments with a persistent SSCP were then subcloned and sequenced. SSCP Analysis There were several variants identified by SSCP analysis. All possible variant patterns were documented and the genomic DNA was PCR amplified in duplicate to determine if the variant was real or an artifact. Several variants were not reproducible and excluded from further investigation. Any variant that persisted in multiple reactions was subcloned into the TA-cloning kit pCRTMII vector (lnvitrogen). Individual clones containing inserts were analyzed by SSCP analysis and clones identified with the variant SSCP patterns were sequenced. Families with characterized mutations MSU3,(153) UGM2,(152) MSU17,(159) MSU5,(320) MSU7(320) and MSU9(320) identified by SSCP analysis prior to this study were included in the mutational screening in this study. The SSCP variants identified in these families were consistently observed and the mutations characterized (TABLE 7; FIGURES 2 and 3). These DNA samples with characterized PAX3 and MITF mutations served as positive controls for the conditions used in this study for SSCP variant detection. There were three families that were kept in the screening set even after SSCP patterns were not reproduced. These included MSU23, MSU26 and in UoM1. MSU23 is a classic WSII family, and MSU26 is a family with hearing 34 loss and multiple anencephalic fetuses. UoM1 is discussed in chapter two. All three of the families had SSCP variants detected in MITF exon one. The PCR products generated during the SSCP analysis were subcloned. Two of the families, MSU26 and UoM1, were of considerable interest, because of their phenotypes. MSU23 was included because of the interest in MITF exon one. SSCP analysis of the clones determined which clones had the observed variant. In MSU23 and MSU26 no variants were identified in fifteen clones from each of the proband's PCR amplified DNA. Multiple PCR amplification of the genomic DNA from these two families did not consistently identify the SSCP variant. Cycle sequencing was also performed using PCR amplified and gel purified DNA from the probands from each family. No sequence changes were observed in the translated region of exon one of MITF for either MSU23 and MSU26. The 5'-UTR was not completely sequenced since the SSCP variants were not consistently observed. There was the possibility that a mutation was missed that fell within the 5'-UTR. The function and relevance of any base change in the 5'-UTR, however, would be difficult to prove. Therefore, further pursuit of this region was not warranted. The variant observed in UoM1 is discussed in chapter two. Cycle Sequencing Along with SSCP analyses Direct Cycle Sequencing protocols were used to directly look for mutations in PCR amplified genomic DNA for the exons 35 0f PAX3 and MITF. Direct sequencing has several potential advantages over the traditional cloning and sequencing protocols. Cycle sequencing reactions are cheaper, faster and theoretically more accurate since the cloning step is eliminated. However, cycle sequencing protocols used in this study were difficult to optimize. Several reactions were done for various exons but the sequencing reactions usually did not produce easily interpretable results. High background in the sequencing reactions made single base substitutions difficult to interpret for most of the exons. In addition, over the course of ten years of gathering samples, genomic DNA was isolated by various methods. The differences in the DNA preparations required individual primer optimization for many of the DNA samples. Cycle sequencing reactions were optimized for each primer of every exon used for the sequencing reaction. Rather than designing new primers, one of the PCR primers was used for the sequencing when possible (TABLE 10 in APPENDIX A). These were the same primers used for the PCR amplification (TABLES 8 and 9) and therefore, were not optimally designed for direct cycle sequencing according to the Amersham protocols. Both ATaq Cycle Sequencing Kit and ThennoSequenase (Amersham) were used. The intensities of the bands for the dd-NTPs varied using the ATaq method. The enzyme preferentially incorporates certain dd-NTPs causing many artifacts and high background. The ThermoSequenase method was designed by the manufacturer to eliminate the preferential incorporation of dd-NTPs. However, 36 this method still did not produce optimal sequencing results. Since many of the primers were those designed specifically optimal for PCR, not sequencing, several of the primers did not have optimal lengths or GC-content recommended by Amersham in the protocols. The sequencing extension reaction requires the exclusion of one of the dideoxynucleotides for the primer to be elongated according to the protocols for cycle sequencing. The length and the GC-content of the elongated primers was not consistent between the different primers for each of the exons used in the study (TABLE 10). Direct sequencing from PCR amplified DNA was suggested to be the most efficient and stringent way to screen for mutations. DNA from several of the WS family members was PCR amplified and cycle sequenced. No new mutations were identified by this method. However, the high background and numerous artifacts made identification of single base substitutions difficult. Further optimization would have added considerable time and material expense to this portion of the study. Therefore considering the technical obstacles this method was abandoned. Mutation Detection Prior to screening the entire data set of thirteen WSI, nine WSII, three WSIV and seven WS-like individuals several families MSU3, UGM2, MSU5, MSU7, MSU9 and MSU17 were screened for mutations in exons two, five and six for PAX3. Seven new WSI mutations were identified and characterized 37 (TABLE 7). The mutational searches were dependent upon the availability of the intron flanking sequence to design PCR primers. The entire proband set was analyzed for mutations once primers for all of the exons of PAX3 and MITF became available. A total of 42 individuals were screened in this study, six mutations in WSI individuals were identified and characterized in PAX3, one mutation in PAX3 in a family with CDHS and one mutation in a WSII family in MITF. Herein one of the six mutations identified in PAX3 was characterized in a WSI family. No new MITF mutations were found in this study among the nine WSII individuals. It is estimated that all WSI families map to PAX3.(5> In a recent study of one hundred and thirty-four families, PAX3 mutations were identified in the coding regions in 20/25 ws1 and WSIII individuals.<155> In the 42 probands in this screening set, there were thirteen WSI individuals. PAX3 mutations were detected in six (46%) of the unrelated WSI individuals. Although this estimate is lower than the expected 80%(155), considering the small sample size the maximum number of mutations in WSI individuals in the known regions of PAX3 may have been identified in this study. MITF mutations causing WSII have only recently been identified. For WSII individuals approximately 20% of the mutations have been identified in MITF. (155) In this screening set, one MITF mutation in nine (11%) unrelated 38 WSII individuals was detected. The results, however, were consistent with the published mutation detection expectation in MITF among WSII individuals. There may be at least one other gene responsible for the W811 phenotype that is yet to be identified. The other WSII gene(s) may be important in the regulation of the genes responsible for normal neural crest cell migration and differentiation. There are six probands in this screening set that were not classified as WSI or WSII. Complete clinical data was lacking for these families, therefore, they could not be characterized. However, they were included in the mutational screening. No mutations were detected in the six unclassified WS individuals. The set also included three WSIV probands that were not included in the WSI or WSII calculations. Also not included in the estimates were the six SOD individuals, MSU17 with CDHS or MSU26. Evaluation of Clinical Data Numerous factors may have contributed to the small number of mutations identified in this analysis. However, the most important depends on accurate collection and interpretation of the clinical data for each family. The data on each family was carefully evaluated to be sure families were classified correctly into the WS sub-categories. My evaluation was based on the data in our files for all the families focusing on the hearing tests, W-index measurements and the mention of any 39 other clinical traits. The data for each family is described in detail in TABLES 15 through 22 in APPENDIX A. Families in this set were ascertained by different clinicians and/or genetic counselors from all over the United States as well as from out of the country (TABLE 11 in APPENDIX A). The data sheets (see APPENDIX B) supplied to them were completed to various degrees, with emphasis on different portions of the phenotype. Often pedigrees were not included or the detail was limited. This made interpretation of the clinical evaluations difficult. For example, many of the families did not have hearing tests, eye exams, inner-canthal, inner-pupillary and outer-canthal measurements. Other clinical traits not classified as WS may or may not have been included. Pictures were rarely available of affected family members. Most of the descriptions of the clinical data were vague and missing actual reports. Families with questionable phenotype or without W—index ratios were not included in the estimates for the approximate mutation detection. Another factor that may influence the number of mutations detected is the improper classification of dystopia canthomm with approximately 98% penetrance. PAX3, when mutant, is responsible for the occurrence of dystopia canthorum; and is thought to play a direct role in skull and facial development.(2°4) This gene may also play an indirect role in development by activating other genes that are responsible for skull organization. Variation in the inner canthi, could be mistaken for WSI when in fact the clinical manifestation is something quite different.‘205 206) Families with craniofacial 40 anomalies should be carefully evaluated before classifying them as WSI or WSII, if they fit the other WS criteria. This does not seem to be the case in our screening set, with the exception of MSU22. There may be skull malformations that are caused by mutations by PAX3 that do not exhibit the other characteristics of WS. An example of this is Craniofacial Deafness Hand Syndrome (CDHS) described originally in 19831158) The clinical manifestation of CDHS is distinct from Waardenburg Syndrome yet a mutation was identified in exon two of PAX3 within the paired domain.(‘59) Mutations Due to Deletions There are several other explanations for a possible lower than expected efficiency of PAX3 mutation detection in WS probands. Cytological analyses could have been done to detect very large deletions in the region of PAX3 or MITF. Deletions of PAX3 have been reported.(2°7) However, a deletion that was submicroscopic could be overlooked. Such deletions could include the regions homologous to one or both of the primers, the entire gene or a large segment of the gene. None of these types of deletion would be detected by the PCR based methods used in this study. There are several methods that can be used to detect deletions, including: competitive quantitative PCR amplification, southern blotting, cytological testing for submicroscopic deletions using fluorescent probes, and possibly identifying excess homozygosity. 41 Mutations in Regulatory Regions Another possible explanation for the low number of mutations detected in this screening set, is that the mutations responsible for the WSI phenotype are within regulatory regions of either PAX3 and/or MITF. These regions may be near the coding sequence or may be hundreds of kilobases away.(2°8'21°) A position effect mutation 85 kilobases away from the 3'-end of the PAX6 gene causes aniridiafz“) Mutations in regions downstream or upstream of the coding region may be difficult to identify.(2‘2) Mutations in a regulatory region of either PAX3 and/or MITF may cause the WS phenotype. Regulatory regions may include promoters, enhancers, silencers or even splicing mutants that create cryptic splice sites within introns or alter the branch point site. Mutations within regulatory regions may affect the function of a gene. Mutations in Alternate Transcripts The existence of alternative transcripts may also explain why more WSI mutations in PAX3 and W811 mutations in MITF were not identified. Several of the PAX genes, including PAX2, PAX8, PAX6 have alternative transcripts that, change the 3'-end of the gene altering the carboxy terminus.(76- 213' 214) Two alternative transcripts of PAX2 are expressed in the human fetal kidney with no observable difference in temporal expression.(214) There are six alternative 42 transcripts identified in murine PAX8 that are temporally and spatially regulated during development in the developing central nervous system (CNS), the thyroid gland and the embryonic kidneys.(76) There are two isoforms of PAX6 mRNA expressed in the developing eye, brain, spinal cord and olfactory epithelium.(213) There are at least two isoforms of PAX3 mRNAs expressed in the human adult cerebellum and skeletal muscle as a result of alternative splicing.(55) The 3'-end of these isoforms would not be screened for mutations by the primers used in this study. In addition to these alternate forms there may be other alternative transcripts of PAX3 that have not been identified. Once these different messages are identified there will be new regions to screen for mutations in classical WS families. Two different forms of Mi have been identified. One expressed in melanocytes (‘37) and the other in heart and skeletal muscle.(215) The difference in the 5'-ends of these two forms may be generated by different promoters.(167) There is the possibility that other forms of MilMITF exist. Technical Obstacles with SSCP The low number of mutant alleles identified in this screening set may be due to the detection methods that were used in this study. A key component of SSCP analysis is primer design. The primers must be specific for the sequence of interest, and the fragment generated by PCR should be within an 43 appropriate size range for optimal variant detection. The optimal size range for PCR fragments used in SSCP analysis are 100-200 base pairs for 80% detection, for fragments of 300-400 base pairs the detection frequency is less than 50%.(2‘6’ Several of the primers used in this study were designed when little was known about the intron sequences flanking the exons of PAX3. Therefore they could not be designed for optimal SSCP analysis. At least one of the primers used with this screening set falls within the 5'-end of the exon (see FIGURE 5 in APPENDIX A). This could account for some of the undetected mutations. The majority of fragments analyzed in this study were between 200 and 400 base pairs, two fragments were greater than 500 base pairs. The fragment sizes detected by the primer pairs utilized in this screen are listed in TABLES 8 and 9 in APPENDIX A. The sub-optimal fragment size could explain why the observed mutations in WSI families were lower than expected. However, it is important to note that the primers used in this study are similar, but not identical to those used in other screens reported in the literature.(15" 155- 203) Deciphering normal and variant conformational patterns can sometimes be difficult. Often there are background bands that vary in intensity and in pattern. Some of this variation can be eliminated by gel purifying the samples after "cold" PCR prior to the "hot" reaction. Very few of the samples were gel purified prior to PCR. A complex pattern of bands may still exist for a variety of other reasons, including various DNA and primer concentrations, overloading 44 the sample, differing PCR amplification efficiencies, electrophoresis conditions and overamplification. Genomic DNA samples not isolated with the PUREGENE kit were often difficult to PCR amplify thus requiring a two step PCR method. Most of the samples were PCR amplified for thirty cycles without isotope and then for twenty-six cycles with isotope. The same primer pairs were used for both the "cold" and "hot" reactions, possibly causing the overamplification. Important variables for SSCP analysis include: electrophoresis temperature conditions, gel composition and wattage. In this study, gels were run at 4°C and at 23°C. They were prepared with MDE and/or native acrylamide with or without glycerol. The power was set at: 8, 15, 20 or 50 Watts with differing electrophoresis times. The time each fragment should be run for adequate separation is dependent on the sequence as well as the gel and electrophoresis conditions and was determined empirically. Although many combinations of the temperature conditions, gel composition and wattage were used in this study, any one of the combinations may have been under- represented. However, SSCP variants identified by one condition were also observed using others conditions. Also, the SSCP variants observed in families with previously documented mutations were consistently demonstrated using a variety of the above conditions. 45 Mutations in Other Genes Mutations in EDNRB, EDN3 and RET have been characterized in individuals with Hischprungs disease (HSCR) and Waardenburg Syndrome. Hirschprung disease or aganglionic megacolon, is associated with the congenital absence of intrinsic ganglion cells in both the myenteric and submucosal plexuses of the distal gastrointestinal tract, leading to the failure of innervation of the colon.(17°) HSCR is estimated to occur in 1/5000 live births with a sibling recurrence risk of 4%.(25) Males are more susceptible than females.(25) HSCR is considered to be a developmental defect stemming from a failure of neural crest cell migration, differentiation or colonization during gestation weeks five to twelvemo) Mapping studies implicated several genes as possible candidates. After the discovery of the genes responsible for the Hirschprung disease phenotype the question of screening our families for mutations needed to be addressed. We have three families, MSU12, MSU13 and MSU28, in our data set that appear to exhibit Hirschprung disease, along with WS (TABLE 7). All three HSCR families have been screened for mutations in PAX3 and MITF by SSCP analysis but not EDNRB, EDN3 or RE T. No apparent SSCP variants were identified in these three families. CONCLUSION Considering the possible errors in experimental design and the missing clinical data there may be more information to be collected from the 31 Waardenburg Syndrome families discussed in this study. One family designated UGM1, has been shown to be linked to PAX3; however, a mutation within the coding region has not been identified (data not shown). There are several aspects that could still be considered for exploration. Direct sequencing with optimal primers of each exon of both PAX3 and MITF for each proband is one possibility. The use of automated sequencers could also eliminate differences in reaction conditions. Several of the families in this study were missing essential clinical data. This made clear classification of WS difficult; thus without accurate and complete clinical data the diagnosis may not be reliable. Prior to undertaking any large scale screening for mutations, a thorough evaluation of the available clinical data is important to ensure that time is not wasted on screening individuals that are unlikely to have mutations in the genes of interest. The identification of submicroscopic deletions may be possible with the use of competitive quantitative PCR amplification, cytological analysis looking for the deletions or identifying an excess homozygosity. All three of the above techniques are time consuming and technically challenging. The results may not be conclusive. Therefore, without linkage data demonstrating the gene of 46 47 interest is responsible for the phenotype, optimizing the techniques may not be cost efficient. In this study markers linked to PAX3 could be used to look for an excess of homozygosity; which may indicate that the alleles are actually hemizygous. The small sample size will not give statistically significant results. MATERIALS and METHODS Family Identification and DNA isolation During the period of this study, WS families were ascertained in various ways and by many different individuals. Several of our families were identified through schools for the deaf, in the United States and in Indonesia. Included in this study were six Indonesian families identified and collected during multiple trips made by Drs. Asher and Friedman to Indonesia (collaborators: SuKarti Moeljopawiro, Sunaryana Wlnata and I Nyoman Arhya, Udayana University, Denpasar, Bali Indonesia). The more recent families used in this study were identified through collaborations with various physicians and genetic counselors (TABLE 11 in APPENDIX A). Most of the clinical data was collected by different physicians and, as a consequence, is not complete in every respect. The entire proband set is outlined in TABLE 5 in Chapter one. Each family is described in TABLES 14-21 to the extent that our records are complete. All family members contacted were informed of the study and signed consent forms in order to participate (see APPENDIX B for copy of blank forms). Patient DNA was obtained from either lymphocyte cells from blood or cheek cells isolated by a saline mouthwash method. DNA obtained from blood was isolated using the PUREGENETM kit (Gentra Systems). The blood sample was incubated with RBC lysis solution, then centrifuged at 2000xg at room temperature. The white cells formed a pellet and the supernatant was 48 49 discarded. The white cells were then incubated with WBC lysis solution at 37°C. Protein precipitating solution was added and the supernatant was collected. Two volumes of ethanol were added to the supernatant. The DNA was precipitated and was washed in 70% ethanol for several minutes and then resuspended in T1oE1 pH 8.0. In families identified in the early 1990's, the DNA was isolated by various other methods including phenol extraction. The mouthwash samples were isolated in 10 ml 0.9% sterile saline. The solution was centrifuged and 500 pl 0.05 N NaOH was added to the pellet. Then the solution was incubated at 95°C for five to ten minutes, stored on ice for zero to five minutes before adding 500 pl T10E1 pH 8.0. Polymerase Chain Reaction DNA amplification was performed on a MJ Research, Inc., Thenno Controller using the Polymerase Chain Reaction (PCR) for each of the known exons of both PAX3 and MITF. See primer list (TABLES 8 and 9). All of the primer pairs for PAX3 and MITF were amplified using the PCR buffer recipe: 10 mM Tris-HCI, pH 8.3, 1.5 mM MgCl2 and 50 mM KCI (Boehringer Mannheim) except exon seven of PAX3. The reaction buffer for exon seven contained: 10 mM Tris-HCI, pH 9.2, 1.5 mM MgCI2 and 75 mM KCI (Stratagene Opti-PrimeTM PCR Optimization Kit, buffer 10). Standard amplifications were performed in a total volume of 25 pl which included: 100 ng genomic DNA, 0.1-0.2 pM of each primer, 0.25 mM dNTP's, 50 2.5 pl 10x PCR buffer, 0.2 units of Thermal Stable DNA Polymerase (TSP). The cycling parameters were: 94°C for 1 minute, a specified annealing temperature (TABLES 9 and 10), 72°C for 3 minutes for 15-30 cycles and then a 10 minute final extension at 72°C. Labeling reactions also included a33P- dATP or a33P-dCTP (Amersham and/or Andotech). Markers linked to PAX3 were used to analyze informative families for linkage. In this study MSU22 was typed for the markers described by Wilcox et al.(183) and Macina et a|.(132) See primer list in TABLE 13. The PCR amplification followed the standard protocol outlined above. Single Strand Conformation Polymorphism Single Strand Conformation Polymorphism (SSCP) was performed on all known coding exons of PAX3 and MITF for all of the probands. The PCR was performed as above. A 2-3 pI aliquot of each amplification was denatured for 3 minutes at 95°C, chilled on ice, then electrophoresed on a MDETM 0.5x Hydrolink® gel (AT Biochem). The MDE gels were prepared with 12.5 ml MDE, 3 ml 10X TBE (Tris base, Boric acid and EDTA), 35 ml dH20, 540 pl 10% ammonium persulfate (APS) and 30 pl TEMED (Tetramethyl-ethylenediamine). Electrophoresis proceeded in 0.6X TBE buffer in both the upper and lower chambers of a NUGENErationTM Sequencing Systems (OWL Scientific models 818 and $28). MDE gels were run at 8 Watts both at room temperature and at 4°C. Gels were also run under the above conditions with and without 10% 51 glycerol. Select gels were run at 15 or 50 Watts at both temperatures. The run time varied from four to 12 hours for each exon due to the size of each fragment and the sequence. Control samples were included for each analysis to distinguish normal patterns from SSCP variants. For DNA samples with a SSCP variant the genomic DNA was PCR- amplified multiple times and run on several gels to determine if the variant pattern was reproducible. The PCR products from individuals with abnormal and reproducible SSCP patterns were either subcloned and sequenced (Amersham SequenaseTM Version 2.0 Kit, or a modified version) or directly sequenced by cycle sequencing protocols (Amersham ATaq CycleTM Sequencer or ThermoSequenaseTM kits). Allele Specific Amplification (ASA) Allele-specific primers were designed so that the single base substitution was at the 3'-end of the primer. An allele specific primer (T F195) was synthesized to verify the base change in family UoM1 in PAX3 exon seven. TF195 was amplified with TF141 for exon seven and in the same reaction tube h a PAX3 exon four control primer pair set TF35 and TF36. The primers were optimized using the Stratagene Opti-PrimeTM Optimization Kit and buffer #2 (10 mM Tris-HCI, pH 8.3, 1.5 mM MgCl2 and 75 mM KCI). The cycling parameters were as above with an annealing temperature of 65°C. The exon four primers (T F35-36) were used for a control fragment, with a product length of 242 base 52 pairs and the allele-specific fragment amplified with TF141 and TF195 produced a 270 base pairs product. (see the primer list TABLE 8). The fragments were separated by electrophoresis on a 4% 3:1 NuSieve for 3 hours and visualized with ethidium bromide staining. Cloning fragments After determining that a SSCP was reproducible, the mutant PCR product containing the SSCP variant was cloned into a pGEM®-T Vector System (Promega) or the TA-Cloning® kit (lnvitrogen). The insert:vector molar ratio was either 3:1 or 1:1. The ligation reactions included: T4 DNA ligase 10X buffer (300 mM Tris-HCI, pH 7.5, 100 mM MgCI2, 100 mM D'I'I' and 10 mM ATP), 50 ng pGEM®-T vector, PCR product, T4 DNA ligase (1 Weiss unit/ml) and dH20 to a final volume of 10 pl. The ligation reaction mix incubated for three to twelve hours at 15°C. The transformation step used a 2 pl aliquot of the ligation reaction mix. After the ligation reactions, the vector was introduced into the Sure Cells by either electroporation or by the cell shock protocol described by Invitrogen®. Cells were grown on LB (Luria-Bertani) plates with ampicillin (50 pg/ml), IPTG (200 mg/ml) and X-Gal (20 mg/ml in dimethylformamide) utilizing the blue-white selection method. LB medium contains 10 grams Bacto®-tryptone, 5 grams Bacto® yeast extract, 5 grams NaCl and 15 grams agar per liter. Inserts cloned into pGEM®-T vector were verified by either restriction digests or PCR. Clones were then propagated in 53 liquid LB with ampicillin (50 pg/ml) for eight to twelve hours at 37°C and shaking at 225 rpm. Plasmid DNA was isolated using the WizardTM Minipreps DNA Purification Systems (Promega). SSCP analysis was performed on individual clones and compared to the patterns generated from the genomic DNA in order to identify clones containing the mutation responsible for the SSCP variant. DNA Sequencing The DNA sequence of the cloned fragments containing SSCP variants was determined using the fonNard and reverse primers from the Sequenase 2.0 kit following the manufacturer's protocol. A modified version of the Sequenase protocol called the Quick Double-Strand DNA Sequencing Protocol was used. This method does not require an ethanol precipitation after the denaturation step and thus saves about one hour. Primer concentrations generally ranged from 1-2 mM however, primer concentrations as high as 20 mM were also used. In the denaturation step, the DNA, primer and 1N NaOH was incubated at 68°C for ten minutes. Freshly prepared TDMN (Tes, concentrated HCI, 1M MgCI2, 4M NaClz and 1M DTT) was added in the annealing step. For the cycle sequencing or direct sequencing reactions the PCR products were separated by electrophoresis on 1-2% low melt (FMC) agarose gels and purified using the VWzardT" PCR Purification Systems (Promega). Cycle sequencing reactions were optimized for each primer for each of the exons of PAX3 and MITF. The annealing temperature varied for each primer 54 (TABLE 10) and either 67°C or 72°C were used for the termination reaction. Cycling was done 50 times at both steps, the overall reaction time was approximately three hours. Cycle sequencing reactions were performed following the manufacturer's protocol using either the ATaq CycleT'V' Sequencing kit (Amersham) or ThermoSequenaseTM Cycle Sequencing kit (Amersham). Sequencing reactions were separated by electrophoresis on 6% acrylamide gels with either flat or wedged spacers. The gel solution was made in 600 ml volumes and stored at 4°C and includes: 90 ml 40% acrylamide (Biorad 19:1 solution), 60 ml 10X TBE, 288 g urea and dH20 to 600 ml. The standard gels use 75-100 ml from the prepared acrylamide stock, 30-50 pl TEMED and 75—100 pl APS. The gels were pre-warmed for 30-45 minutes in 0.5X TBE buffer prior to denaturing the PCR fragments at 94°C for two to ten minutes. Electrophoresis proceeded at 55 or 95 watts in order to maintain a constant temperature of 55°C. Gels were fixed in 20% methanol and 10% acetic acid for 45 minutes, dried and exposed to HyperfiImW-MS (Amersham). CHAPTER 2 A Waardenburg Syndrome type I family with the proband exhibiting WSI and Septo-Optic Dysplasia. INTRODUCTION Septo-Optic Dysplasia Septo—Optic Dysplasia (SOD) also known as Septo-Optic-Pituitary Anomaly (SOPA) has a highly variable phenotype.(217'223) Key characteristics of SOD are optic nerve abnormalities, partial or complete absence of the septum pellucidumm" 225’ and endocrine dysfunctionmwzs) The pituitary dysfunction can be highly variable and change throughout life. Some of the key characteristics of pituitary dysfunction are: short stature, neonatal hypoglycemia, seizures, apnea, cyanosis, jaundice, thermal instability and fever, CNS abnormalities and mental retardation.(226' 227' 229233) The primary diagnostic characteristic of SOD is visual impairment, including amblyopia and nystagmus.(234' 235) The cause of SOD is not known. There are some correlations between a young age of the mother and even possible drug use with the occurrence of SOD. There are also examples of various infections during pregnancy including Rubella, viral and urinary tract infectionsfzza' 2357-39) There are two 55 56 cases with relatives displaying SOD suggesting the possibility of a genetic component, yet all other reported occurrences of SOD have been sporadic.(24°- 241) SOD arises early in gestation and represents a mild _form of holoprosencephaly.(227' 242’246) The approximate time in embryogenesis is at about four to six weeks gestation when the anterior wall of the diencephalon invaginates and the optic nerve ganglion cells develop.(247) Findings of widespread calcification and glial nodules in the septal region and the anterior hypothalamus suggest that a destructive process with necrosis and neuronal loss between eighteen totwenty weeks has occurred?“ 243) This is approximately the time when the septum pellucidum forms (242' 243) (FIGURE 7). Septum Pellucidum Figure 7 Medial surface of a 4 month human embryo brain. The arrow is pointing to the newly forming septum pellucidum, the broken line indicated the future expansion of the corpus callosum. (modified from Langman's Medical Embryology, Sixth Edition)(322) 57 The morphological role of the septum pellucidum is to divide the two telencephalic ventricles and to permit the adhesion of the fornix to the corpus callosum‘217- 218) (FIGURE 8). When agenesis of the septum pellucidum occurs, the mass of embryonic neuralgia tissue which forms the commissural plaque between the origin of the corpus callosum and the anterior commissure does not forrn<217- 2"” (FIGURE 9). Thus the fornix is not attached to the corpus callosum. If there is a distinct function of the septum pellucidum it is not known.(249) 58 corpus callosum fornix septum pellucidum Figure 8 Normal frontal view of the brain. Arrows pointing to the corpus callosum, septum pellucidum and the fornix. (modified from The Human Brain and Spinal Cord, Lennart Heimer).(323) Figure 9 Frontal view of a brain with Septo-Optic Dysplasia. Notice the absence of the septum pellucidum which causes an enlargement of the ventricles. The corpus callosum and fornix are not attached and seem malformed. (modified from de Morsier)(242) 59 Ascertainment of an Individual with WSI and SOD UoM1 is a four generation family (FIGURE 10) that was ascertained at the University of Michigan Pediatric Genetics clinic by Dr. Jeffrey Innis. Some members of the family exhibited a typical WSI phenotype (TABLE 18 in APPENDIX A). The proband in this family has Septo—Optic Dysplasia (SOD) and WSI, however, the other six individuals with WS do not have SOD. There are no reports in the literature of an individual or family with WS and SOD. There are reports of other clinical associations with SOD including digital anomalies,(25°) cleft face,(251' 252) craniofacial anomalies such as Apert Syndrome‘253) and other severe brain anomalies not including WS.(254) Six additional individuals with SOD and/or optic nerve hypoplasia (SOD1-SOD6) but not WS were ascertained by Dr. Innis in collaboration with Dr. Nancy Hopwood. All of these individuals were sporadic cases of SOD, identified at the University of Michigan genetics clinic. A description of the phenotype for each individual is in TABLE 21 in APPENDIX A. Although none of these individuals demonstrated any characteristics of Waardenburg Syndrome they were included to investigate a possible connection between a PAX3 or a MITF mutation and SOD. There are no reports in the literature that individuals with SOD have been examined for mutations in either PAX3 or MITF. Until ascertaining UoM1 there would have been no reason to suspect such a connection. 60 2i 5! 5 . é @- <2 <2 Ill é Figure 10 The pedigree of UoM1. The symbols are divided into two halves, the right portion being shaded if the WS phenotype is present and the left portion if SOD is present. The WS phenotypeincluded: dystopia, premature graying and deafness. RESULTS UoM1 was screened for all of the exons of PAX3 and MITF. There were three SSCP variants identified in the analysis, one in PAX3 exon seven, and two in MITF, one in exon one and the other in exon nine. SSCP Analysis of PAX3 The SSCP variant found in PAX3 exon seven was not identified in the any of the other 42 probands (FIGURE 13). The SSCP was reproducible and seen in all WS affected individuals in the family. The PAX3 exon seven SSCP variant was not seen in the unaffected mother of the proband. The PCR fragment was gel purified and cloned into the p-Gem-T Vector. The materials and methods are described for all experiments in Chapter one. The clones were analyzed by SSCP analysis and clones with the variant SSCP and the normal pattern were sequenced using the Sequenase version 2.0. A guanine (G) to cytosine (C) transversion was identified in exon seven predicting an amino acid change at codon 391 changing a glutamine (Q) to histidine (H) (FIGURE 11). PCR amplified genomic DNA from several WS individuals from this family were directly sequenced and the same base substitution was identified. An allele-specific primer was designed (T F195) and used to amplify genomic DNA in combination with a normal upstream exon seven primer (TF140). The PAX3 exon four primers (TF35-36) were included in 61 62 the same PCR amplification as a control (see TABLE 8 in APPENDIX A for primer description). All WS individuals in the family amplified the mutant allele- specific fragment while the normal mother only amplified the control band (FIGURE 12). A set of 60 random individuals were screened by PCR amplification with the mutant allele-specific primer set and the control set. None of the random individuals had the allele specific fragment yet all amplified the control fragment. Figure 11: 63 PAX3 exon 7 normal and the mutant DNA sequence and the normal and mutant protein sequence. The normal sequence is on top for both the DNA and protein and the mutant is on the bottom. There is a G to C transversion, that is boxed in with an arrow. This change alters the amino acid sequence substituting a glutamine (Gln) with a histidine (His) which occurs at codon 391. This is the 3'-end of exon seven and may alter splicing since the splice consensus is changed from Aggtcagt to Acgtcagt. 64 1 10 20 30 40 so 60 70 S' AGAAAACATGATGGTTGACAATCTTTTTCATTTCAGCTGTGTCAGATCCCAGCAGCACCGTTCACAGACC *************************************************************t******** 5' AGAAAACATGATGGTTGACAATCTTTTTCATTTCAGCTGTGTCAGATCCCAGCAGCACCGTTCACAGACC 1 10 20 30 40 50 60 70 71 80 90 100 110 120 130 140 TCAACCGCTTCCTCCAAGCACTGTACACCAAAGCACGATTCCTTCCAACCCAGACAGCAGCTCTGCCTAC ***********************************************t*t******************** TCAACCGCTTCCTCCAAGCACTGTACACCAAAGCACGATTCCTTCCAACCCAGACAGCAGCTCTGCCTAC 71 80 90 100 110 120 130 140 141 150 160 170 180 190 200 210 TGCCTCCCCAGCACCAGGCATGGATTTTCCAGCTATACAGACAGCTTTGTGCCTCCGTCGGGGCCCTCCA *************i*****************i***1********************************** TGCCTCCCCAGCACCAGGCATGGATTTTCCAGCTATACAGACAGCTTTGTGCCTCCGTCGGGGCCCTCCA 141 150 160 170 180 190 ' 200 210 211 220 230 240 250 260 270 280 ACCCCATGAACCCCACCATTGGCAATGGCCTCTCACCTC TCAGTCCCGTGTTTCTAGACAGACGATT **************************************** ****************************t ACCCCATGAACCCCACCATTGGCAATGGCCTCTCACCTC TCAGTCCCGTGTTTCTAGACAGACGATT 211 220 230 240 250 \\, 260 270 280 281 290 TGCTGTATACC 3' *********** TGCTGTATACC 3' 281 290 ValSerAspProSerSerThrVaIHisArgProGlnProLeuProProSerThrValHisGlnSerThrllePro ValSerAspProSerSerThrValHisArgProGInProLeuProProSerThrValHisGlnSerThrllePro SerAsnProAspSerSerSerAIaTerysLeuProSerThrArgHisGlyPheSerSerTerhrAspSer SerAsnProAspSerSerSerAlaTerysLeuProSerThrArgHisGlyPheSerSerTerhrAspSer PheValProProSerGlyProSerAsnProMetAsnProThrlleGlyAsnGlyLeuSerPro In PheValProProSerGlyProSerAsnProMetAsnProThrIleGlyAsnGlyLeuSerPro is Figure 12: 65 ASA of the PAX3 exon 7. The allele specific amplification of the PAX3 exon 7 mutation identified in UoM1. Five of the seven WS affected individuals were analyzed by ASA. The WS affected individuals are have the allele-specific fragment which is 270 base pairs. The unaffected mother (#10) does not have the allele-specific band but does have the control fragment which is 242 base pairs. The four random individuals only have the control fragment. 66 Allele-specific Amplification 4109611R1R2R3R4 67 Figure 13: SSCP variants identified in UoM1. The top panel demonstrates a portion of the proband screening set analyzed by SSCP for PAX3 exon 7, number 27 represents the proband of UoM1. The lower panel demonstrates a sample of probands analyzed by SSCP for MITF exon 1, number 27 represents UoM1. 68 SSCP Analysis 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 26 27 28 29 30 69 Mutation Identification in MITF The exons of MITF were also analyzed in UoM1 for mutations. Two SSCP variants were identified in MITF. In order to determine if the observed SSCP variants identified in MITF were reproducible, the genomic DNA from the proband was amplified six times along with both of the proband's parents. The SSCP variant that was identified in exon nine was not consistently observed, therefore it was not further analyzed. An obvious SSCP was identified in exon one (FIGURE 13) however, the variant was difficult to identify in separate PCR amplifications electrophoresed multiple times on different gels made with the same recipe. In order to determine if there was a sequence change responsible for the SSCP variant in exon one of MITF cycle sequencing was performed on the PCR- amplified fragment for the proband and his parents. No sequence variations were observed in the mother, father or proband within the coding region of exon one of MITF. The proband's PCR amplified DNA fragment of 290 base pairs was subcloned and 60 clones were screened by SSCP analysis. None of the clones showed the obvious SSCP variant identified originally in the proband. Two clones with two different SSCP variants, different from the one observed in the genomic DNA from the proband, were sequenced. The only sequence change 70 was within intron one beyond the splice site junction. No other sequence changes were observed in either clone. Other Individuals with SOD Six individuals with SOD were ascertained from the University of Michigan Pediatric Endocrinology clinic. All six individuals were screened for mutations in PAX3 and MITF by SSCP analysis. Two different, subtle SSCP variants in exon seven of PAX3 were detected in two of the SOD individuals, SOD2 and SOD4, but were not reproducible. The PCR fragments were cloned and 25 clones were analyzed by SSCP. No variant clones were detected. DISCUSSION Description of UoM1 UoM1 is a four generation family with Waardenburg Syndrome Type I and one individual with both WSI and Septo—Optic Dysplasia (SOD) or de Morsier Syndrome. Individuals in this family present a typical WSI phenotype with dystopia canthorum, pre-mature graying and deafness. The proband however, also has SOD. The proband has optic nerve hypoplasia and absence of the septum pellucidum. The father was examined by magnetic resonance imaging (MRI)(255- 256) and found to have a normal septum pellucidum and other intracranial structures. He has no vision loss and no apparent endocrine dysfunction. Therefore, there is no indication of SOD in the father of the proband. Due to the possibility of some connection between the presence of SOD and either a PAX3 or MITF mutation, several additional individuals were ascertained with SOD. This was the first opportunity to explore a possible genetic basis for Septo—optic dysplasia. One hypothesis is that mutations in either PAX3 or MITF were responsible for both the WS phenotype and/or the SOD. 71 72 Mutational Analysis of PAX3 and MITF The UoM1 proband was screened by SSCP analysis for PAX3. A SSCP variant was detected in PAX3 exon seven that was not observed in the 40 other probands (FIGURE 13). There was a glutamine (G) to cytosine (C) transversion identified in the third position of the last codon of exon seven (FIGURE 11). The substitution was verified in three other WS affected individuals in the family by directly sequencing the products from PCR-amplified genomic DNA. This transversion mutation predicts an amino acid change at codon 391 changing a glutamine (Q) to histidine (H) (FIGURE 11). This single base substitution also predicts a splice site mutation that may create a truncated protein due to the premature stop approximately 70 nucleotides downstream, which would possibly eliminate a portion of the PAX3 transcriptional activation domain. This putative splice site mutant is predicted by the consensus splice sites.‘257- 258) Although there are now more than 50 mutations causing WSI, this is the first example of an exon seven mutation of PAX3 identified in patients with WSI (TABLE 5). In order to verify the sequence change in the genomic DNA an allele- specific primer was designed (TF195) and used to amplify genomic DNA in combination with a normal upstream exon seven primer (T F140). The PAX3 exon four primers (T F35-36) were included in the same reaction mix as a control (see TABLE 8 in APPENDIX A). The expected fragment sizes were 270 73 base pairs for the allele specific fragment and 242 base pairs for the control fragment. The PCR-amplified DNA was separated by electrophoresis on a 4% NuSieve agarose gel and stained with ethidium bromide. All WS individuals in the family had the allele specific band while the normal mother only amplified the control fragment (FIGURE 12). A set of 60 random individuals were screened by PCR amplification with the allele specific primer set and the control set (data not shown). None of the random individuals had the allele specific fragment yet all amplified the control band. This indicates that the mutation identified in UoM1, in PAX3 exon seven, was most likely the WS associated mutation and not a common polymorphism. The proband was also screened by SSCP analysis for MITF. Two variants were identified in the proband exhibiting both WS and SOD, one in MITF exon one and the other in exon nine. Neither parent, the normal mother or the WS affected father, had either of the two SSCP variants seen in the proband. The variant in exon nine was not reproduced in multiple PCR amplifications, and therefore was not further investigated. The MITF exon one variant was an obvious pattern difference (FIGURE 13) compared to the other 40 probands but was not observed in all experiments. The PCR amplified fragment from exon one was cycle sequenced. No sequence changes were identified in the coding region of exon one (data not shown), however the entire 5'-UTR was not readable by this method. The fragment was subcloned into a plasmid vector and 60 clones were analyzed by 74 SSCP. There were two clones with SSCP patterns that were different from the original SSCP variant identified in the proband. These clones were sequenced, the only sequence variation was within intron one beyond the splice site. No other variant SSCP patterns were identified in the 58 clones (data not shown). Other individuals with SOD Due to the possibility of a connection between WS and SOD in this family and the SSCP variants identified in both PAX3 and MITF, several individuals with SOD were ascertained. There were two individuals with SOD, designated families SODZ and SOD4, that had two different SSCP variants identified in PAX3 exon seven. The genomic DNA was PCR-amplified in multiple sets along with control samples and the SSCP variants were not reproduced in SOD2. The variant pattern in $004 was subtle. Considering the importance of these data the genomic DNA from SOD2 and SOD4, was PCR- amplified and the fragments were subcloned. A total of ten clones were screened by SSCP analysis for each proband. No SSCP variants were detected in any of the clones. No other SSCP variants were identified in the remaining exons of PAX3 or MITF for either proband. There were no SSCP variants identified for any of the exons of PAX3 or MITF for SOD1, SOD3, SOD5 and SOD6. 75 SOD1 through SOD6 did not exhibit any WS characteristics (TABLE 21 in APPENDIX A). However, a diagnosis of a mild or subtle form of SOD may be easily missed in WS individuals due to the high variability of the clinical manifestations.(259‘261) It is possible that some individuals with WS may also have very mild SOD that was not diagnosed. This was why the father of the proband in UoM1 was examined by MRI. Individuals with only mild endocrine dysfunction, an absent septum pellucidum and without any nerve hypoplasia may not be identified. Verifying the absence of a septum pellucidum is expensive and would not be done without good reason. Therefore, it is possible that WS patients may have SOD with only mild characteristics and would not be identified. There is a possibility that the PAX3 mutation in exon seven is responsible for both the WS and SOD phenotypes. The connection may not have been observed before due to a bias of ascertainment, individuals that did not have dystopia canthorum. Ascertaining other SOD individuals with dystopia canthorum or other WS characteristics may further elucidate a possible connection between the SOD and the WS phenotypes in the presence of a PAX3 mutation. CONCLUSION Considering the involvement of PAX3 with neural crest cell migration and role of MITF in melanocyte differentiation it is reasonable to propose that other neural tube defects or melanocyte-deficient diseases may be related to mutations in either of these genes. Identifying families with WS phenotypes associated with other clinical traits may help further characterize the clinical characteristics of the WS phenotype. Although the connection between WS and SOD could not be established in this study, this observation may alter the guidelines set for ascertaining and characterizing disorders. Whether the occurrence of SOD is sporadic or inherited is yet to be determined. 76 CHAPTER 3 Waardenburg Syndrome co-segregating with other severe craniofacial anomalies. INTRODUCTION Genes Causing Craniofacial Anomalies Several genes have been associated with syndromes that are characterized by craniofacial and limb anomalies. The molecular control of embryogenesis and differentiation is regulated by a system of coordinated genes expressed both spatially and temporally. Some of these genes encode DNA-binding proteins that in turn regulate other genes. Several families of genes fall into this category including HOX, PAX, POU and zinc finger genes. In the initial mapping studies for Crouzon Syndrome a candidate gene approach was taken that included several genes important in early development, including the entire PAX gene family.(262) Fibroblast growth factor receptors (FGFRs) are members of the transmembrane tyrosine kinase receptor family with three extra cellular immuno—globulin like (lg) loops. The FGFRs bind fibroblast growth factors (FGFs). The FGF family is made up of related polypeptides that function in various aspects of embryogenesis, growth and homeostasis. Three of the four human FGFRs (FGFR1, FGFR2 and FGFR3) have been implicated in several disorders. Mutations in FGFRs have been found associated with three skeletal 77 78 dysplasias including: achondroplasia‘zsa' 264) (ACH), thanatophoric dysplasia type "(265166) (T DI I) and hypochondroplasia‘ze") (HCH); and four craniosynostotic syndromes‘zs‘” including: Apert (MIM 101200), Crouzon (MIM 123500), Jackson-Weiss‘zsg) (MIM 123150) and Pfeiffer (MIM 101600) Syndromes. Description of MSU22 A five generation family (FIGURE 14), MSU22 was identified with both WS and craniofacial anomalies described in TABLE 23 in APPENDIX A. The craniofacial anomalies in MSU22 are similar to those observed in the other craniosynostotic syndromes including: Apert, Saethre-Chotzen (MIM 101400), Crouzon, Pfeiffer and Jackson-Weiss Syndromes. The craniofacial abnormalities in this family appeared to include the typical WS phenotype including dystopia canthorum and broad nasal root, along with craniosynostosis and dysostosis. Craniofacial anomalies like this have not been observed in WS individuals prior to this study. The goal of this study was to determine if the craniofacial anomalies in MSU22 were due to a PAX3 or MITF mutation. Craniofacial Syndromes Pfeiffer Syndrome (acrocephalosyndactyly type V) is inherited as an autosomal dominant disorder. The condition is caused by coronal craniosynostosis creating a tall and narrow skull (clover leaf heads). The 79 individuals exhibit midface hypoplasia, hypertelorism, proptosis, downslanting fissures, thumb abnormalities (broad), syndactyly, fusion of hands and elbows.(27°' 271) Some sporadic cases also display hearing loss.(272'275) Mutations in FGFR1, on chromosome 8976) and in FGF R2, on chromosome 10q‘276' 277) have been identified as at least two of the genes associated with Pfeiffer Syndrome in some individuals. Crouzon Craniofacial Dysostosis Syndrome (acrocephalosyndactyly type II) segregates as an autosomal dominant disorder. The condition is caused by cranial synostosis or premature fusion of the bone sutures. Crouzon is clinically characterized by an abnormally short skull, protrusion of the anterior fontanel (oxycephaly). hypertelorism, external strabismus, exophthalmos, parrot-beaked nose, short upper lip, hypoplastic maxilla, relative mandibular prognathism, hearing loss and visual loss.(278‘23°) Mutations have been identified in both FGFR2‘268' 277- 231486) and FGFR3, on chromosome 4p,(237) in individuals with Crouzon Syndrome. Apert Syndrome (acrocephalosyndactyly type 0,983) is yet another craniosynostosis syndrome. Clinical characterization includes hypopigmentation, CNS malformationsfzwzg‘) cleft palate, cervical vertebral fusion, syndactyly, bone fusion and nail abnormalities of the hands and feet.(292'3°2) Hearing loss has been observed in individuals with Apert Syndrome.(3°3' 3°" The only gene identified with mutations causing Apert Syndrome patients thus far is FGFR2.(3°5) 80 Saethre-Chotzen Syndrome (acrocephalosyndactyly type III) is an autosomal dominant disorder characterized by premature fusion of the cranial sutures in association with mild cutaneous syndactyly, brachydactyly and clinodactyly.(318) Individuals may have short stature, skin abnormalities of the fingers and toes. The clinical characteristics include brachycephaly, microcephaly, skull asymmetry, hypertelorism, ptosis, strabismus and unusually shaped ears.(3°6'315) The gene responsible for the development of Saethre- Chotzen Syndrome has not been identified; however, deletions and linkage studies have implicated 7p21.2 as the candidate region.(313- 315- 317) In MSU22, the hypothesis that either a PAX3 or MITF mutations was responsible for both the WS and some or all of the craniofacial phenotypes was tested. However, due to the fact that there were only a few individuals with a clear WS phenotype and with obvious craniofacial anomalies, the possibility that two syndromes were co-segregating in this family was a possibility. 81 cabocmca m>> 9: Lo :m: 6:0 98 3632520 E NF? ovv vwm mum DIIO wow mwvwvvmwvmvv va Orv 3% #11 own arm arm .2”? mom NON 2.305% .3092ch 5 3m new mom Sm 5m 05 all. its mahococa 93 m- 4vllmV flbv mov vow mow Nov row I aDiAm Di F O ('0 mom Now 4:13 9140114 «Fa NON Pom mu. 11 row E 82586:: 13883520 28 m3 3me u: charm RESULTS Mutational Analysis MSU22 was included in the SSCP analysis of PAX3 and MITF. No SSCP variants were identified for any of the exons of PAX3 or MITF. Sequencing was done for PAX3 exons two and six with no sequence changes identified. These two exons included portions of the paired domain and the homeodomain, respectively, which is why they were examined directly. Linkage Analysis to PAX3 A linkage simulation (SLINK) was performed by Dr. J. H. Asher, Jr. that predicted that it would be possible to determine whether or not this was a single disorder or if either of the two clinical entities were linked to PAX3. The simulation indicated the need for additional family members’ DNA. DNA samples were obtained for fifteen of the thirty necessary individuals to obtain a LOD score of greater than 3.0. The DNA was typed for two markers linked to PAX3: the 5'-marker described by Wilcox et al.(183) and an intron seven marker described by Macina et al.(132) The primers, similar to those described in VIfilcox et al.(183) and Macina et al.(132) were designed, and are listed on TABLE 12 in APPENDIX A. The assay used is described in the Materials and Methods in chapter one. 82 83 The intron seven marker showed at least two recombinations in individuals that clearly had Waardenburg Syndrome. The data from the 5'- marker also had at least two obligate recombinants. The analysis was done assuming one syndrome including both the WS phenotype and the craniofacial abnormalities, WS alone (FIGURE 15) and the craniofacial anomalies alone (FIGURE 16). The DNA from all the necessary family members” was not available, therefore a formal linkage analysis was not possible. Figure 15: 84 MSU22 pedigrees: linkage analysis with WS phenotype. This includes individuals with single WS traits that would not be classified as WS by the consortium criteria according to the WS consortium clinical criteria, see TABLE 4. Included is a table with the individuals genotyped for the STRs described by Wilcox et al.(183) and Macina et al.(132) and the genotypes. 85 36298 of? EEoEoEooo; o F m m NFv on va vvF MSW; m F m m m F m N va MNm ONm oFm mFm em: mONH ooN NON ’ oON N9 6 : O 0 EEozEmo $62.96 3m. 8m N8 3m 853. So is o p m v v N m m o N v m m m m N m N N N 9 v mov vov mov Nov Fov I a Duo His m M v m m F N v o v oFm vam va :nom moon mom vom m E 0110.1 NON FoN Nom Fom FoF N oEEchm wusnnogaag NNDm—Z umF 23E Figure 16: 86 MSU22 pedigree: linkage analysis with the CA phenotype. Those family members that are do not exhibit the phenotype and do not have offspring with the phenotype are not included in this pedigree. All individuals with the craniofacial anomalies have had surgery except 317, this individual has been not been seen by our collaborator. Included is a table with the individuals genotyped for the STRs described by Wilcox et al.(183) and Macina et al.(132) and the genotypes. 87 o F m m \. Fv O Fv O F O F va ONO OON o o N N vom mom Nom Fom w o w v v N m m o N v m m m m N m N o N O Fv mov vov mov Nov Fov m m m N N N v m O F N F m N v N m N n v v o v m ONO oFm OFm NFO OFO mFm va mom mom mom vow mom Nom Fom o F v m OON NON OON m NON FON NOF . FOF Q N mom—«ESE 3683520 NNDmE 6F PEER DISCUSSION Description of MSU22 MSU22 was ascertained due to an inherited form of ulnar neuropathy by Dr. Robert Spinner (Duke University Medical Center). Seven members of the family clearly have a typical Waardenburg Syndrome phenotype including: deafness, heterochromia irides and white forelock. There was a definite correlation between the WS and craniofacial anomalies. Seven family members exhibited only one of the major WS characteristics and would be classified unaffected by WS diagnostic criteria.(5) Seventeen of the family members exhibited severe craniofacial anomalies, with all but one individual needing surgery in infancy. The proband, 405, and his affected grandmother, 202, exhibit the Waardenburg Syndrome and the craniofacial abnormalities. A high resolution chromosomal analysis was done by the Greenwood Genetic Center for both individuals. No obvious chromosomal rearrangements were identified in either individual. The W-index developed by Arias and Mota in 1978“” uses inner-canthal (a), outer-canthal (c) and interpupillary (b) eye measurements. The W-index is a quantitative measure of dystopia canthorum. Three individuals, identified as 305, 315 and 405, had both WS and craniofacial anomalies and had a W-index that indicated non-apparent (NAD) dystopia canthorum‘e) (TABLE 13). All three of these individuals had multiple extensive facial surgeries throughout early 88 89 childhood. One other family member, identified as 202, with extensive craniofacial surgeries clearly had dystopia canthorum (TABLE 13). No other members of this family had any suggestion of dystopia canthorum. The pre- surgery photographs are not available. Due to the severe craniofacial anomalies in this family a reliable diagnosis of dystopia canthorum could not be made, therefore, both PAX3 and MITF were analyzed for mutations. However, we could not ignore the possibility that two distinct clinical disorders were segregating in this family with mutant alleles in both gene(s). 90 Table 13: W-index for members of MSU22 ; I :MSU22. .....--....u_-_._. ”.7- ._——— -,._—..- _.——-.— -— I L___._..,'__.._-,-z_._.___.--__._-_-....u -- .. -. , Id 1 A9" I i r b cX__ 11W, 99011939? - 303 z 39 I 33 1 60 _ 96 0.4.349 0.7852 177003 ._ __ fl- __.._ _-,5_0_€_:___ 11 _,; 30 a 55‘ 95 T03765L 0.7701' T169412T 307 i 40 40 70 120 ".'0.4'2'22T 0.6373163093 409 2 26 46 _g_ 85 T'0.',"'4009' L'LT06'355 1.6196 ' 1 501 L 3 26 40 . 76 {0.4735 T10526 222608 note age ' 323 i 40 36 66 3 110 :_0.4071T' 0.7516 166616: 416 i 16 37 66 ‘ 100T 0469 '0.7611T.161416 417 i 16 36 75 3 110 T134435 07115166165 ' 315 g 39 , 40 e 65 1151-1054196 09209 "1.96606T NAD-craniofaCiaI surgery T _410T 6 i 30 L 55 , _90L_'0'.4113 T0.7701T1'.72692T , , _. “f*‘ ”11]”; §--.1__§9-__i_§§_.._.80 , 0.4692 0.7T7'01T1.60463T _ 305 i 43 ' i 43 : 72 116 0.4958 0.8905 1983461NAD-craniofaaal surgery —...-__...4__‘.._.-9_ _. .—_..__- -——i T405 . ”16615 f 43 72 T: 116 10.4956 0.6905 1963461NA0-cranioiaciai surgery __392 1—60's;__4_1____u6_7_. , 100 r 0.569 09156 209679ioygtopiacramoraTéia'iTsiTr'g'eiy' __306 ;_40_'s _L 32 L- 62 . 'TT93 0.4342 0.7195 166966 , __ 403 1 30's I 32 E, 63 ; "T93 Lo.43'42 0.7041 1.6463? Y Y ...----.._._ H“ .-._._.__. . _ _ .:_ .. _...._.__._4 J 1 1 -‘—L‘v --_. 4.. -.-__,.- -. ..._-. Table 13 The table includes the a, b and 0 measurements along with the values for X, Y and W. The individual identification number and approximate ages are also included. The individuals with dystopia canthorum and NAD all have had craniofacial surgery prior to the measurements. NAD = non apparent dystopia canthorum. W = X + Y + alb = [2a - (0.2119 c + 3/909)]/c Y = [28 - (0.2497b +3I909)]/b W 2 2.07 = dystopia canthorum 1.875 W _>_ 2.07 = non apparent dystopia canthorum (NAD) W < 1.87 = normal 91 SSCP and Sequence Analysis SSCP analysis was performed for the eight exons of PAX3 and the nine exons of MITF. No SSCP variants were detected in the proband for any of the exons of PAX3 or MITF. The sequence analysis for exons two and six of PAX3 did not demonstrate any sequence variations. Mutations in either PAX3 or MITF cannot be ruled out by SSCP analysis or by sequencing the coding regions alone. Linkage Analysis Due to the size of the family and the availability of the two closely linked loci within or adjacent to PAX3, a simulation linkage analysis identified individuals that needed to be typed for a LOD score 2 3.0. A maximum LOD score of 5.97 at 9 = 0.0 could be obtained with thirty family members. DNA samples were received from fifteen of the thirty individuals necessary; thus a formal linkage analysis could not be performed. However, the data indicated that there were at least two different obligate recombinants for both loci, for affected WS individuals (FIGURES 15 and 16). This data strongly suggested an exclusion of linkage to the PAX3 locus. Linkage to the MITF locus was not possible due to a lack of informative markers closely linked to the gene. The SSCP analysis did not indicate any possible mutations in MITF. However, without the availability of informative 92 linked loci, linkage to MITF cannot be ruled out. Once other genes are identified that cause Waardenburg Syndrome at least a portion of this family should be reconsidered for mutation screening or linkage analysis. CONCLUSION The linkage data for the two loci linked to PAX3 demonstrate at least two obligate recombinants therefore, linkage to PAX3 was excluded. Linkage analysis could not be performed for MITF since polymorphic markers linked to MITF have not been identified. The WS phenotype and the craniofacial abnormalities in MSU22 are a likely to be two distinct disorders. Therefore, screening the members of this family with the craniofacial anomalies for mutations in FGF R1, FGFR2 or FGFR3 may identify the gene associated with this anomaly. These genes are implicated in Crouzon, Apert, Jackson-Weiss and Pfeiffer Syndromes. The region of chromosome 7p linked to Saethre- Chotzen is another candidate region that could be screened in this family. 93 APPENDICIES APPENDIX A 94 Table 8: PAX3 PCR Primers EXON PRIMER SEQUENCE 7 SIZE TEMP COMMENT ‘ .- .._.-, _ 'TT1T_L - TF167 cccmccccrrCAccrccA TL ' "153' :3 __6T_'__'IL_T_LPcRIT5§cP T— ) rr=59 ececrcAcecccrcccjrjritc __ L _4 _ -_--_-- u_ ___-_-_ .._ .s- -. _ __.-_...___I-__. w... 2 W30 Armcccccmrrecrcrc ___-___ L: 535. _ 61L I PCR, sscp rr=32 CCGGTCT'I'CCCCAACACAGG I- __ - L - TI T L 3 TF33 ccrcccccccrer'rcrc'TLT:L" T197 '_ '_ 6:0": ITifcn, sscr> Ti=34~ ccAcreAcrcrcccechL __ ; - . -- . . .- L - _ _ _ _- "' T4 TTTT 71335 AGCCCTGCTTGTCTCAACCA‘I’ET ' 'L 242 ' I 56 ' P0636012 TF36 TGCCCTCCAAGTCACCCAGCAAGT g I 3 - A. “i" . I ”I -- -_- 5 TF100 TCACTGTAATGGTGTCTTGO T L _3'55'1 55 “7 PCR, sscp TF101 TCCTGTCTGGACTGAAGTAG _. ? L L_ -- ._-I-----,,_- 6 TF98 AGMGCCTCTAAIETE - I I' _ ... , . - 39°- 55-3953912- T_r=99 GTI'CGGACAACCIGATGTAT _ '- ___ I “T" * ‘1‘“‘7“—‘“‘* “‘“fi” “I 7 TF140 GGATATCAGCAAATCGTCTGTCTmm?" 290" L49'TT ' PCR, sscp TF141 AGAAAACATGATGGTTGACAATC I TTTT' TF156 cccccnerercecrrAArcLL_, -_ L'_:'s'_6'5"'" "'50T'T'1'50R, ssCPT1 TF157 GCTC‘ITITITI'AGGTAATGGG -- .- -- _ ._- 1 7 TF195 CTAGAAACACGGGACTGACG "TIT 64 I ASA UoM1 Table 8 The primers used for PCR amplification, SSCP analysis and Allele- specific amplification (ASA) are listed. The size of the expected fragment, the annealing temperature for each primer set, the exon number and the primer identification number are included. 95 Table 9: MITF PCR Primers EXON PRMB-‘i SEQUENCE SEE TEMP COMMENT 1 T F1 20 GGATACCTI'GTTTATAGTACCTTC 2 7 0 5 5 PCR. SSJP T F1 2 1 AAAAG AGCAGATITATACTTA'ITG 2 T F1 22 TATGAAACTCACAAATAACAGCGC 3 4 3 5 5 PCR. SSCP TF123 TATTCAACAGACAAGTTAT'ITAGC 3 TF124 CCATCAGCTITGTGTGAACAGGTC 2 4 5 5 5 PCR. SSCP TF125 TITCAGGAAGGTGTGATOCACCAC 4 TF1 26 AACTAAAGACCATTATTGC I I I GO 2 64 5 5 PCR. SKIP T F1 27 AGAAAAGAACCCTGGAAACACCT C 5 T F1 2 8 ATAAATCCTAGAGTAGGATATAGG 2 7 O 5 5 PCR. SSCP TF129 ACTITGTCTTATCAGGAAATGGAC 6 TF130 TCAAGTCAAATAAGC'ITCTGTATG 280 5 5 PCRSSCP TF131 GTAGGAATCAACTCTCCTCTACAG 7 TF132 GTGCTAAATGCATACATGGCACT G 264 5 5 Pcasecp TF1 33 TTAGGAATAGAACCAAAGGGAGAG 8 TF134 'ITCATTGAGCCTCAAATCCTAAAG 264 5 5 PCR. 83)? TF135 CT GTTI'CT ACT GTCTT GAAGTCGG 9 TF136 AGTOCTCTGTGCTCGTCCTA'ITI'C 71 5 5 5 P0118331” TF137 AAGCTAAAGTCTGTGGTGAAT'I'C Table 9 The primers used for PCR amplification and SSCP analysis. The size of the expected fragment, the annealing temperature for each primer set, the exon number and the primer identification number are included. 96 3m 8m Ean—oooEoI nmvfl EmEou 66563360 I EmEoc vegan. mg .16 .N2 h s: o 2955 FEB .2303? ocoo 83¢ um 3.59.... 97 F632 2.56m I came. 53% 656.32-14.15 m... OOF vnF NIv ., «.62 F mi? O Fme 295.5 5:5 232:? ocoo “2.3 ”O 3.59”. ONF ONF NNFuh «NF F FNEF 98 Table 10: Cycle Sequencing Primers LABELING REACTIQl----_ __ -_ IEXIENSIQE 654511011 --_.__----_._ _-_.._- _------.--._.-- -- .L-__._ _ .9299;6595-9569199953969 34-6491 :66:14241663111 " . . : ’7‘ “"".“"'-“- : “-1 Eli-X3 2 _-.-_.~!A§----39_-;'3_§'1139:1116"T97"? T537 7476"” PAX3 3_ TF_3__4 16 L60L____5_7___L 26T 72'T'IT'7T1 : 6772' -._'.’_6§§-----4--IE§§--2?; 5‘? -. _.,..5.5--_- ”T 55 ”"72T7'TT65TT TEKTITST PAX3 5 TF101 __20L ‘43-- ---§.°_- 30 ' '67 T' TsfiTiTafiF'j PAX3 6___ TF103 _ 20 46 45 L'T'31" T" 67'" ' 42" : T116713 3613*] -._T-'_:151-_-__2§-_.-_ 52 s _35 T; 31571272” ~35‘TW—3'GTFTJ ___-_-----_-_--_.--_-_-_-,-_;_ --_-_.-- _w'_ _31 ,,__ , - TTTTT Jle-___1_-_.-_I§!39-_-_34-- .§£---‘-_--§_3 r31 I’ ‘72 ’42"""'dAfP' _MITF __2 TF122 24 50 L42TTL'jT'T3'6T1T7‘2 ""'36""""64715T MITF 6 TF131 24 . 50 LL46 T. 31 .T72T'IT'39' “*“35115” MITF 7 - _ri=132 24L _§9_L‘_L46'TTT'T53TI7'2’TT “36" T6672" MITF 6 TF134 24-_-...§9-.--- 3'6"_'L"""'T30' 1T7T2'T'T33TTT116TT‘9TT I I Table 10 The gene, exon and primer identification number are included along with the length of the primer before and after the extension reaction, the annealing and extension temperatures used, the % G+C before and after the extension reaction and the dideoxynucleotide omitted in the annealing step. 99 FAMILY Physician/examiner :‘ Location I MSU 1 James H. Asher Jr., PhD Michigan MSU 2 James H. Asher Jr., PhD Michigan State Speech and Hearing Clinic MSU 4 James H. Asher Jr., PhD Michigan MSU 5 James H. Asher Jr., PhD lMichigan MSU 6 James H. Asher Jr., PhD {Michigan MSU 7 Jessica Davis, MD iDivision of Human Genetics New York Hospital MSU 8 James Higgins, PhD [Michigan State University Clinical Center MSU 9 Saroj Kapur lMichigan StaTemU—niversity GeneticsLClinicL _.._-_. “LL-m— MSU 10 Paula Czarnecki, GC Henry Ford Hospital Michigan -.-.-..__-._..___-.__._-.: MSU 11 7??? . Arizona UGM 1 ArhyalWinata/SuKarti Indonesia MSU 3 Karol Christenson, GC lMichigan_ State University Clinical Center MSU 12 William B. Dobyns, MD lDivision Pediatric Neurology University of Minnesota MSU 13 Ephrat Levy-Lahad, MD Children' 5 Hospital 8. Medical Center, Washington MSU 14 Paula Czarnecki, GC Henry Ford Hospital, Michigan MSU 15 Paula Czarnecki, GC Henry Ford Hospita, Michigan MSU 16 Susan Kirkpatrick, GC Waisman Center Univer_sity of Wisconsin- Madison MSU 17 Annemarie Sommer, MD Children' 5 Hospital, Ohio -_- -————- ——————-.-——o- - ——c—--—---...—.o_~_-.--_4 MSU 18 John Pierpont iArizona__ Health Sciences Center _ _____ MSU 19 Personal contact by family Indiana ....... '— MSU 20 Uta Francke, MD Children's Hospital at Stanford California MSU 21 Erawati Bawle, MD Children's Hospital, Detroit Michigan MSU 22 Robert Spinner, MD ' Genetic Clinic Veteran's Hospital, N. Carolina UGM 3 ArhyaNVinata/SuKarti Indonesia UGM 4 ArhyaNVinata/SuKarti Indonesia UGM 5 IArhyaNVinata/SuKarti |lndonesia ___... ..-_-. _ UGM 6 IArhya/Winata/SuKarti |lndonesia --___- .. .. -... “__OH—“fl MSU 23 Lester Weiss, MD Henry Ford Hospital Michi_g_aEL L "— UofM 1 Jeff Innis, MD, PhD Universfiy.o1-Mi0higan.GenethsElinpal Center “O ___... MSU 25 Harry Ostrer, MD . New York UhNeE-it—y-Human Genet1s PFogramm -...._ --- MSU 24 David Wargowski, MD Clinical Genetics Center University of Wisconsin MSU 26 Tanya Dien, GC Michigan State _University Clinic SOD 1 Nancy Hopwood, MD University of Michigan Clinic— UofM 4 Jeff Innis, MD, PhD University of Michigan Genetics Clinic UofM 3 Jeff Innis, MD, PhD University of Michigan Genetics Clinic SOD 2 Nancy Hopwood, MD University of Michigan Clinic SOD 3 Nancy Hopwood, MD University of Michigan Clinic SOD 4 Jeff Innis, MD, PhD University of Michigan Clinic MSU 27 Kambouris, PhD Hen-ry- F016Hos-01H'Michigan ._ W “W _ .-_.-__ __- SOD 5 Nancy Hopwood, MD [University of Michigan CITnTEu --_-._--___.-_._----.__- MSU 28 Lynne Bird, GC Children' 5 Hospital, San Diego Calif0m1a SOD 6 Nancy Hopwood, MD University of Michigan Clinic MSU 29 Erawati Bawle, MD Children's Hospital, DetroitMichigan MSU 30 Jeff Innis, MD, PhD lUniversity of Michigan Clinic Table 11 Collaborators for each WS and WS-like family. A listing of the clinicians and genetic counselors that identified the families as well as the approximate location of the families in the proband set. One individual from each collaboration is listed. GC = genetic counselor. If the clinic was not indicated the state was mentioned. 100 Table 12: PAX3 Linked Markers 65 PM W SIZE TEMP COMMENT PAX3 TF 1 61 TITATATGTGGGTGGAATGCGAT 2 5 5 5 0 Macina et al. TF162 OCTCTGATGAAACOCAGACTG PAX3 TF175 AGTTGCTGAGGGOGGAGAAG 208 50 Chatkupt et al. TF1 76 GAAATCACAAGAGGATAGAGGCT PAX3 JA32 GGGAGATGGCAG'ITCIDTGAG 183 5 8 Wilcox et al. JA33 CACACAGAGGCACAGAAAGA PAX3 TF26 CAGGGAGATGGCAG'IT 227 50 Wilcox et al. TF38 CAGAGGCACAGAAAGA Table 12 The list of primers for PCR amplification of the markers linked to PAX3. These markers include the marker described by Wilcox et al.(183) at the 5'-end and the marker described by Macina et al.(132) at the 3'-end of PAX3. The primer identification number, the size of the PCR fragment and the annealing temperature are included. The primer pair described by Chatkupt amplifies the 5'-end marker described by Wilcox et al.(183) 101 Table 14: Phenotypes for MSU1-MSU7 PHENOTYPE Dystopia canthorum Broad nasal root Deafness Heterochromia Pee-mature graying Mite forelock Hypopigmentation Symphrys Hirschprung's disease Cleft palatelllp Ocular albinism Vitiligo Blindness Gil deficiency Telecanthus Hypertelorlsm Hypoplastic blue eye Missing nasal bone syndactylv Craniofacial anomalies Ptosis Heart defects Neuropathles Septo-Optic Dysplasia Endocrine dysfunction Hypoplasia of optic nerve absent septum pellucidu Hypoplasia of nasal bone Developmental delay Anencephaly 18q Syndrome Brachycephaly 'Kidney disfunction Nystagmus Strabismus Vestibular disturbances Ataxia Mental Retardation Otosclerosis Tarsal coalition Tear duct aplasla Craniofacial surgery \ MSU I +++.. MSU 2 ++++ MSU 3 o+n+ 4. MSU 4 +++|t 4- msus _ .++.+ g. MSU 6 -'o-o-e*"' ' MSU 7 +++ +4. 102 Table 15: Phenotypes for MSU8-MSU14 PHENOTYPE MSU 8 MSU 9 MSU 10 MSU 11 MSU 12 MSU 13 MSU“ Dystopia canthomm Broad nasal root Deafness Heterochromia Pro-mature graying White forelock Hypopigmentation Synophrys Hirschprung‘s disease Cleft palatellip Ocular albinism Vitiligo Blindness Gil deficiency Telecanthus Hypertelorlsm Hypoplastic blue eye Missing nasal bone Syndactvlv Craniofacial anomalies Ptosls Heart detects Neuropathies Sepia-Optic Dysplasia Endocrine dysfunction Hypoplasia of optic nerve absent septum pellucidu Hypoplasia of nasal bone Developmental delay ' Anencephaly ‘ 18q Syndrome Buchvmhflv Kidney dlsfuncllon Nystagmus Strablsmus Vestibular disturbances Ataxia Mental Retardation Otosclerosls Tarsal coalition Tear duct aplasia Craniofacial surgery +I+|QIQ .+.++++ ? + 7 + 0+.++..- titr‘I'iI +*+.*II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I + I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I + I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I + I I I I + I IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII+I IIIIIIIIIIIIIIIIIIIIIIIII+*+IIIIIII I + I I I I I I I I I I I I I I I I I I + I I I I I I I I I I I I I I 103 Table 16: Phenotypes for MSU15-MSU21 PHENOTYPE MSU 15 MSU 16 MSU 17 MSU 18 MSU 19 MSU 20 MSU 21 Dystopia canthorum 7 7 - - - - - Broad nasal root - 7 - - - - - Deafness + 7 + + + + + Heterochromia - 7 - + + - - Pro-mature graying - 7 - - + + - White forelock + 7 - + - - + Hypoplgmentation + 7 - - + - + Synophrys - - - - - . . Hirschprung‘s disease - - - - - - - Cleft palamlllp - - - . . - - . Ocular albinism - - . . . - - Vitiligo - - . _ . - - . - Blindness ‘ - - - - - . - GH deficiency - - - . - - - Telecanthus - - - - - - - Hypertelorism - - - - - - - Hypoplastlc bue eye - - - - - + - Missing nasal bone - - + - - - - syndactvlv - - + - - - - Craniofacial anomalies - - - - - ' - - Ptosis - - - - - - - Heart defects - - - - - - .. Neuropathies - - - - . - - Septo-Optic Dysplasia - - - - - . - Endocrine dysfunction - - - . - . - Hypoplasia of optic nerve - - - - - - - absent septum pellucidu - — - . - - - Hypoplasia of nasal bone - - - - . . - Developmental delay - - - - . - . Anencephaly - - - . - . - 18q Syndrome - - - - . - . Brachycephaly - - - - - . - Kidney dlsfunction - - - - . - - Nystagmus - - - - - - - Strabismus - - - - - . - Vestibular disturbances - - - - - - . Ataxia - - - - . - . Mental Retardation - - - - - - . Otosclerosis - - - - - + . Tarsal coalition - - - - . - . Tear duct apiasla - - - - . . - Craniofacial surgery - - - - - . . 104 Table 17: Phenotypes for MSU22-MSU28 PHENOTYPE c MSU 22 MSU 23 MSU 24 MSU 25 MSU 26 M8027 MSU 28 Dystopia canthorum 7 - - - - - - Broad nasal root - - - + - - - Deafness + + + - + + + Heterochromia + + + - + - + Pro-mature graying - Y.. This was a 5.4% increase in the skull half width. Because 41 of the 42 comparisons indi- cated that skull landmarks were symmetric. further analyses were restricted to the leit side of the skull (Table 4). . Displacements of skull landmarks. The null hypothesesforthesecompariaonswere-t-l-r-X.- Sp‘l+ X. and +l+ Y. :- Sp‘l+ Y. and were similar for landmarks 7 and 8. As six comparisons were made between pairs of groups. a = 0.008 was chosen as the level of significance. In B. mice. there are no differences in the locations of skull landmarks caused by Sp‘ (Ta- ble 4). This is also true on the F. genetic background (Table 4). Among BC. skulls. Sp‘ causes landmark 8 to be displaced 3.2% anteriorly (Table 4). Some mouse skull landmarks for Be. SPR. F.. and BC. differ significantly in their location (Table 4). As a consequence of the displacements of these landmarks. the relative size and shapes of the interfrontal bones differ significantly. From these measurement differ- ences. the size and shape of the interfrontal bone is controlled by genes with a number of different rmdes of inheritance. The relative lengths of the left interfrontal bones (Kg-X.) were compared with a - 0.05/10 - 0.005. The order of the group means with equivalent means underscored is g BC. in r. so. Sp‘l+ SPR The ranking of the above means indicates that genes that cause the interfrontal bone to be short are domi- nant (P < 0.005). For the F.-SPR comparison. P a- A MURINE MODEL NR WAARDENBURG SYNDROME VARIATION 0‘23! 5.7x FIG. 2. Donal views of three skulls from BC. mice. (A) A scan- ning photmnacmgraph ofa normal HIM InouacgtBlA drawing ofa . - .. ‘ ‘ t :1 Ina-“t. . charac- terize the shape ofthe interfrontal bone. Landmarks 1. 2. d. 5, 6. and 8 are defined by the intersections ofsuturea. Landmarks 3 and ‘laredelincduuiepointaofthenamwcddistance ween orbital ridges. (C) A scanning photomaaograph of a mutant (Sp‘lol 0.00051. The relative posterior half widths of the inter- frontal bones 0’.) were compared with a = 0.05/6 - 0.008. The rank order of the means is SPR s F. < BC. I 8.. For the F.—BC. comparison. P I: 0.00045. This result indicates that genes that produce posteriorly narrowed interfrontal bones appear to be domimnt file relative left distances between the posterior aspect of the interfrontal bone and the narrowest interorbital distances (X1-Xg) were compared with a I: 0.05/6 - 0.008. A single comparison was found to be a' 'fi- cantly different: BC. < B. (P = 0.00021). The relative interorbital half widths (Y1) were compared with a - 0.05/6 - 0.008. The rank order of means is F. < SPR - BC. - B.. For the F.—SPR comparison. P = 8.84 x 10“. These results indicate that the interorbital half width exhibits negative heterosis. Finally. the relative anterior half widths of the interfrontal bones 0’.) were compared with a - 0.05/6 = 0.008. The rank order of means is F. = BC. = B. < SPR For the B.—SPR comparison. P = 5.86 x 10". This result indicates that narrow anterior half width interfrontal bones are in- herited as a dominant trait. 291 orphcnrelu'c wriabiluy. 11.. above analysis indi- cates that craniofacial morphology of mouse skulls' Is influenced by genes that control the location of given landmarks. Craniofacial morphology Is also influenced by genes that may independently control the variability of the location of a given landmark without the average location of the landmark To evaluate this aspect of the craniofacial variability. the slope (6.) of the linear regression line through a given mean landmark location (X., Y.) was determined (Table 4). The null hypothesis was 6.- = 0 for a given landmark. For each group (8.. SPR, F.. BC. +/+. and BC. Sp‘/+). there were three slopes-tested and as a consequence. a = 0.05/3 = 0.017. With the exception of a single landmark (F. +14» X., Y.), all slopes for B.. SPR. and F. Sp‘l+ were not significantly different from zero (Table 4). As B. and SPR are highly inbred strains and as the Sp‘ mutation occurred spontaneously on the B. strain, B. mice with a given P013 genotype (Sp‘/+ or 444-) should lack genetic variability. Since Sp‘l+-containing litters were stored as frozen embryos following nine genera- tions of backcrossing to B. to minimize the accumula- tion of newly arising mutations linked or unlinked to Sp‘. Sp‘/+ and +/+ mice should differ at very few other loci. The significant slope of the regression line through landmark 6 of +l+ F. mice cannot be explained by segregating genes. F. .3 ‘ ll' gunners. . mal loci. F. males should be identical genetically and physiologically with a SPR Y chromosome and a B.X chromosome. F. females should be identical genetically with a SPR X chromosome and a B.X romosome. However. it may be possible that the significant regres- sion slope at landmark 6 of F. mice could be related to X chromosome inactivation. If F. mice are pooled into males and females regardless of genotype (Table 4). we note that the regression line through landmark 6 has a slope significantly different from zero in females (b. = -0.773 1 0.199. t. = 3.884. df = 15. P = 0.0014. c = 0.05/6 - 0.008) and a slope not significantly different from zero in males (b. - 0.217 t 0.461. t. - 0.470. df = 6. P = 0.655) BC. mice are segregating M. muscular and M. spretus alleles at all loci including Paul. The slopes for all three landmarks in BC. mice show significant differences from zero (Table 4). Paired comparisons be- tween slopes using 1 tests indicated that fortwooftheae landmarks (6 and 7) the slopes are significantly difi'er— ent between +l+ and Sp‘ /+ (Table 4). again usinge - 0..017 The slopes at landmark 6 indicate that BC. Sp‘ l+ mice have both broader and narrower posterior interfrontal bone width compared to their +I+ siblings. The slopes at lasidmark 7 indicate that BC. +l+ mice have both broader and narrower interorbital widths compared to their Sp‘l+ siblings. Finally. the slopes at landmark 8 are roughly equivalent for BC. +l+ and Sp‘l+ siblings, indicating that the anterior interfrontal measurements are influenced by the genetic back- ground in the BC. mice but are not influenced by Sp‘. 122 292 ThuSp‘inaeaaastlIerangeofvariabilityobserved in craniofaa'al measurements in a. systematic manner. Sp‘l+ micecanhaveverynarrowaswellasverybroad interfrontal bones. The shape ofthe interfrontal bone dependsupon the interaction betweenSp‘andthege- netic background. Toillustrate. Figs.3Aand3B presentplotsofthe relative shape coordinates for all +14» and Sp‘l+ BC. mouse skulls. respectively. The shape coordinates are normalized using the distance between landmarks l and 5. Notice that the shape coordinates for landmarks 2. d. 6. and 8 (Fig. 2) diverge from the skull midline represented by the line from landmark l (1.0) to land- mark 5 (0,0). The t tests in Table 4 indicate that this divergence is significant for each genotype (P < 0.017 - a) and that Sp‘l+ mice have a greater divergence . than do 444- mice for landmark 6 (P B 5.57 x 10"). The Pax3 mutation Sp‘ causes the posterior aspect of the interfrontal bones of Sp‘l+ mice to be significantly wider and narrower than that of +l+ mice (Table 4 and Fig. 2C). Selection arm“ with sun Shape Extremes To determine if the genes controlling the average location and variability of a given landmark are the same for all landmarks. a subset of selected skulls were plotted. Using the coordinates of landmark 6 (Fig. 3 and Table 4), 10 animals of each genotype with either. the widest or the narrowest skulls were selected (6th and 8th percentiles. respectively. Figs. 3A and 3B). The coordinates of these animals appear in Figs. 30 and. 30. Note that animals selected in this manner produce. as expected. two separate nonoverlapping clusters of points for landmarks ( and 6 but produce a single clus- ter of overlapping points for landmarks 2 and 8. On theotherhand. when 10 animalsarechosenfromthe extremes of the distributions for landmark 8. two sepa- rate nonoverlapping clusters are observed for land- marks 2 and 8 and a single cluster is observed for land- marks 4 and 6 (Figs. 38 and 3!"). The animals that exhibited the extreme values for landmarks 4 and 6 were not the same animals that exhibited the extreme values for landmarks 2 and 8. These observations sug- gest that the location. and variability of the location of these two pairs of landmarks. 2/8 and W. and thus the anterior and posterior width ofthe head. are under independent genetic controls. 1f the sexes of the animals chosen because of their extreme position within the distribution are pooled across genotypes. there is asignificantescess ofmales among mice with the narrowest faces with-respect to landmark 6 (the lower 6th and 8th percentiles for +14- and Sp‘/+. respectively). There are 17 males and 3 fe- males(x’ - 9.80,df- 1.1’ -0.002).T‘hereareequal numbers of males and females among mice with the widest faces with respect to landmark 6 (the upper 6th and 8th percentiles for 44+ and Sp‘l+. respectively). There are 12 males and 8 females (x2 -= 0.80. df - 1. ASHERETM. P - 0.371). On the other hand. if the sexes of animals chosen from theextremesofthedistributions forland- mark 8 are pooled. the numbers of males and females are not significantly different. with 13 males and 7 females with the narrowest faces and 7 males and 13 females with the widest faces. These observations sug- gest that one of the genes controlling the posterior shape of the skull is X-linked or sex-influenced. while the genes controlling the anterior shape of the skull are autosomal. To see if the distortion in sex ratio would persist with an increase in the number of selected skulls. 10 additional skulls were chosen from the extremes of the distributions of each genotype. A summary of the phe- notypic characteristics of the selected animals appears in Table 5. With respect to +I+ mice. there are more males than females with nanow skulls regardless of the selected landmark (Table 5). With respect to Sp‘l + mice. there are many more males than females with narrow skulls at landmark 6. Following independent selection at landmarks 7 and 8. there are. an equal number of males and females with narrow skulls. Thus. mice with narrow skulls have different sex ratios de- pending on the landmark selected and the P013 geno- type. Unselected mice have a significant distortion in the transmission ratio with respect to theA-locus. A contin- gency x' analysis indicates that mice with narrow skulls have the same segregation ratios regardless 'of the landmark selected (Table 5). In addition. the pooled ratio is not significantly different from the distorted transmission ratio seen in unselected mice. Thus. the A-locus or a gene closely linked to it does not appear to influence the shape of the skull for the narrowest of these skulls. , Mice with the most narrow skulls do not exhibit the aamedegrceofpenetnnceofthewhitebellyspotde— pending upon the landmark selected. Penetrancs is high in mice selected for narrow skulls at landmark 6 and lower in mice selected for narrow skulls at land- marks 7 and 8 (Table 5). Comparisons of the skull landmarks. sex ratio. A- locus segregation ratios. and penetrance yields quite different results when considering mice selected for very wide skulls. First. the sex ratios among mice with the widest skulls are not different from each other and are not different from 1:1. contrary to what is observed for mice with very narrow skulls (Table 5). Second. transmission ratio distortions for the A-locus among, mice with the widest skulls do not differ with respect to the selected landmarks but these distorted ratios are different from the transmission ratio distortion seen in the unselected data set (Table 5). Finally. penetrance of the white belly spot is not different in mice with wide skulls regardless of the landmark selected. contrary to what is observed in mice with very narrow skulls (Table 5). Penetrance among the selected mice is not different from the penetrance in the unselected data set In addition to the segregation analysis performed on 123 A “URINE MODE NR WAARDENBURG SYNDROME VARIATION 293 0.6 . 0.6 , A : e B .0. ‘ e s . . . . . s 0 . . O Q . 0.; . w‘}. 0... h‘. ~ . assists . . ‘ ‘1‘? 2’30 0 t c 4‘ O 4‘ A _ s 0 cs 1.2 r) 0.6 12 ' «$13; «east 4.; es sewage... 4’ . «3:. *' '3 «$23.. .I‘ ° ' .g‘duf a 83:2; g“. r0 “d a. 0 :0 if. g:- . ‘ r :. ‘ as . 416 ~ 0.6 . 0.6 T C . i D . e 0 . 0:. 0. ‘ _ o r : ‘ 0.4 . .3} e:- o‘ “ :- 5, «(c .afr s . O . .‘ 6 . 02 .. é ‘*., 0.2 . m‘. o . ¢ V ; 0 4. : r : . t 0.2 0 4 0.6 0.8 l 1.2 l) 0.2 0.4 0.6 0.8 l 1.2 -02 . ‘02 ‘r s a s‘ 4?‘ 1g... . :4; . 2.: ‘3‘. .. e .i.’ -0.4 .. 3... . 0?. '0" ‘L ‘05. ..:d: -o'.6’ -o.s .i 0.5 . . 0.6 . E 1 F 0.4 a: '- 0.4 . fl . , : .0 o J .;;.: e 1‘ 9.. e.‘ ‘0. . .. e ..s .t s 02 . ”4‘1“ oz . 3‘3“ o 1 w' 3 C ‘ 4‘ 0 fig 4 g —:-...-...-.. -l t D 0.2 0.4 0.6 0.8 1 1 2 0 02 0,4 0.5 0,. r r 2 '02 a. 0.2 . ' «t a“. ‘ '.‘M .:.O‘ 0 Cf. . .0. ’ 5 ‘. or .‘V' . or .s; 3 ‘ . 4. -0.6 «0.6 316.3. undistn’budossfdrspemudinatesfsrhndmukszltttanddfromBC.msuseaksllsssillustntodinFig.1w1he dlstributlonsfshapecsordinstelandmarksforlfl 01+ “(Elfindktribufionsfshapecoordinstelandmarksforlfifi‘le mice.(Cl 'l'hsdistributionsfslrspscsordinatslandmsrksfsr” 41+ miossslsctedbscsusetheyarefromthetwoextremesofthcdisuibution inA (upperandlowerGth perourts’lekSelsdionwssbssedsnlandmsrkGJIllThe distribution sfshapecoordinste landmarksfcrNSp‘le mics selectsdbecsusetheyarefromthetwoextremesofthedistributioninBtuppersndlowerBthpcroenu’le).8eloctionwasbsaedonlandmsrk 6.(E)‘lhedistn‘butioncfshapecoordinatelandmarksfcr20+l+ miceselectedbeauselheyare fromthetwoestremessfthcdistributlsn lnA(upperandlowerfithmwlmmbmdumdmmmdshammdimuhMmmwfl Sp‘lemiceselectedbecausetheysrefromthetwoestromsssfthsdistributiooinntupperandlower8thpercentilei8clectionwasbsssd on landmark 8. these selected mice (Table 5). the analysis of the loca- tion and variation of the location for landmarks for each selected group is presented in Table 6. Sp‘/+ mice with narrow skulls. with regards to landmarks 6 and 8. have broader skulls than +/+ mice selected for narrow skulls (Table 6). On the other hand. mice selected for the widest skulls do not differ in landmark location regardless of genotype or landmark of selection (Table 6). Thus. the structure of the mouse face is controlled in part by the interaction ofSp‘ with a number of different 123 A “URINE MODEL FOR WAARDENBURG SYNDROME VARIATION 0.6 . A : . o e . . C e . 5 . . e1. 0 c : 4. q as 12 saogag‘g m . 35.1.; msgg‘ . O ‘ fi“.,,,‘é5.r- .531: C . . -O.6 L 0.6 i C « 0.4 . ‘g- . 3- o $- é' . 3" s .3. 02 . '1» 0 : c c . ‘ 0 0.2 0.4 0.6 0.8 1 12 oz. , . e 2'? , :3... ‘0‘ 2:0 . .? as 0.6 .. E . a: :- 04 ‘ . .. . .x :0. “0 as 0 oz . 34f” 0 a : e : : J. t) 0.2 0.4 06 08 1 1.2 02 ”C's. . o‘e’ 0?.0 -0.4_ ‘3‘: Q: -0.6 293 eo‘ o ‘ e 3%.: : "n‘ ' 03 L . 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' :3?" 0 :- fl : :-----~---- ‘1 0 02 0.4 0.6 0.8 1 12 0.2 . «‘33,». .. .53 3‘33: 0.8 316.3. ”redistributisnsfslrspecsudinstesfsrlsndmarksLSJJJJnd8fromBQmoussskullsasillmatodinl-‘ig.2.w1ho distributionofshapecsordlnstelandmsrksforlfl +I+mim.(3)1hsdistributiosofshspeeoordinstclandmsrksforfl$$p‘l+ mics.(Cl MdktfibutionsfshspecssrdinstelandmsrksforloelemiceselsctedbsausetheyarefmmthetwoextremesofthcdisuibudoninA (upperandlowerGthMkmmhasdsnhndmsrkfimflhedistfibuuonofshapemdinau landmarksfcr208p‘le mice sslededbecsusetheyarefromthetwoestremessfthedistributioninBtupperandlowerOthperoentilel.Selcctlonwssbaacdonlandmsrk 6.(E)'l'hedistn'buticnofslupecsordinatslandmsrksform+l+ miceselededbeauseuieyarefromthetwoestremsscfthcdisuibutisn inAtupperandlowerGthmmelSeWnsbudmhndmsrk8mfludidfibufimddmmdimuhndmrksbrfl Sp‘l+miceselectedbscsusetheyarefromtlwtwoestremssofthedistributioninfltupperandlowerOthpsrcentileLSelectionwasbassd on landmark 8. these selected mice (Table 5). the analysis of the loca- tion and variation of the location for landmarks for each selected group is presented in Table 6. Sp‘/+ mice with narrow skulls. with regards to landmarks 6 and 8. have broader skulls than +I+ mice selected for narrow skulls (Table 6). 0n the other hand. mice selected for the widest skulls do not differ in landmark location regardless of genotype or landmark of selection (Table 6). Thus, the structure of the mouse face is controlled in part by the interaction of Sp‘ with a number of different 124 294 ASHER er 111. TABLE 5 The Sex. Genotype. and Penetranoe of White Belly Spot for 20 Animals Selected from the Extremes (Narrowed or Widest) of the Skull Measurements Narrowest Widest L“ G d 9 As as P N? d 9 As on P NP 6 - 44+ 15 5 7 13 - -- 8 12 13 7 — _ 5.1% 11 a 15 5 16 1 s 11 16 4 11 s 7 «ble [4 6 10 10 -— - l l 9 15 5 -—- .. Sp‘le l3 7 10 10 10 10 9 ll 15 5 13 7 a 11+ ' 13 r 11 6 — — 11 9 15 s _ _ Sp‘h 9 11 9 ll 8 12 12 8 17 3 15 6 x' 441- 0.48‘ 2.04 -— 1.20 0.66 - x' Sp‘l+ 7.03‘ 4.21 7.067 1.20 0.63 0.48' r’ Pooled 9.13' 0.00 11.11‘ ‘1..landmark; G.Pax3genotype;A/e.Agorui:sls. mwi.‘ P. penetrantforawhite bellyspot;Nf’.notpcnetr-antfora white belly spot.x'for +I+orSpl+ uacsnungerrcyx’wrthdf-I2;x’forthcpooledssmples1saconungcncyx‘w1thfivedegreesoffrecdom ‘1‘hcsea ratios among the +I+ mice with narrow skulls and landmarks6. 7. snd8sre notdifferent but are significantly different from thecxpcctcd 1:1 ratiode I 42/18. x’ I9..60 de1. PI I.0002.a I0.05l2).1‘hcre aresignificantiymore +I+ males with narrow skulls than there are females with narrow skulls. "l‘hcsex ratiosarnongthcselectedSp‘M micewithnsrrowskullsaronotequivslenttPI I..003 a I0..05) WithrespccttolandmsrkG. therearemoreSp‘Hmaleswithnsrrowskullsfdl?I17I:l.x'I.I9.8.dII1PI...00018nI005I3I 00171W1th1espectto8plem1ce with narrow skulls at landmarks 7 and 8. the sex ratios do not differ. “The penetrance of the white belly spot differs between the landmarks of narrow skulls (P I 0.03. or I 0.05). SIP/4 mice with narrow skulls at landmark 6 have greater penetrance than animals with narrow skulls at landmarks 7 and 8. "The penetrance of the white belly spot among mice with wide skulls does not differ with respect to landmarks. This penetrance (l‘lNl’ I 42/18) 1s not. different from the penetrance in the unselected data setfPlNPI 93/44. contingency x' I 0..09 dIIl. PI 0.77) "The transmission ratiosoonlorrannngselectedsamplesdonotdiffertdlI I.5 PI 009) and the pooled ratiofAe/oo _- 65I55)is not significantly different from the distorted transmission ratio seen in the unselected sample Melee I 192/125: contingency x' I 1 .47 d] s l. P I 0.22). “DreAln transmission ratiosaronotdifferentinmioe withwideskullsfdl- 5.P I0.79)butthcdistortioninthepoolcdtransmissisn ratiossftheselected sompleszs/es I 91/29)isdifferentfronrtheAIe transmission distortion intheunsclccted ssmplctx' I 8.89.de l.l’ I 0.003.11 I 0.05“ I 0.012511‘here is a higher number efAc among mice with wide skulls. genetic elements. By performing genome-wide disequi- librium mapping using animals from the extremes of these distributions. the chromosomal regions con. taining the genes that interact with Sp‘ controlling the shape of the face should be identified. Genetic Models for Waardenbra'g Syndromes Currently. PAX3. MITF. and EDNRB. when mu- tated. are capable of causing Waardenburg syndrome (Foyel al.. 1990; Asherct al.. 1991;11ughes‘et al.. 1994; TassabehjietaL. 1994; PuffenbergeretaL. 1994). There are over 50 PAX3 mutations that cause Waardenburg syndrome type 1 (Farrer et al.. 1994; Read. 1995) and all cause dystopia canthorum. a craniofacial anomaly. Only three of these mutations segregating in WS fami- lies have penetrance for deafness between 75 and 100% (Baldwin et al.. 1992; Morell et al.. 1992. 1993). Cranio- facial deafness hand Syndrome. CDHS. is also caused by a PAX3 mutation (Asher et al.. 1996). In an admit- tedly small family. this mutation is fully penetrant for both deafness and craniofacial abnormalities. CDHS shares many characteristics with W83. including pro— found deafness and skeletal anomalies. yet they are clinically distinct (Asher et aL. 1996; Goodman ct al.. 1982; Sommer el al.. 1983; Klein. 1983; Sheffer and 2101080“. 1992). The phenotypic similarities and high levelsofpenetranceofW83andCDHS mightboex- plained by the molecular nature of their PAX3 muta- tions. Mutant alleles of MITF cosegregate with some in- stances of Waardenburg syndrome type 2 (W82). Three MITF mutations have been characterised (Tassabehji at al.. 1994; Morell et al.. unpublished results). As with PAX3 mutations. the molecular defects caused by MITF mutations alone are not sufficient to account for the phenotypic variability observed in W82 families. The phenotypic variability observed both within and. between families with Waardenburg syndromes can be explained by at least three different but not mutually exclusive genetic models: (1) different mutant alleles at a single locus. (2) mutant alleles at more than one locus affecting the same developmental processes. and (3) a single mutant allele at one locus interacting with modifying genes at other loci (Asher and Friedman. 1990). Identibring Genes Interacting with Par3 Mouse mutants have been used to help identify the causes of WS variability. Evidence presented here dem- 125 A MURle-Z MODEL FOR WAARDENBURG SYNDROME VARlA‘l‘lON 295 TABLE 6 Mean Coordinaws (X). Y.) for Landmarks (i I 6. 7. and 8) for the Left Side of Mouse Skulls Comparing BC. Mice with the 20 Most Extreme Measurements with the Slope (6.) of the Regression Line Passing through the Landmark (L. 16). the Standard Deviations (1). and the t-Tests (1.) Comparing Individual Means and Slopes a Comparison 8 Xe 1's 54 X. Yr 01 X. Y. b. 20 N 41+ I 0.4” 0.35 0.060 0. 570 0.195 0.073 0.909 0.277 4.0” 8 0.039 0.010 0.“1 0. 049 0.019 0.088 0.042 0.“ 0.044 20 N Sp‘l+ x 0.472 0131 4.115 0. 704 0.207 -0. 000 0 955 0.295 0.007 1 0.023 0.0” 0.205 0. 035 0.010 0. 067 0. 028 0.01 1 0.0“ 20 w +I+ 2 0.398 0.414 -0.183 0. 732 0.292 0.181. 0. 990 0 .412 0.544. 6 0W1 0.035 0.128 0.125 0.033 0. 045 0.129 0 .053 0. 054 20 w Sp‘l+ I 0.373 0.419 4559' 0.892 0.294 -0.004 0.983 0. 401 ' -0.127 5 0.044 0.042 0.177 0.047 0.027 0. 133 0.042 0 .mo 0.165 N (4’4 vs $177+) 4. 0.988 5.000 2.525 2. 500 4.075 5.918 P. 0.330 113-5 0.018 0. 017 2.384 7.43-7 w (+14 vs 5pm) 1. 1.400 0.409 1.340 0 210 07.31 one Pg 0.145 0.685 0.188 0.835 0.819 0.424 1. 1.721 0342 2.000 p, 0.090 0.734 0.010 Note. a. sample size. 8. statistics. including the mean (11. the standard deviation (1). and calculated 1 (L). P... the probability of obscrvirm avalueofl. that largeorlargcrbychancealoncwithdlI 38.ParX.orY.ofagivengroupcompan’sontobeconsideredsignificanLP < 0.008 (0.05/6). For 6. within a given group or between two groups to be coruidered significant. P < 0.017 (0.05/3). 8 Values and 1 values meeting this criterion are in boldface. N. the narrowest 20 BC. skulls of a particular genotype. W. the widest 20 BC. skulls of a particular genotype "The slope ofthc regression 1m.- passing through landmark a a. significantly different from zero with 1, - 4.022. 41 - 10. P - 0.0000. “The slope of the regression line passim through landmark 8 is significantly different from zero with t. I 6.370. d! I 18. P I 5.33 x 10 ". 'The slope ofthe regression line passing through landmark 6 is significantly different from zero with l. I 3.159. d! I 18. P I 0.0054. onstrates that when Sp‘ (11 P013 mutation) is segregatp' ing on the highly inbred and coisogenic mouse strain 8.. heterozygotes have white belly spots but rarely ex- hibit dysmorphic features. On the other hand. when Sp‘ is segregating in a very diverse genetic back- ground. i.e.. in an interspecifrc BC. with Mus muscular and Mus spretas alleles. it is associated with pheno- typic variability similar to that observed within large W8 families. Mice with craniofacial abnormalities and very broad interfrontal bones are heterozygous for Sp‘ and likely carry alleles for at least two other loci that interact with Pax3 to influence skull shape. Wildtype +l+ BC. mice do not exhibit craniofacial abnormalities but can have broad interfrontal bones and are likely to carry the same alleles at other loci that interact with a Pax3 mutant allele producing a very broad interfrontal bone. Because of the sex distribution (Table 51 among mice with extreme skull shapes and because of the sex-asso- ciated differences in regression slopes of the F.. one of these loci is either sex-linked or sex-influenced. 'lhis locus appears to help control the posterior shape of the mouse interfrontal bone. A second locus appears to be autosomal and control the anterior shape of the inter- frontal bone. Extensive variability was observed with respect to white belly spots in these BC. mice. Approximately 32.1% of the BC. Sp‘l+ mice were nonpenetrant for a white belly spot. Sp‘l+ on the inbred C57BU6J strain~ are 100% penetrant for a white belly spot. in addition. there is a significant nonrandom association between the sex of the mouse and the presence of a white spot. Male Sp“/+ mice more frequently have white belly spots than do female Sp‘l+ mice. Thus. both skull shape and the presence of a white belly spot are in some way influenced by the sex of the mouse. Sp‘l+ mice with the narrowest heads and white belly spots are generally males. One simple explanation 1s the ex- istence of an X-linked allele fixed in the 057815.! strain that modifies the effects of Sp‘l-r- with respect to the production of a white belly spot and the shape of the mouse skull.’ Alternatively. these two effects may be controlled by two different X-linked loci. In addition to the influence of sex on the presence of white belly, spots. mice with the ole genotype are more frequently penetrant with respect to the white belly spot. The shape of the face of mice is also related to the A-locus. This effect may be directly influenced by the 4: allele or a gene closely linked to the 0 allele. In addition to the phenotypic variability associated with craniofacial morphology and pigmentation. Sp‘ segregating in the BC. mice also reduces viability. This effect. appears to be enhanced by the A-locus genotype as well as the sex of the BC. progeny. It has been dem- onstrated that there is distortion of the 1:1 transmis~ sion ratio when B. and M. spretus mice are used to make an interspecific backcross (Siracusa er al.. 1989. 1991). This distortion in transmission ratio involves genes on chromosomes 2 (containing the A-locus). 4. and 10 but does not involve chromosome 1 (containing 126 296 Pax3). The distortion of transmission ratios reported by Siracusa er al. (1989; Aa/aa = 77140) is not different from the distortion reported here (Ac/ac = 185/132. contingency x’ = 1.98. df - 1. P - 0.16). In both cases. the distortion of Ala transmission ratios is not differ- ent when considering males and females separately. Among BC. progeny. however. there is a significant deficiency of Sp‘l+;a/a progeny. As chromosome 1 transmission ratio distortion has not been noted pre- viously (Siracusa el al.. 1989. 1991). this suggests a unique interaction between Sp‘ and the a allele or a gene closely linked to the A-locus. This interaction might take place during embryonic development or fol. lowing birth (Siracusa et al., 1991). An analysis of our breeding data suggests that both are possible. Eighteen +I+ B. females produced 35 litters with an average litter size of 6.9 t 2.3 pups/litter. Fifteen Sp‘l+ 8. females produced 30 litters with an average litter size of 6.7 t 2.5 pups/litter. These litter sizes do not differ (to = 0.34. (a = 2.41, P ‘3 0..05l For 15 SP‘/+ BC. females producing 92 litters. the average litter size was 4.1 1 2.3 pups/litter. BC. litters are significantly smaller than B.‘ litters by nearly 3 pups/litter (t. x 6.13. 1,. = 3.60. P = 0.001). Although BC. mice are exceptionally vigorous and mature rapidly and females are very active breeders. their litters are smaller than B. litters. In addition. of the 381 pups born to BC. females. 28 died between birth and weaning. Thus. both in utero and neonatal losses could account for the decreased viability of Sp‘l+ progeny. Because allelic variation at. the A-locus can cause widely disparate phenotypic effects including embryonic lethality. obe- sity. diabetes. and tumor formation (Bultman et al.. 1992). it is possible that the M. musculus 0 allele and Sp‘ might interact directly to lower the viability of Sp‘l+ embryos and/or neonates. This could happen through the action of these genes on the neural crest cells. Genetic modifiers play a major role in the final deter- mination of a phenotype. Coleman (1978) observed that two mouse mutants. ab and db. on the C57BU6J ge- netic background caused obesity but not diabetes. 0n the CS7BUSK genetic background. these mutations caused both obesity and type II insulin-dependent dia- betes. A murine Ape (adenomatous polyposis coli) mu. tant allele is virtually benign on the AKR genetic back. ground but causes intestinal neoplasias on the C5781] 6.! genetic background (Dietrich el al.. 1993). In a re- cent finding relevant to the determination of craniofa- cial and hand phenotypes of humans. Rutland et al. (1995) identified two sporadic mutations of fibroblast growth factor receptor 2 (FGFR2) in exon 7. T to C at nucleotide 1036 and G to A at nucleotide 1037, Cya342Arg and Cys342‘1‘yr. respectively. that cause Pfeiffer syndrome (craniosynostosis with hand anoma- lies). In different families. these same two mutations cause Crouzon syndrome (craniosynostosis without hand anomalies). A possible explanation for this pheno- typic heterogeneity is the segregation of modifier genes ASHER ET AL that interact with the ob, db. Ape. and FGFR2 muta- tions (Reardon el al.. 1994; Rutland et al.. 1995). Lander and Schork (1994) reviewed the nature of com- plex phenotypes and outlined a number of strategies to identify-these modifier genes. Pavan er al. (1995). using such a strategy. identified six loci that appeared to modify the expression of the sls genotype with re- spect to white spotting. We suggest that the phenotypic variability associ- ated with Waardenburg syndrome requires a mutant allele at PAX3, MITF. or EDNRB interacting with other genes. Therefore. to understand the phenotypic variation associated with PAX3 mutations. the genes interacting with PAX3 must be identified and cloned and their functions determined. The Sp‘ interspecilic backcross mouse model described here offers one oppor- tunity for mapping and eventually cloning these mod- ifier genes ACKNOWI£DGM£NTS We thank Darwin Dale. Computer Photografx. Lansing. MI. for the scanning photcnucrographs of the mouse skulls and Marlene Cameron for the graphic art. We thank Dr. Dennis Gilliland and Ms. Lei Chen. Statistical Consulting Service. Department of Statistics and Probability. Michigan State University. for their advice. This project was supported in part by grants to J.H.A. and T.B.F. from the Deafness Research Foundation and Grant DC 01160-04 from the National Institute on Deafness and other Communication Disorders. National Institutes of Health. Dr. James H. Asher. Jr.. the senior and communicating author on this article. died on May 13. 1996. He was our colleague and friend and will be sorely missed. This paper is dedicated to his memory. REFERWCB Aasvod. It. (19621. Waardenburg‘s syndrome. Acre Ophucbnol. «I: 622-628. Ambani. L. It. (19831. Waardenburg and Hirachspruu Syndromes. J. Pediatr. 102: ear. Anderson. 1‘. W.. and Sclove. 8. 1.419861% Statistical Analysis of Date.” 2nd ed.. Scientific Press. Palo Alto. CA. Arias. S. (1971). Genetic heterogeneity in the Waardenburg Syn- drome. Birth Defects Orig. Art. Ser. 7: 87-101. Arias. 8. (1M). Waardenburg Syndrome—N distinct types. Am. J. Med. Gard. 6: 99- 100. Asher. J. H. Jr., and Friedman. T. B. (1990). Horses and hamster mutants as models for Waardenburg syndromes in humans. J. Med. Genet. 27: 618-626. Asher. J. 11.. Jr., Morell. IL. and Friedman. T. B. (19911. Waarden- ,burg syndrome (W3): The analysis of a single family with a WSI mutation showing linkage to RFLP markers on Human chromo- some 2Q. Am. J. Hum. Genet. 48: “-52. Asher. J. 11.. Jr., Pierpon., _ and Friedman. T. B. (19931. A War- denburg syndrome type 2 (W82) mutation not linked to PAX3. Am. J. Hum. Genet. 58(Suppl.l: 1685. (Abstract) Asher. J. 141.. Jr., Sommer "A.. Morell. 1L. and Friedman. T. B. (19961. Misaenee mutation in the paired domain of PAX3 causes Craniofa- cial-deafness-hand syndrome. Hum. Mat. 7: 30-35. Baldwin. C. T.. Hath. C. F.. Amos. J. A.. daSilva. E. 0.. and Milun~ sky. A. (1992). An exonic mutation in the HuP2 paired domain gene causes Waardenburg‘s syndrome. Nature 355: 637-638. Bookstein. F. L. (19911. ‘Morphometric Tools for Landmark Data: Geometry and Biology.“ Cambridge Univ. Press. England. 127 A MURINE MODEL FOR WAARDENBURG SYNDROME VARIATION Bultman. S. J.. Michaud. E. J.. and Woychik. R. P. (1992). Molecular characterisation ofthe mouse agouti locus. Cell 71: 1195-1204. Calinikos. J.(1963). Waardenburg’s syndrome. J. Laryngol. Otol. 77: 59-62. Coleman. D. L. (1978). Obese and diabetics: Two mutant genes caus- ing diabetes-obesity syndromes in mice. Diobctolqio 14: 141- 148. Davis. L 6.. Dibner. M. D.. and Battey. J. F. (1986). 'Methods in Molecular Biology.‘ Elsevier. New York. Delleman. J. W.. and Hageman. M. J. (1978). Ophthalmological find- ings in 34 patients with Waardenburg syndrome. J. Pediatr. OM: (helmet. Strobis. 15: 341-345. Dickie. M. M. (1964). New splotch alleles in the mouse. J. Herod. 55: 97 -101. - Dietrich. W. F.. Lander. E. 8.. Smith. J. 8.. Maser. A. R.. Gould. K. A.. Luongo. C.. Borenstein. N.. and Dove. W. (1993). Genetic identification of Mom-I. a major modifier locus affecting Min-in- duced intestinal neoplasia in the mouse. Cell 75: 631—639. DiGcorge. A. M.. Olmsted. R. W.. and Robinson. D. H. (1960). Waar- denburg Syndrome. J. Pediatr. 57: 649-699. Divekar. M. V. (1957). Waardenburg's syndrome. J. All India Oph- thalmol. Soc. 5: 1-5. Epstein. D. J.. Vekemans. M.. and Gros. P. (1991). Splotch (Sp’"). a mutation affecting development of the mouse neural tube. shows a deletion within the paired homeodomain of Pax3 Cell 67: 767- 774. Farrier. L A.. Grundfast. K. M.. Amos. J.. Arnos. K. S.. Asher. J. 11.. Jr.. Brighton. F.. Dichl. 8.. Fox. J.. Fey. 0.. Friedman. T. B.. Grecnberg. J.. Hoth. C.. Marazita. M.. Milunsky. A.. Morell. R.. Nance. W.. Newton. V.. Ramcsar. R.. San Augustin. T. B.. Skare. J.. Stevens. C. A.. Wagner. R. G.. Wilcox. E. 8.. Winshrp. 1.. and Read. A. 1". (1992). Waardenburg syndrome (WS) type 1 is caused by defects at multiple loci. one of which is near ALPP on chromo- some 2: First report of the WS Consortium. Am. J. Hum. Genet. 50: 902-913. Farrer. L. A.. Arnos. K. S.. Asher. J. 11.. Jr.. Baldwin. C. T.. Drchl. S. It. Friedman. T. B.. Greenberg. J.. Grundfast. K. M.. 1100). C.. Lalwani. A. K.. Lands. 8.. Leverton. K.. Milunsky. A.. Morell. R.. Nance. W.. Newton. V.. Ramesar. R.. Rae. V. S.. Reynolds. J. E.. San Agustin. T. 8.. Wilcox. E. R.. Winship. 1.. and Read. A. P. (1994). Locus heterogeneity for Waardenburg syndrome is pre- dictive of clinical subtypes. Am. J. Hum. Genet. 55: 728-737. Fey. C.. Newton. V.. Wellesley. D.. Harris. R.. and Read. A. P. (1990). Assignment of the locus for Waardenburg syndrome type 1 to hu- man chromosome 2q37 and possible homology to the splotch mouse. Am. J. Hum. Genet. 46: 1017-1023. Goldberg. M. I". (1966). Waardenburg syndrome with fundus and other anomalies. Arch. Ophthalmof. 76: 797-810. Goodman. It M.. Lewithal. 1.. Solomon. A.. and Klein. D. (1982). Upper limb involvement in the Klein-Waardenburg Syndrome. Am. J. Med. Genet. 11: 425-433. Hageman. M. J.. and Delleman.J. W. (1977). Heterogeneity in Waar- denburg Syndrome. Am. J. Hum. Genet. 29: 468-485. Hoth. C. F.. Milunsky. A.. Lipsky. N.. Sheffer. R.. Clarren. S. K.. and Baldwin. C. T. (1993). Mutations in the paired domain of the hu- man PAX3 gene cause Klein-Waardenburg syndrome (WSIII) as well as Waardenburg syndrome type I (WS-I). Am. J. Hum. Genet. 52: 455-462. Hughes. A. 8.. Newton. V. 8.. Liu. X. Z. and Read. A. P. (1994). A gene for Waardenburg syndrome type 2 maps close to the human homologue of the microphthalmia gene at chromosome 3p12- p14.l. Nature Genet. 7: 509-512. Klein. D. (1983). Historical background and evidence for dominant inheritance of the Klein-Waardenburg Syndrome (Type 111). Am. J. Med. Genet. 14: 231-239. Lander. E. 8.. and Schork. N. J. (1994). Genetic dissection ofcomplex traits. Science 265: 2037—2040. 297 Learny. L. (1974). Heritability of osteometric traits in a random bred population of mice. J. Hated. 65: 109-120. Liu. X.. Newton. V. 8.. and Read. A. P. (1995). Wairdenburg syn. drometype ll: Phenotypic findings and diagnostic criteria. Am. J. Med. Genet. 55: 95- 100. Lyon. M.. and Scarle. A. B. (1989). ‘Genetic Variants and Strains of the Laboratory Mouse.‘ 2nd ed.. Oxford Univ. Press. NY. Meijer. R.. and Walker. J. C. (1964). Waardenburg's syndrome. Plas- tic Reconstr. Surg. 34: 363—367. Meire. 1" .. Standaert. L.. DeLaey. J. J.. and chg. L. H. (1987). Waar- denburg syndrome. Hirschsprung megacolon. and Marcus Gunn Ptoais. Am. J. Med. Genet. 27: 683-687. Morell. R.. Friedman. T. 8.. Moeljopawiro, S.. Hartono. Soewito. and Asher.J. H.. Jr. (1992). A frameshifl. mutation in the HuP2 paired domain of the probable human homolog of murine Fed is responsi- ble for Waardenburg syndrome type 1 in an Indonesian family. Hum. Mol. Genet. 1: 243-247. Morell. R.. Friedman. T. 8.. and Asher. J. H.. Jr. (1993). A plus-one frameshift mutation in PAX3 alters the entire deduced amino acid sequence of the paired box in a Waardenburg syndrome type I (WSI) family. Hum. Mal. Genet. 2: 1487-1488. . Nance. W. E. and McConnell. F. E. (1973). Status and prospects of research in hereditary deafness. Adu. Hum. Genet. 4: 173-250. Nance. W. 15.. and Sweeney. A. (1975). Genetic factors in deafness of early life. Ota. Clm. N. Am. 8: 19-48. Natcr. J.. and Wasserrnan, W. (1974). “Applied Linear Statistical Models: Regression. Analysis of Variance and Experimental De- sign." Richard D. Irwin. Inc. liomewood. IL. Omcnn. G. S.. and McKusick. V. A. (1979) The association of Waar- denburg syndrome and liirschsprung megacolon. Am. J.. Med. Genet. 3: 217-233. Pantkc. O. A.. and Cohen, M. M. l 1971 l. The Waardenburg syndrome. Birth Defects: Orig. Art. Srr. 7: 147-152. Partington. M. W. (1964). Waardenburg's syndrome and heteroch- romia iridis in a deaf school population Can. Med. Assoc. J. 90: 1008- 1017. Pastcris. N. G.. Trask. 11. J.. Sheldon. 8., and Gorski. J. L (1993). Discordant phenotype of two overlapping deletions involving the PAX3 gene in chromosome 2q35. Hum. Mol. Grorct. 2: 953-959. I’avan. W. J.. Mac. 8.. Cheng. M.. and Tilgliman. S. $111995). Quan- titative trait loci that modify the severity of spotting in piebald mice. Genome Res. 5: 29-41. Puffenbcrger. E. 6.. Hosoda. K.. Washington. 8. 8.. Nakao. K.. deWit. D.. Yanagisawa. M.. and Chakrnvarti. A. r 1994 l. A missense muta» tion of the endothelin B receptor gene in multigenic Hirschsprung's disease. Cell 79: 1257-1266. Ray. D. K (1961). Waardenburg's syndrome. Br. J. Ophthalmol. 45: 568-569. Read. A. 1’. (1995). Par: genes—Paired feet in three camps. Nature Genet. 9: 333-334. Reardon. W.. Winter. R. M.. Rutland. I’.. Pullcyn. L. J.. Jones. B. M.. and Malcolm. S. (1994). Mutations in the fibroblast growth factor 2 gene cause Crouzon syndrome. Nature Genet. 8: 98-103. Reed. W. 8.. Stone. V. M.. Boder. E.. and Ziprokowslti. L. (1967). Pigmentary disorders in association with congenital deafness. Arch. Dermatol. 95: 176-186. Rugel. S. J.. and Keates. E. U. (1965). Waardenburg's syndrome in six generations offine family. Am. J. Dis. Child. 109: 579-583. Rutland. P.. Pulleyn. L. J.. Reardon. W.. Barsitser. M.. Haywood. R.. Jones. 8.. Malcolm. 5.. Winter. R. M.. Oldridge. M.. Slaney. S. F.. Poole. M. D.. and Wilki. A O. M. (1995). Identical mutations in the FGFR2 gene cause both Pfeiffer and Crouzon syndrome pheno- types. Nature Genet. 9: 173-176. Shah. K. N.. Dalal. S. J.. Desai. M. P.. Sltcllt. l’. N.. Joshi. N. C.. and Ambani, L M. (1981) White forelock. pigmentary disorder of 128 298 irides. and long segment Hiracltsprung disease’ : Possible "MM of Waardenburg syndrome. J. I’ediatr. 99: 432-435. Sheffer. R.. and Zlotogora. J. (19921. Autosomal dominant inheri- tance of Klein-Waardenburg syndrome. Am. J. Med. Genet. 42: 320-322. . Siracuss. L D.. Buehherg. A. M.. Copeland. N. G.. and Jenkins. N. A. (1989). Recombinant inbred strain and interspecific back- croas analysis of molecular markers flanking the murine agouti coat color locus. Genetics 122: 669-679. Sirocusa. L. D.. Alvord. W. 6.. Bicltmore. W. A.. Jenkins. N. A.. and Copeland. N. G. (1991). Inter-specific baeltcrnss mice show sex- specific differences in allelic inheritance. Genetics 128: 813-821. Snedecor. G. W.. and Cochran. W. G. (1967). “Statistical Methods." 6th ed.. Iowa State Univ. Press. Ames. IA. Sommer. A.. Young-Wee. T.. and Frye. T. (1983). Previoust unde- scribed syndrome of craniofacial. hand anomalies and sensorineu- ral deafness. Am. J. Med. Genet. 15: 71 -77. ASHER ET AL Shelter. 1". M. (1962). A deaf-mute with two congenital 8’“me- Arcli. Otoloryngol. 76: 42-46. Tassabehji. M.. Read. A. P.. Newton. V. E.. Harris. 11.. Belling. 11.. Cross. P.. and Strachsn. T. (1992). Waardenburg's syndrome pg. tients have mutations in the human homologue of the Pox-J paired box gene. Nature 355: 635—636. Tassabehji. M.. Newton. V. E.. and Read. A. P. (1994). Waardenburg syndrome type 2 caused by mutations in the human microphuul. mia (MITF) gene. Nature Genet. 8: 251 -2.55. Vegan. K. J.. Epstein. D. J.. Trasler. D. 0.. and Gros. P. (1993). The swatch-Delayed (Sp‘) mouse mutant carries a point mutation within the paired box of the Pas-3 gene. Genomics 17: 364-369. Waardenburg. P. J. (19511. A new syndrome combining develop- mental anomalies of the eyelids. eyebrows and nose root with pig- mentary defects of the iris and head hair and with congenital deafness. Am. J. Hum. Genet. 3: 195-253. Wang. 1... Ksrmody. C. 8.. and Pashsysn. H. t 1981). Wasrdenburg‘s syndrome: Variations in expressivity. Otololyn. Head Neck 8mg. 89: 666-670. 129 :4? Mums: (sweetening/ma 54 3 Hum Hered 178,3Morell Mai-“pages: Q “de.fio.landubggwiflheddetedmpagm 046 Mark: ‘ 047 MPAXJMMMNWSI 048 Short Communication 049 Hum Hered 50 Rajmmrell' Three Mutations in the Paired M in]. Carey' . 51 mm Mm” Homeodomam of PAX3 That Cause Thoma: . riedman“ - g: Jamesflitgrerlr.“ Waardenburg Syndrome Type 1 059 ; Department «Miriam.» 060 ‘__ State Universith'East [numb 061 ‘ Mich, 062 ‘ Devanment ofOtolaryneolou. _ _063 Head and Neck Surgery, 064 University of California. 065 San Francisco. Calif.. and 066 ‘ Graduate Program in Genetics, 067 Michigan State University, 068 East Lansing. Mich” USA 07 1 Key Words Abstract 072 PAX3 Genomic DNA from probands of various Waardenburg syn- 073 Waardenburg syndrome ' dromc (W8) families were PCR-amplified using primers 074 WSl flanking the 8 exons of PAX3. The PCR fragments were -080 screened for sequence variants, and subsequently cycle se- 08l queneed. Mutations were detected in exon 6 for 3 probands of 082 W8 type 1 families. These mutations all occur in the paired 083 homeodomain DNA-binding motif. or: KARGER onset-madame Manna-arm _ m :3 mama mmafififlm mam mm, m rumnmuu mmmununsrusu man”. on W.W.d Accepted: Apri 12. I,“ IQ Id! 102 -106 107 108 109 110 .111 .112 -113 n114 nllS nll6 n11? nll8 ..119 n120 n121 n122 n123 -124 n125 126 127 —128 -129 130 131 -132 133 134 ~135 136 137 138 139 140 141 -142 143 144 145 146 147 -148 149 .150 151 202 203 - 204 - 205 206 Wmsrlmxlmslmsslm I 130 Hum Hered 178, Morell 2 Waardenburg syndrome (W8) is an autoso- mal dominant condition characterized by deafness and various defects of neural-crest- derived tissues [1]. It accounts for over 2% of the congenitally deaf papulation [21. At least four types are recognized (types 1, 2, 3 and 4) on the basis of clinical attributes [31. Muta- tions in the PAX3 gene have been demon- strated in individuals with type 1 and type 3 |4-7I 9, 10! and with craniofacial deafness hand syndrome (CDHS) m. PAX3 encodes a transcription factor containing two DNA- binding motifs, a paired domain (exons 2, 3 and 4) and a paired-type homeodomain (ex- ons 5 and 6) [11). It is expressed in developing neural cresteells and in the brain [21. Until the availability of sequence information on exons 5-8, mutation screening was confined to exons 1-4; thus. almost all of the W8 muta- tions reported so far have been in the paired domain. WS-associated mutations have been demonstrated recently within the paired-type homeodomain [12). Here we report three ad- ditional mutations in the paired homeod main of PAX 3. ' Methods for isolation of DNA from blood. PCR primers and cycling parameters for am- plifying and sequencing exon 6 of PAX3, labelling PCR products by incorporation of ”P a-CI'P. and detection of single-strand con- formation variants (SSCVs) are described elsewhere [7, 10, 13, 14]. We screened for SSCVs of PCR products for all eight exons of the PAX3 gene from 34 different individuals (68 chromosomes). These individuals were either probands or obligate mutation carriers from different W8 families. SSCVs were de- tected in exon 6 PCR products amplified from the DNA of probands for 3 W8 type 1 families designated: MSUS, MSU7 and MSU9. PCR products from these individuals were gel-purified by electrophoresis through 2% GT0 low-melt agarose (FMC). eluted us- ing Wizard PCR prep columns (Promega), and sequenced using the A1214 cycle sequenc- ing kit (USB). Two nucleotide substitutions (in MSUS and MSU7), and one nucleotide insertion re- sulting in a frameshifl (in MSU9) were de- tected on sequencing gels. For the two substi- tutions, the sequence change was confirmed by PCR amplification of genomic DNA using allele-specific primers, referred to as amplifi- cation refractory mutation system (ARMS) (g). In MSUS a substitution 810 C-rT would create an Arg271Cys mutation, and is con- firmed by substituting TF149 (5’-TC1‘G- GTI'TAGCAACOGCI‘J‘) for 1365’ 10 as the forward primer for PCR amplification of DNA from members of the family (fig _1_A). Allele-specific products (290 bp) were amplio fied only from DNA of affected members and not from unaffected members. Mutant allele specific PCR products were not detected when genomic DNA from 50 random individ- uals (100 chromosomes) was amplified using the ARMS primers. In MSU7 an 820 G-iA substitution would create a Trp274Trm non. sense mutation, and is confirmed by substi- tuting TF1 13 (5'-AGCAACCGCCGTGCAA— GATA-3’) as the forward primer for PCR am- plification. Again, allele-Speciftc products (250 bp) were amplified from DNA of af- fected members only (fig .1_ B). Allele-specific products and control products were waived on 2% NuSeive 3:1 gels and visualized by ethidium bromide staining. The 874ins‘G‘ in MSU9 was detected on sequencing gels of PCR fragments as a consis- tent duplication of bands resulting from the ovedap of cycle-sequence products generated from the normal allele and the mutant allele (fig 1C). The duplicate sequencing bands oc- curred downstream of the insertion site. while sequencing band patterns upstream of the site were normal. A similar pattern was generated in reactions using a sequencing primer in the reverse direction as well, and was seen only in reactions generated from the DNA of affected individuals. We confirmed that this pattern was due to the insertion of a ‘G‘ in one of the alleles by cloning PCR fragments into the pGEM-T vector (Promega) and sequencing representative clones. Two varieties of clones could be distinguished by both sequencing and by subjecting PCR products generated from cloned DNA as template to SSCV analy- sis: those comprising the normal exon 6 se- Aarnnnn and 0km“ na.~u—.-. 20! 110 211 - 214 215 -216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 — 232 233 234 235 236 237 ‘ 238 239 240 241 242 243 244 245 - 246 247 248 249 250 253 254 255 -256 257 158 259 316 .321 -32r 329 -330 331 .332 333 334 335 WM’IIMIMMIM I 131 Hum Hered 178, Morell C Thae three mutations are important addi- tienrs to» tin: fiueratuue: ref 1541(32 Tilre ArgZ7les mutation in family M805 is iden- tical tcvtlue ruse runeurrhng,in rut arnrarranthy unrelated British family [WS.IO; A. Read, [aerst rxrnnnnuurJ. 1fhds is tin: first resonates! occurrence of a shared mutation among the more than 25 W8 mutations described [9:10]. Haplotype analyses should reveal whether the mutant alleles segregating in families MSUS and WS.10 have the same origin, or if the nucleotide substitution occurred at least twice in history. This mutation also occurs at the same position as the one described in family NTH8 (Arg27lGly) [LO] The differences in penetrance of deafness between family NIH8 (516) and WS.10 plus MSUS (217 and 3/11 respectively) are potentially informative as to the etiology of deafness in W8. MSU7 is segre- gating for a nonsense (T rp274Trrn) mutation. Yet like WS.10. MSU5, and NlH8, all of which have missense mutations in exon 6. the affected individuals in'MSU7 have typical W8 type 1 features. Three of the four affected individuals in MSU7 display profound senso- rineural hearing loss. Another family with typical W81 features. MSU9, has a frameshifl mutation (874ins‘G') in cson 6. In MSU9. the framesth mutation is detected only among thc3children,eechwithtypicelW8typel features. but not detected in either parent, who are clinically normal. An earlier study using RFLP markers demonstrated that indi- vidual MSU9-1 is the biological father of the three affected children [1_51. This confirms the hypothesis that W81 in this family is the result of a germline mosaicisrn [1;]. Acknowledgements 11riswcrkwassupportedinpertbyaresearchgrant number ROI D011 160 from the National institute on Deafness and Other Communication Disorders. Na- tional lnstitutescf Health toll-1A and TBF. We would liketothanltmembcrsofthefarniliesfortheirhelpand initiative. ‘1 References Waardenburg Plenewsyndrotne ambining develops-sud anoma- lies of the eydids. eyebrows and noserootwithpkmentarydefeusof Id deafness. Am i_i-lura Genet 1931;):195-133. hrtingtcn MW: Waardenburg's duo: in a deaf sehcd poptdation. Cenad Med Are .1 "64:90:10“- 1017. McKusick VA: Mendelian inheri- tance in Man - CD. Million. Johns floptins University Press, 1994. Baldwian.11cthG.AmcslA.da Silva £0. Milunsty A: An exonie mionittbefluflpaireddo ' main gene causes Wurdenburgs syndrome Nature 1992;355:637- 63!. $TassabchiiM.lodAl’.Newton d 10 VF. Harris ft. Ming ll. Guns 1’. Stradran T: Waardenburg's syn- drome patients have mutations in tbebuman homolognecflhefAX-J paired boa gene. Nature 1992:3352 635-636. Morell K Friedman TB. Moeljopa- wiroS. Harmon. Sowic.AsherJl'1 Jr: A frameshift mutation in the paired domain offlun is responsi- bleforWaar-denburgsyndrornetypc I 'm an Indonesian far-3y. Hum Molechnet 199131243447. Morell IL Friedman TI. Asher 1H Jr: Aplusene framedtitl mention '- I’AXJ alters the entire deduced syndrome type I (WSI) family. Hum Melee Gena 19932:!487-1488. Asher 1H 1r. Sommer A Mord R. FriedmanTnzMirsensernutsticn in the paired dorne'u cf PAX3 ceases .... ' ‘.-‘ drone. lit-n Moat 1996:7330-35. Tl. Greenberg J. Grundfaat L MCLIIflniALundaBJevo crtcn & Minsky A Morell 1L Nance WE. Hem V. llameear ll. Ran V3. WJESIIAugustin TI, Wilcox Ell. Winship 1. Read AP: hosts beterogenert' y for Waar- derliurg syndrome is predictive of dish! subtypes. Ara J Hum Genet 1994;55:728-737. Lalwani AK Brirter JR. Fe: 1. Grundfast KM.Plcplis 8.8anAgus tin T8, Wilcox ER: Furtherelucida- tion of the genomic structure of PAXLar-d identifrestioncftwo dif- ferent point mutations within the PAX! homeoboa that cause Waar- denburg syndrome type I in too families. Am 1 Hum Genet 1995;56' ‘( 1‘1 132 336 ”7 wmszlmxlmlmlme 3338 Hum Hered 178, Morell 6 341 11 Ootdding MD. Chalepakis G. 342 Detach U. Emeline 11L Grins P: .343 PAX-3. a novel marine DNA hind- 344 kg protein W during early 34$ netsrogenesis. £14130 3 1991;10: 346 1133-1147. 347 12 Snort 131'. Kioussi C. Gross P: -348 Mammalian PAX gene: in Camp- 349 hell A Anderson W. Jones E (eds): 330 Annual Review of Genetic; Pale a 351 Allo.Annua11teviews.1994.vo128. 332 ”219—236. 333 13 AsherJHJrJ-icrelLI-‘riedrnen 354 TI: Waardenburg syndrome (W8): 33$ Theanalysisofasindefsrnilywithe 356 W81 mination showing linkage to -337 RFU’ markers on hurnan chm 338 somehAmJHrnnGenetl”l;48: 339 43-32. 360 14 MorellkljengY.Asher3HJr,We- -361 hamflinnaurfJVinata 8.Ar- 362 hya 1N. Friedman TB: Analysis of -363 shorttandesnrepeetmmlefre 364 queney distributions in a Balinese 365 population. Hum Motor: Genet 366 1995313341. 361 ISKapurS.1(arun8:Gerrn-linemo- 368 saieisu 'nr Waardenburg syndrome 369 Gin Genet 1991;39:194-198. 133 Hum Hered 178, Morell Hg. ‘ ‘ IIBI It 1‘ ‘I I symbolscortespoodtothenumhersoverlanesofthe u] f' template tn PCR metions containing primers for both exon 2 and themutantsequcnce(810 C—eT)ofexon6 of PAX3. All motions generated the $35 hp control fngnent the ARMS exon 6 fragnentwas enter! only from DNA of affected individuals. i- groe. The lea-Inna lane contains PCR products used' In our lab as molecular weight nurkers. A 535-bp frag- ment containing exon 2 of PAX3 Is generated from all genomic DNA templates in a PCR reaction whfle the mutant allele (820 G-rA)-specific primers amplify DNA from affected individuals only. C MSU9 pedi- G‘inserrion at nucleotide 874 of PAX3 The arrowhead indicates the first occurrence (at nt 874) of the extra band seen in insertion show the same pattern in all lanes The same pattern is seen when sequencing reactions are per- I‘- M” -‘A ,. I 616 7176181911122013114152 I I 0 232425 26 27 262930 31 32333435 3637 2 3 9 41016 62.324257172618827 exon 2 , ARMS ’ "M 6 19112930122031 3213213334 14 rs sear exon 2; ARMS exon 6 h- e LIST OF REFERENCES LIST OF REFERENCES Waardenburg, P.J. (1951) A new syndrome combining developmental anomalies of the eyelids, eyebrows and nose root with pigmentary defects of the iris and head hair with congenital deafness. Am J Hum Genet 3, 195-253. Wmship, l. and Beighton, P. (1992) Phenotypic discriminants in the Waardenburg syndrome. Clin Genet 41 (4), 181-188. Farrer, L.A., Grundfast, K.M., Amos, J., Amos, K.S., Asher, J.H., Jr., Beighton, P., Diehl, S.R., Fex, J., Read, A.P., et al. (1992) Waardenburg syndrome (WS) type I is caused by defects at multiple loci, one of which is near ALPP on chromosome 2: First report of the WS consortium. Am J Hum Genet 50(5), 902-913. Partington, MW. (1964) Waardenburg's Syndrome and heterochromia irides in a deaf school population. Can Med Assoc J 90, 1008-1017. Farrer, L.A., Arnos, K.S., Asher, J.H., Jr., Baldwin, C.T., Diehl, S.R., Friedman, T.B., Greenberg, J., Grundfast, K.M., Hoth, C., Lalwani, A.K., et al. (1994) Locus heterogeneity for Waardenburg syndrome is predictive of clinical subtypes. Am J Hum Genet 55(4), 728-737. Arias, S. and Mota, M. (1978) Apparent non-penetrance for dystopia in Waardenburg syndrome type I, with some hints on the diagnosis of dystopia canthorum. J Genet Hum 26(2), 103-131. Arias, S. (1980) Waardenburg syndrome—two distinct types. Am J Med Genet 6(1), 99-100. Arias, S. (1984) Diagnosis and penetrance of dystopia canthorum in Waardenburg syndrome type I (W81) [letter]. Am-J-Med-Genet 17(4), 863-867. 134 10. 11. 12. 13. 14. 15. 16. 135 Liu, X.Z., Newton, V.E., and Read, AP. (1995) Waardenburg syndrome type II: phenotypic findings and diagnostic criteria. Am J Med Genet 55(1), 95-100. Varughese, S., Kumar, A., Rao, 8., and Puliyel, J.M. (1988) Type II Waardenburg syndrome. Indian Pediatr 25(4), 384-386. Klein, D. (1983) Historical background and evidence for dominant inheritance of the Klein-Waardenburg syndrome (type III). Am J Med Genet 14(2), 231-239. Goodman, R.M., Lewithal, l., Solomon, A.. and Klein, D. (1982) Upper limb involvement in the Klein-Waardenburg syndrome. Am J Med Genet 1 1 (4), 425-433. Klein, D. (1947) Albinisme partiel (Ieucisme) accompagne de surdimutite, d'osteomyodysplasie, de raideurs articulaires congenitales multiples et d'autres malformations congenitales. Arch Klaus Stifl‘ Vererb Forsch 22, 336-342. Klein, D. (1950) Albinisme partiel (Ieucisme) aves surdimutite, blepharophimosis et dysplasias myo-osteo-articulaires. Helvet Paediat Acta 5, 38-58. Hoth, C.F., Milunsky, A.. Lipsky. N.. Shaffer, R., Clarren, SK, and Baldwin, C.T. (1993) Mutations in the paired domain of the human PAX3 gene cause Klein-Waardenburg syndrome (WS-lll) as well as Waardenburg syndrome type I (WS-I). Am—J—Hum-Genet 52(3), 455- 462. WIIbrandt, HR. and Ammann, F. (1964) [New observation of the severe form of the Klein-Waardenburg syndrome]. Arch Julius Klaus Stift Vererbungsforsch Sozialanthropol Rassenhyg 39(1-4), 80-92. 17. 18. 19. 20. 21. 22. 23. 24. 25. 136 Sheffer, R. and Zlotogora, J. (1992) Autosomal dominant inheritance of Klein-Waardenburg syndrome. Am J Med Genet 42(3), 320-322. Perrot, H., Ortonne, J.P., and Thivolet, J. (1977) Ultrastructural study of leukodermic skin in Waardenburg-Klein syndrome. Acta Derm Venereol Stockh 57(3), 195-200. Patrizi, A.. Colombati, S., and Valenti, L. (1985) [A case of Waardenburg-Klein syndrome]. G Ital Dennatol Venereol120(4), 277- 279. Partsch, C.J. and Schleyer, K.H. (1971) [Chromosome aberrations in the Waardenburg-KIein-Syndrome]. HNO 19(4), 121-123. Ortonne, J.P., Perrot, H., Beyvin, A.J., Revol, L., and Thivolet, J. (1976) [The Waardenburg-Klein syndrome]. Ann Dermatol Syphiligr Paris 103(3), 245-56. Nutman, J., Nissenkom, l., Varsano, l., Mimouni, M., and Goodman, RM. (1981) Anal atresia and the Klein-Waardenburg syndrome. J- Med-Genet 18(3), 239-241. Zlotogora, J., Lerer, |., Bar David, S., Ergaz, Z., and Abeliovich, D. (1995) Homozygosity for Waardenburg syndrome. Am J Hum Genet 56(5), 1173-1178. Ariturk, E., Tosyali, N.. and Ariturk, N. (1992) A case of Waardenburg syndrome and aganglionosis. Turk J Pediatr 34(2), 111-114. Badner, J.A. and Chakravarti, A. (1990) Waardenburg syndrome and Hirschsprung disease: Evidence for pleiotropic effects of a single dominant gene. Am J Med Genet 35(1), 100-104. 26. 27. 28. 29. 30. 31. 32. 33. 137 de Lumley Woodyear, L., Boulesteix, J., Rutkowski, J., and Umdenstock, R. (1980) Waardenburg syndrome associated with Hirschsprung disease and other abnormalities [letter]. Pediatrics 65(2), 368-369. Omenn, GS. and McKusick, VA. (1979) The association of Waardenburg syndrome and Hirschsprung megacolon. Am J Med Genet 3(3), 217-223. Shah, K.N., Dalal, S.J., Desai, M.P., Sheth, P.N., Joshi, N.C., and Ambani, L.M. (1981) White forelock, pigmentary disorder of irides, and long segment Hirschsprung disease: possible variant of Waardenburg syndrome. J Pediatr 99(3), 432-435. Meire, F ., Standaert, L., De Laey, J.J., and Zeng, L.H. (1987) Waardenburg syndrome, Hirschsprung megacolon, and Marcus Gunn ptosis. Am J Med Genet 27(3), 683—686. Arias, S. (1971) Genetic heterogeneity in the Waardenburg syndrome. Birth Defects 07(4), 87-101. Hageman, M.J. (1980) Heterogeneity of Waardenburg syndrome in Kenyan Africans. Metab Pediatr Ophthalmol 4(4), 183-184. Darwin, C. (1 859) The Orgin of Species. 1859, Oxford: Oxford University Press. Brown, K.S., Bergsma, DR. and Barrow, M.V. (1971) Animal models of Pigment and hearing abnormalities in man. Birth Defects Original Article Serios(Vll), 102-109. Fraser, GR. (1 976) The Causes of Profound Deafness in Childhood. 1976, Baltimore: Johns Hopkins University Press. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 138 Kapur, S. and Karam, S. (1991) Germ-line mosaicism in Waardenburg syndrome. Clin Genet 39(3), 194-198. Mallory, S.B., Vlfiener, E. and Nordlund, J.J. (1986) Waardenburg's syndrome with Hirschsprungs disease: a neural crest defect. Pediat Dermat 3(2), 1 19-124. McKusick, VA. (1973) Congenital deafness and Hirshsprung's disease. New England Journal of Medicine 288, 691. Auerbach, R. (1954) Analysis of the developmental effects of a lethal mutation in the house mouse. J Exp Zoo/127, 305-329. Lyon, M.F.a.S., A.G. (1989)Genetic Variants and Strains of the Laboratory Mouse. 1989, Oxford: Oxford University Press. Webster, W. (1973) Embryogenesis of the enteric ganglia in normal mice and in mice that develop congenital aganglionic megacolon. Journal Embryo! Exp Morphol 30, 573-585. Franz, T. (1992) Neural tube defects without neural crest defects in splotch mice. Teratology 46(6), 599-604. Epstein, D.J., Malo, D., Vekemans, M., and Gros, P. (1991) Molecular characterization of a deletion encompassing the splotch mutation on mouse chromosome 1. Genomics 10(1), 89-93. Epstein, D.J., Vekemans, M., and Gros, P. (1991) Splotch (Sp2H), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Fax-3. Cell 67(4), 767-774. Baker, S.J. and Reddy, E.P. (1995) B cell differentiation: role of E2A and Pax5/BSAP transcription factors. Oncogene 11(3), 413-426. 45. 46. 47. 48. 49. 50. 51. 52. 53. 139 Franz, T. (1993) The Splotch (Sp1 H) and Splotch-delayed (Spd) alleles: differential phenotypic effects on neural crest and limb musculature. Anat Embryol Berl 187(4), 371-377. Franz, T., Kothary, R., Surani, M.A., Halata, Z., and Grim, M. (1993) The Splotch mutation interferes with muscle development in the limbs. AnatEmbron Bert 187(2), 153-160. Franz, T. and Kothary, R. (1993) Characterization of the neural crest defect in Splotch (Sp1 H) mutant mice using a lacZ transgene. Brain Res Dev Brain Res 72(1), 99-105. Goulding, M.. Sterrer, S., Fleming, J., Balling, R., Nadeau, J., Moore, K.J., Brown, 8.0., Steel, K.P., and Gruss, P. (1993) Analysis of the Pax-3 gene in the mouse mutant splotch. Genomics 17(2), 355-363. Moase, CE. and Trasler, D.G. (1989) Spinal ganglia reduction in the splotch-delayed mouse neural tube defect mutant. Teratology 40(1), 67-75. Moase, CE. and Trasler, D.G. (1990) Delayed neural crest cell emigration from Sp and Spd mouse neural tube explants. Teratology 42(2), 171-182. Underhill, D.A., Vogan, K.J., and Gros, P. (1995) Analysis of the mouse Splotch-delayed mutation indicates that the Fax-3 paired domain can influence homeodomain DNA-binding activity. Proc Natl Acad Sci USA 92(9), 3692-3696. Vogan, K.J., Epstein, D.J., Trasler, D.G., and Gros, P. (1993) The splotch-delayed (Spd) mouse mutant carries a point mutation within the paired box of the Pax-3 gene. Genomics17(2), 364-369. Tremblay, P. and Gruss, P. (1994) Pax: genes for mice and men. Pharmacol Ther 61 (1 -2), 205-226. 54. 55. 56. 57. 58. 59. 60. 61. 62. 140 Asher, J.H., Jr. and Friedman, T.B. (1990) Mouse and hamster mutants as models for Waardenburg syndromes in humans. J Med Genet 27(10), 618-626. Tsukamoto, K., Nakamura, Y., and Niikawa, N. (1994) Isolation of two isoforms of the PAX3 gene transcripts and their tissue-specific alternative expression in human adult tissues. Hum Genet 93(3), 270- 274. lshikiriyama, S., Tonoki, H., Shibuya, Y., Chin, S., Harada, N., Abe, K., and Niikawa, N. (1989) Waardenburg syndrome type I in a child with de novo inversion (2) (q35q37.3). Am J Med Genet 33(4), 505-507. lshikiriyama, S. (1993) Gene for Waardenburg syndrome type I is located at 2q35, not at 2q37.3. Am J Med Genet 46(5), 608. Foy, 0., Newton, V., Wellesley, D., Harris, R., and Read, AP. (1990) Assignment of the locus for Waardenburg syndrome type I to human chromosome 2q37 and possible homology to the Splotch mouse. Am J Hum Genet 46(6), 1017-1023. Asher, J.J., Morell, R., and Friedman, T.B. (1991) Confirmation of the location of a Waardenburg syndrome type I mutation on human chromosome 2q. Tight linkage to F N1 and ALPP. Ann N YAcad Sci 630, 295-297. Asher, J.H., Jr., Morell, R., and Friedman, TE. (1991) Waardenburg syndrome (VVS): the analysis of a single family with 3 W81 mutation showing linkage to RFLP markers on human chromosome 2q. Am J Hum Genet 48(1), 43-52. Burri, M., Tromvoukis, Y., Bopp, D., Frigerio, G., and Noll, M. (1989) Conservation of the paired domain in metazoans and its structure in ' three isolated human genes. EMBO J 8(4), 1183-1190. Noll, M. (1993) Evolution and role of Pax genes. Curr Opin Genet Dev 3(4), 595-605. 63. 64. 65. 66. 67. 68. 69. 70. 141 Frigerio, G., Burri, M.. Bopp, D., Baumgartner, S., and Noll, M. (1986) Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network. Cell 47(5), 735-746. Bopp, D., Burri, M.. Baumgartner, S., Frigerio, G., and Noll, M. (1986) Conservation of a large protein domain in the segmentation gene paired and in functionally related genes of Drosophila. Cell 47(6), 1033-1040. Bopp, D., Jamet, E., Baumgartner, S., Burri, M., and Noll, M. (1989) Isolation of two tissue-specific Drosophila paired box genes, Pox meso and Pox neuro. EMBO J 8(11), 3447-3457. Stapleton, P., Weith, A., Urbanek, P., Kozmik, Z., and Busslinger, M. (1993) Chromosomal localization of seven PAX genes and cloning of a novel family member, PAX-9. Nature Genet 3(4), 292-298. Chalepakis, G. and Gruss, P. (1995) Identification of DNA recognition sequences for the Pax3 paired domain. Gene 162(2), 267-270. Schnittger, S., Rao, V.V., Deutsch, U., Gruss, P., Balling, R., and Hansmann, l. (1992) Pax1, a member of the paired box-containing class of developmental control genes, is mapped to human chromosome 20p11.2 by in situ hybridization (ISH and FISH). Genomics 14(3), 740-744. Wallin, J., Mizutani, Y., lmai, K., Miyashita, N.. and Moriwaki, K. (1993) A new Pax gene, Pax-9, maps to mouse chromosome 12. Mammal Genome 4(7), 354-358. Dressler, G.R., Deutsch, U., Chowdhury, K., Nomes, HO, and Gruss, P. (1990) Pax2, a new murine paired-box-containing gene and its expression in the developing excretory system. Development 109(4), 787-795. 71. 72. 73. 74. 75. 76. 77. 78. 142 Dressler, GR. and Douglass, EC. (1992) Pax-2 is a DNA-binding protein expressed in embryonic kidney and Wilms tumor. Proc Natl Acad Sci USA 89(4), 1179-1183. Nomes, H.O., Dressler, G.R., Knapik, E.W., Deutsch, U., and Gruss, P. (1990) Spatially and temporally restricted expression of Pax2 during murine neurogenesis. Development 109(4), 797-809. Czerny, T., Schaffner, G., and Busslinger, M. (1993) DNA sequence recognition by Pax proteins: Bipartite structure of the paired domain and its binding site. Genes Dev 7(10), 2048-2053. Asano, M. and Gruss, P. (1992) Pax-5 is expressed at the midbrain- hindbrain boundary during mouse development. Mech Dev 39(1-2), 29- 39. Zannini, M.. Francis Lang, H., Plachov, D., and Di Lauro, R. (1992) Pax-8, A paired domain-containing protein, binds to a sequence overlapping the recognition site of a homeodomain and activates transcription from two thyroid-specific promoters. Mol Cell Biol 12(9), 4230-4241. Kozmik, Z., Kurzbauer, R., Doerfler, P., and Busslinger, M. (1993) Alternative splicing of Pax-8 gene transcripts is developmentally regulated and generates isoforms with different transactivation properties. Mol Cell Biol 13(10), 6024-6035. Plachov, D., Chowdhury, K., Walther, C., Simon, 0., Guenet, J.L., and Gruss, P. (1990) Pax8, a murine paired box gene expressed in the developing excretory system and thyroid gland. Development 1 10(2), 643-651. Tamura, T., lzumikawa, Y., Kishino, T., Soejima, H., Jinno, Y., and Niikawa, N. (1994) Assignment of the human PAX4 gene to chromosome band 7q32 by fluorescence in situ hybridization. Cytogenet Cell Genet 66(2), 132-134. 79. 80. 81. 82. 83. 84. 85. 86. 87. 143 Ton, C.C., Miwa, H., and Saunders, GP. (1992) Small eye (Sey): cloning and characterization of the murine homolog of the human aniridia gene. Genomics 13(2), 251-256. Walther, C. and Gruss, P. (1991) Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 113(4), 1435-1449. Schafer, B.W. and Mattei, MG. (1993) The human paired domain gene PAX7 (Hup1) maps to chromosome 1p35-1p36.2. Genomics 17(1), 249-251. Schaefer, B.W., Czerny, T., Bemasconi, M.. Genini, M.. and Busslinger, M. (1994) Molecular cloning and characterization of a human PAX-7 cDNA expressed in normal and neoplastic myocytes. Nuc Acids Res 22(22), 4574-4582. Shapiro, D.N., Sublett, J.E., Li, B., Valentine, M.B., Morris, SW, and Noll, M. (1993) The gene for PAX7, a member of the paired-box- containing genes, is localized on human chromosome arm 1p36. Genomics 17(3), 767-769. Jostes, B., Walther, C., and Gruss, P. (1990) The murine paired box gene, Pax7, is expressed specifically during the development of the nervous and muscular system. Mech Dev 33(1), 27-37. Chalepakis, G., Goulding, M.. Read, A., Strachan, T., and Gruss, P. (1994) Molecular basis of splotch and Waardenburg Pax-3 mutations. Proc Natl Acad Sci USA 91 (9), 3685-3689. Read, AP. and van Heyningen, V. (1994) PAX genes in human developmental anomalies. Semin Dev Biol 5(5), 323-332. Read, AP. (1995) Pax genes: Paired feet in three camps. Nat Genet 9(4), 333-334. 88. 89. 90. 91. 92. 93. 94. 95. 144 Stoykova, A. and Gruss, P. (1994) Roles of Pax-genes in developing and adult brain as suggested by expression patterns. J Neurosci14(3 Pt 2), 1395-1412. Strachan, T. and Read, AP. (1994) PAX genes. Curr Opin Genet Dev 4(3), 427-438. Chalepakis, G., Stoykova, A., lenholds, J., Tremblay, P., and Gruss, P. (1993) Pax: gene regulators in the developing nervous system. J Neurobiol 24(10), 1 367-1 384. Gerard, M., Abitbol, M.. Delezoide, A.L., Dufier, J.L., Mallet, J., and Vekemans, M. (1995) PAX-genes expression during human embryonic development, a preliminary report. C R Acad Sci III 318(1), 57-66. Timmons, P.M., Wallin, J., Rigby, P.W., and Balling, R. (1994) Expression and function of Pax 1 during development of the pectoral girdle. Development 120(10), 2773-2785. Wallin, J., Wilting, J., Koseki, H., Fritsch, R., Christ, B., and Balling, R. (1994) The role of Pax-1 in axial skeleton development. Development 120(5), 1109-1121. Grindley, J.C., Davidson, DR, and Hill, RE. (1995) The role of Pax-6 in eye and nasal development. Development 121(5), 1433-1442. Hill, R.E., Favor, J., Hogan, B.L., Ton, C.C., Saunders, G.F., Hanson, l.M., Prosser, J., Jordan, T., Hastie, ND, and van Heyningen, V. (1991) Mouse small eye results from mutations in a paired-like homeobox-containing gene [published erratum appears in Nature 1992 Feb 20;355(6362):750]. Nature 354(6354), 522-525. 96. 97. 98. 99. 100. 101. 102. 1 03. 1 04. 145 Quiring, R., Walldorf, U., Kloter, U., and Gehring, W.J. (1994) Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans [see comments]. Science 265(5173), 785- 789. Matsuo, T., Osumi Yamashita, N., Noji, S., Ohuchi, H., Koyama, E., Myokai, F.. Matsuo, N., Taniguchi, 8., Doi, H., lseki, S., et al. (1993) A mutation in the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells. Nat Genet 3(4), 299-304. Hanson, l.M., Seawright, A., Hardman, K., Hodgson, S., Zaletayev, D., Fekete, G., and Van Heyningen, V. (1993) PAX6 mutations in aniridia. Hum Mol Genet 2(7), 915-920. Davis, A. and Cowell, J.K. (1993) Mutations in the PAX6 gene in patients with hereditary aniridia. Hum Mol Genet 2(12), 2093-2097. Glaser, T., Walton, BS, and Maas, R.L. (1992) Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene. Nature Genet 2(3), 232-239. Glaser, T., Jepeal, L., Edwards, J.G., Young, S.R., Favor, J., and Maas, R.L. (1994) PAX6 gene dosage effect in a family with congenital cataracts, aniridia, anophthalmia and central nervous system defects. Nature Genet 7(4), 463-471. Jordan, T., Hanson, l., Zaletayev, D., Hodgson, S., Prosser, J., Seawright, A., Hastie, N.. and van Heyningen, V. (1992) The human PAX6 gene is mutated in two patients with aniridia. Nature Genet 1(5), 328-332. DeRespinis, PA. and Wagner, RS. (1987) Peters' anomaly in a father and son. Am-J-Ophthalmol104(5), 545-6. Eiferrnan, RA. (1984) Association of VVIlms' tumor with Peter's anomaly. Ann-Ophthalmol16(10), 933-934. 105. 106. 107. 108. 109. 110. 111. 112. 113. 146 Hanson, l.M., Fletcher, J.M., Jordan, T., Brown, A., Taylor, 0., Adams, R.J., Punnett, H.H., and van Heyningen, V. (1994) Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly. Nat-Genet 6(2), 168-173. Ivanov, l., Shuper, A., Shohat, M., Snir, M., and Weitz, R. (1995) Aniridia: recent achievements in paediatric practice. Eur J Pediatr 154(10), 795-800. Fantes, J.A., Bickmore, W.A., Fletcher, J.M., Ballesta, F., Hanson, l.M., and van Heyningen, V. (1992) Submicroscopic deletions at the WAGR locus, revealed by nonradioactive in situ hybridization. Am J Hum Genet 51(6), 1286-1294. Schwartz, F.. Neve, R., Eisenman, R., Gessler, M., and Bruns, G. (1994) A WAGR region gene between PAX-6 and FSHB expressed in fetal brain. Hum Genet 94(6), 658-664. Mirzayans, F.. Pearce, W.G., MacDonald, l.M., and Walter, MA. (1995) Mutation of the PAX6 gene in patients with autosomal dominant keratitis. Am J Hum Genet 57(3), 539-548. Sanyanusin, P., Schimmenti, L.A., McNoe, L.A., Ward, T.A., Pierpont, M.E., Sullivan, M.J., Dobyns, W.B., and Eccles, MR. (1995) Mutation of the PAX2 gene in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux. Nat Genet 9(4), 358-363. Balling, R., Deutsch, U., and Gruss, P. (1988) Undulated, a mutation affecting the development of the mouse skeleton, has a point mutation in the paired box of Pax1. Cell 55(3), 531-535. Balling, R. (1994) The undulated mouse and the development of the vertebral column. Is there a human PAX-1 homologue? Clin Dysmorphcl 3(3), 185-191 . Chalepakis, G., Fritsch, R., Fickenscher, H., Deutsch, U., Goulding, M., and Gruss, P. (1991) The molecular basis of the undulated/Pax-1 mutation. Cell 66(5), 873-884. 114. 115. 116. 117. 118. 119. 120. 121. 147 Stuart, ET. and Gruss, P. (1995) PAX genes: what's new in developmental biology and cancer? Hum Mol Genet, 1717-1720. Stuart, E.T., Yokota, Y., and Gruss, P. (1995) PAX and HOX in neoplasia. Adv Genet 33, 255-274. Maulbecker, CC. and Gruss, P. (1993) The oncogenic potential of Pax genes. EMBO J 12(6), 2361-2367. Shanna, P.M., Bowman, M., Yu, BF, and Sukumar, S. (1994) A rodent model for erms tumors: embryonal kidney neoplasms induced by N-nitroso-N'-methylurea. Proc Natl Acad Sci USA 91(21), 9931- 9935. Poleev, A.. Fickenscher, H., Mundlos, S., Wmterpacht, A., Zabel, B., Fidler, A., Gruss, P., and Plachov, D. (1992) PAX8, a human paired box gene: Isolation and expression in developing thyroid, kidney and erms' tumors. Development 116(3), 61 1-623. Poleev, A., Wendler, F.. Fickenscher, H., Zannini, M.S., Yaginuma, K., Abbott, C., and Plachov, D. (1995) Distinct functional properties of three human paired-box-protein, PAX8, isoforms generated by alternative splicing in thyroid, kidney and Vlfilms' tumors. Eur J Biochem 228(3), 899-91 1. Hazen Martin, D.J., Re, G.G., Garvin, A.J., and Sens, DA. (1994) Distinctive properties of an anaplastic Wllms' tumor and its associated epithelial cell line. Am J Pathol 144(5), 1023-1034. Eccles, M.R., Yun, K., Reeve, A.E., and Fidler, A.E. (1995) Comparative in situ hybridization analysis of PAX2, PAX8, and WT1 gene transcription in human fetal kidney and WIImS' tumors. Am J Pathol 146(1), 40-45. 122. 123. 124. 125. 126. 127. 128. 129. 130. 148 Tagge, E.P., Hanson, P., Re, G.G., Othersen, H.B., Jr., Smith, CD, and Garvin, A.J. (1994) Paired box gene expression in Vlfilms' tumor. J Pediatr Surg 29(2), 134-141. Kozmik, 2., Sure, U., Ruedi, D., Busslinger, M., and Aguzzi, A. (1995) Deregulated expression of PAX5 in medulloblastoma. Proc Natl Acad Sci USA 92(12), 5709-5713. Adams, B., Doerfler, P., Aguzzi, A., Kozmik, Z., Urbanek, P., Maurer Fogy, l., and Busslinger, M. (1992) Pax-5 encodes the transcription factor BSAP and is expressed in B lymphocytes, the developing CNS, and adult testis. Genes Dev 6(9), 1589-1607. Stuart, E.T., Kioussi, C., and Gruss, P. (1994) Mammalian Pax genes. Annu Rev Genet 28, 219-236. Bennicelli, J.L., Fredericks, W.J., VVIISOI'I, R.B., Rauscher, F Jr., and Barr, F.G. (1995) erd type PAX3 protein and the PAX3-FKHR fusion protein of alveolar rhabdomyosarcoma contain potent, structurally distinct transcriptional activation domains. Oncogene 11(1), 119-130. Biegel, J.A., Nycum, L.M., Valentine, V., Barr, F.G., and Shapiro, D.N. (1995) Detection of the t(2;13)(q35;q14) and PAX3-FKHR fusion in alveolar rhabdomyosarcoma by fluorescence in situ hybridization. GenesChromosom Cancer 12(3), 186-192. Davis, R.J., CM, 0.0., Lovell, M.A., Biegel, J.A., and Barr, F.G. (1994) Fusion of PAX7 to F KHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma. Cancer Res 54(11), 2869-2872. Pappo, A.S., Shapiro, D.N., Crist, WM, and Maurer, HM. (1995) Biology and therapy of pediatric rhabdomyosarcoma. J Clin Oncol 13(8), 2123- 2139. Pappo, AS. (1994) Rhabdomyosarcoma and other soft tissue sarcomas of childhood. Curr Opin Oncol 6(4), 397-402. 131. 132. 133. 134. 135. 136. 137. 138. 149 Sublett, J.E., Jeon, IS, and Shapiro, D.N. (1995) The alveolar rhabdomyosarcoma PAX3/FKHR fusion protein is a transcriptional activator. Oncogene 11(3), 545-552. Macina, R.A., Barr, F.G., Galili, N., and Riethman, H.C. (1995) Genomic organization of the human PAX3 gene: DNA sequence analysis of the region disrupted in alveolar rhabdomyosarcoma. Genomics 26(1), 1-8. Barr, F.G., Galili, N., Holick, J., Biegel, J.A., Rovera, G., and Emanuel, BS. (1993) Rearrangement of the PAX3 paired box gene in the paediatric solid tumour alveolar rhabdomyosarcoma. Nature Genet 3(2), 113-117. Galili, M, Davis, R.J., Fredericks, W.J., Mukhopadhyay, S., Rauscher, F.J., lll, Emanuel, B.S., Rovera, G., and Barr, F.G. (1993) Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nature Genet 5(3), 230-235. Shapiro, D.N., Sublett, J.E., Li, 3., Downing, JR, and Naeve, CW. (1993) Fusion of PAX3 to a member of the forkhead family of transcription factors in human alveolar rhabdomyosarcoma. Cancer Res 53(21), 5108-5112. Fredericks, W.J., Galili, N., Mukhopadhyay, S., Rovera, G., Bennicelli, J., Barr, F.G., and Rauscher, F.J., Ill (1995) The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3. Mol Cell Biol 15(3), 1522-1535. Goulding, M.D., Chalepakis, G., Deutsch, U., Erselius, JR, and Gruss, P. (1991) Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. Embo J 10(5), 1135-1147. Goulding, M. (1992) Paired box genes in vertebrate neurogenesis. Semin Neurosci 4(5), 327-335. 139. 140. 141. 142. 143. 144. 145. 146. 147. 150 Goulding, M.D., Lumsden, A.. and Gruss, P. (1993) Signals from the notochord and floor plate regulate the region-specific expression of two Pax genes in the developing spinal cord. Development 117(3), 1001- 1 016. Goulding, M. and Paquette, A. (1994) Pax genes and neural tube defects in the mouse. Ciba Found Symp 181, 103-113. Mansouri, A., Stoykova, A., and Gruss, P. (1994) Pax genes in development. J Cell Sci Suppl 18, 35—42. Pruitt, SC. (1992) Expression of Pax-3- and neuroectoderm-inducing activities during differentiation of P19 embryonal carcinoma cells. Development 116(3), 573-583. Gruss, P.a.W., C (1992) Pax in development. Cell 69, 719-722. Goulding, M.. Lumsden, A., and Paquette, A.J. (1994) Regulation of Pax-3 expression in the derrnomyotome and its role in muscle development. Development 120(4), 957-971. Bober, E., Franz, T., Arnold, H.H., Gruss, P., and Tremblay, P. (1994) Pax-3 is required for the development of limb muscles: A possible role for the migration of dermomyotomal muscle progenitor cells. Development 120(3), 603-612. Buckingham, ME. (1994) Muscle: The regulation of myogenesis. Cun' Opin Genet Dev 4(5), 745-751. Epstein, J.A., Lam, P., Jepeal, L., Maas, R.L., and Shapiro, D.N. (1995) Pax3 inhibits myogenic differentiation of cultured myoblast cells. J BioIChem 270(20), 11719-11722. 148. 149. 150. 151. 152. 153. 154. 151 Williams, BA. and Ordahl, CF. (1994) Pax-3 expression in segmental mesoderm marks early stages in myogenic cell specification. Development 120(4), 785-796. Baldwin, C.T., Hoth, C.F., Macina, RA, and Milunsky, A. (1995) Mutations in PAX3 that cause Waardenburg syndrome type I: ten new mutations and review of the literature. Am J Med Genet 58(2), 115- 122. Baldwin, C.T., Lipsky, N.R., Hoth, C.F., Cohen, T., Mamuya, W., and Milunsky, A. (1994) Mutations in PAX3 associated with Waardenburg syndrome type I. Hum Mutat 3(3), 205-211. Lalwani, A.K., Brister, J.R., Fex, J., Grundfast, K.M., Ploplis, B., San Agustin, T.B., and VVIICOX, ER. (1995) Further elucidation of the genomic structure of PAX3, and identification of two different point mutations within the PAX3 homeobox that cause Waardenburg syndrome type 1 in two families. Am J Hum Genet 56(1), 75-83. Morell, R., Friedman, T.B., Moeljopawiro, S., Hartono, Soewito, and Asher, J.H., Jr. (1992) A frameshift mutation in the HuP2 paired domain of the probable human homolog of murine Pax-3 is responsible for Waardenburg syndrome type 1 in an Indonesian family. Hum Mol Genet 1(4), 243-247. Morell, R., Friedman, T.B., and Asher, J.H., Jr. (1993) A plus-one frameshift mutation in PAX3 alters the entire deduced amino acid sequence of the paired box in a Waardenburg syndrome type 1 (W81) family. Hum Mol Genet 2(9), 1487-1488. Read, A.P., Foy, C., Newton, V., and Harris, R. (1991) Localization of a gene for Waardenburg syndrome type I. Ann N YAcad Sci 630, 143- 151 . 1 55. 1 56. 1 57. 1 58. 159. 160. 161. 162. 152 Tassabehji, M., Newton, V.E., Liu, X.Z., Brady, A., Donnai, D., Krajewska Walasek, M.. Murday, V., Norman, A., Obersztyn, E., Reardon, W., et al. (1995) The mutational spectrum in Waardenburg syndrome. Hum MoIGenet 4(1 1), 2131-2137. Kissinger, C.R., Liu, 88., Martin Blanco, E., Kornberg, T.B., and Pabo, CO. (1990) Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution: a framework for understanding homeodomain-DNA interactions. Cell 63(3), 579-590. Xu, W., Rould, M.A., Jun, 8., Desplan, C., and Pabo, CO. (1995) Crystal structure of a paired domain-DNA complex at 2.5 angstrom. Cell 80(4), 639-650. Sommer, A., Young-Wee, T. and Frye, T. (1983) Previously undescribed syndrome of craniofacial, hand anomalies and sensorineural deafness. Am J Med Genet 15, 71-77. Asher, J.H., Jr., Sommer, A., Morell, R. and Friedman, T.B. (1996) Missense mutation in the paired domain of PAX3 causes Craniofacial- deafness-hand syndrome. Human Mutation 7, 30-35. Arias, S. (1993) Mutations of PAX3 unlikely in Waardenburg syndrome type 2 [letter]. Nat Genet 5(1), 8. Hughes, A.E., Newton, V.E., Liu, X.Z., and Read, AP. (1994) A gene for Waardenburg syndrome type 2 maps close to the human homologue of the microphthalmia gene at chromosome 3p12-p14.1. Nat Genet 7(4), 509-512. Tachibana, M.. Perez Jurado, L.A., Nakayama, A.. Hodgkinson, C.A., Li, X., Schneider, M.. Miki, T., Fex, J., Francke, U., and Arnheiter, H. (1994) Cloning of MITF, the human homolog of the mouse microphthalmia gene and assignment to chromosome 3p14.1-p12.3. Hum Mol Genet 3(4), 553-557. 163. 164. 165. 166. 167. 168. 169. 170. 153 Tassabehji, M.. Newton, V.E., and Read, AP. (1994) Waardenburg syndrome type 2 caused by mutations in the human microphthalmia (MITF) gene. Nat Genet 8(3), 251-255. Burley, S.K., Clark, K.L., Ferre, D.A.A., Kim, J.L., and Nikolov, DB. (1993) X-ray crystallographic studies of eukaryotic transcription factors. Cold Spring Harb Symp Quant Biol 58, 123-132. Ferre, D.A.A.R., Prendergast, G.C., Ziff, EB, and Burley, SK. (1993) Recognition by Max of its cognate DNA through a dimeric b/HLHIZ domain [see comments]. Nature 363(6424), 38-45. Hemesath, T.J., Steingrimsson, E., McGill, G., Hansen, M.J., Vaught, J., Hodgkinson, C.A., Arnheiter, H., Copeland, N.C., Jenkins, NA, and Fisher, DE. (1994) Microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family. Genes Dev 8(22), 2770-2780. Steingrimsson, E., Moore, K.J., Lamoreux, M.L., Ferre D' Amare, A.R., Burley, S.K., Sanders Zimring, D.C., Skow, L.C., Hodgkinson, C.A., Jenkins, MA, et al. (1994) Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences. Nat Genet 8(3), 256-263. Rao, V.V., Loffler, C., and Hansmann, l. (1991) The gene for the novel vasoactive peptide endothelin 3 (EDN3) is localized to human chromosome 20q13.2-qter. Genomics 10(3), 840-841. Attie, T., Till, M., Pelet, A., Amiel, J., Edery, P., Boutrand, L., Munnich, A. and Lyonnet, S. (1995) Mutation of the endothelin-receptor B gene in Waardenburg-Hirschsprung disease. Human Molecular Genetics 4(1 2), 2407-2409. Puffenberger, E.G., Hosoda, K., Washington, S.S., Nakao, K., deVlflt, D., Yanagisawa, M., and Chakravart, A. (1994) A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung's disease. Cell 79(7), 1257-1266. 171. 172. 173. 174. 175. 176. 177. 178. 154 Edery, P., Lyonnet, S., Mulligan, L.M., Pelet, A.. Dow, E., Abel, L., Holder, 8., Nihoul Fekete, C., Ponder, BA, and Munnich, A. (1994) Mutations of the RET proto-oncogene in Hirschsprung's disease [see comments]. Nature 367(6461), 378-380. Romeo, G., Ronchetto, P., Luo, Y., Barone, V., Seri, M.. Ceccherini, |., Pasini, B., Bocciardi, R., Lerone, M., Kaariainen, H., et al. (1994) Point mutations affecting the tyrosine kinase domain of the RET proto- oncogene in Hirschsprung's disease [see comments]. Nature 367(6461), 377-378. Ceccherini, l., Zhang, A.L., Matera, l., Yang, G., Devoto, M.. Romeo, G., and Cass, D.T. (1995) Interstitial deletion of the endothelin-B receptor gene in the spotting lethal (sl) rat. Hum Mol Genet 4(1 1), 2089-2096. Ceccherini, l., Bocciardi, R., Luo, Y., Pasini, B., Hofstra, R., Takahashi, M.. and Romeo, G. (1993) Exon structure and flanking intronic sequences of the human RET proto-oncogene. Biochem Biophys Res Commun 196(3), 1288-1295. Attie, T., Edery, P., Lyonnet, S., Nihoul Fekete, C., and Munnich, A. (1994) [Identification of mutation of RET proto-oncogene in Hirschsprung disease]. C R Seances Soc Biol Fil 188(5-6), 499-504. Attie, T., Pelet, A.. Sarda, P., Eng, 0., Edery, P., Mulligan, L.M., Ponder, B.A., Munnich, A.. and Lyonnet, S. (1994) A 7 bp deletion of the RET proto-oncogene in familial Hirschsprung's disease. Hum Mol Genet 3(8), 1439-1440. Attie, T., Till, M.. Pelet, A.. Edery, P., Bonnet, J.P., Munnich, A., and Lyonnet, S. (1995) Exclusion of RET and Pax 3 loci in Waardenburg- Hirschsprung disease. J Med Genet 32(4), 312-313. Attie, T., Pelet, A., Edery, P., Eng, C., Mulligan, L.M., Amiel, J., Boutrand, L., Beldjord, C., Nihoul Fekete, C., Munnich, A.. et al. (1995) Diversity of RET proto-oncogene mutations in familial and sporadic Hirschsprung disease. Hum Mol Genet 4(8), 1381-3186. 179. 180. 181. 182. 183. 184. 185. 186. 155 Lyonnet, S., Edery, P., Mulligan, L.M., Pelet, A., Dow, E., Abel, L., Holder, 8., Nihoul Fekete, C., Ponder, BA, and Munnich, A. (1994) [Mutations of RET proto-oncogene in Hirschsprung disease]. C R Acad Sci III 317(4), 358-362. Baynash, A.G., Hosoda, K., Giaid, A.. Richardson, J.A., Emoto, N., Hammer, RE, and Yanagisawa, M. (1994) Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 79(7), 1277-1285. Hosoda, K., Hammer, R.E., Richardson, J.A., Baynash, A.G., Cheung, J.C., Giaid, A., and Yanagisawa, M. (1994) Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice. Cell 79(7), 1267-1276. Gariepy, C.E., Cass, D.T., and Yanagisawa, M. (1996) Null mutation of endothelin receptor type B gene in spotting lethal rats causes aganglionic megacolon and white coat color. Proc Natl Acad Sci USA 93(2), 867-872. VVlICOX, E.R., Rivolta, M.N., Ploplis, B., Potterf, SB, and Fex, J. (1992) The PAX3 gene is mapped to human chromosome 2 together with a highly informative CA dinucleotide repeat. Hum Mol Genet 1(3), 215. Carezani Gavin, M.. Clarren, SK, and Steege, T. (1992) Waardenburg syndrome associated with meningomyelocele [letter] [see comments]. Am J Med Genet 42(1), 135-136. Calmettes, L., Deodati, F., Bec, P., and Labro, J.B. (1968) [Waardenburg-Klein syndrome with blind lacrimal fistulas]. Bull Mem Soc Fr Ophtalmol 81, 144-155. Char, F. (1971) Cleft lip/palate in the Waardenburg syndrome. Birth Defects 7(7), 258. 187. 188. 189. 190 191. 192. 193. 194. 195. 156 Hol, F.A., Hamel, B.C., Geurds, M.P., Mullaart, R.A., Barr, F.G., Macina, RA, and Mariman, EC. (1995) A frameshift mutation in the gene for PAX3 in a girl with spina bifida and mild signs of Waardenburg syndrome. J Med Genet 32(1), 52-56. Kaplan, P. and de Chaderevian, JP. (1988) Piebaldism-Waardenburg syndrome: histopathologic evidence for a neural crest syndrome [see comments]. Am J Med Genet 31(3), 679-688. Laor, N. and Korczyn, AD. (1978) Waardenburg syndrome with a fixed dilated pupil. Br J Ophthalmol 62(7), 491 -494. . Pierpont, J.W., Doolan, L.D., Amann, K., Snead, GR, and Erickson, RP. (1994) A single base pair substitution within the paired box of PAX3 in an individual with Waardenburg syndrome type 1 (W81). Hum Mutat 4(3), 227-228. Silverman, G.A., Schneider, S.S., Massa, H.F., Flint, A.. Lalande, M., Leonard, J.C., Overhauser, J., van den Engh, G., and Trask, B.J. (1995) The 18q- syndrome: analysis of chromosomes by bivariate flow karyotyping and the PCR reveals a successive set of deletion breakpoints within 18q21.2-q22.2. Am J Hum Genet 56(4), 926-937. Strathdee, G., Zackai, E.H., Shapiro, R., Kamholz, J., and Overhauser, J. (1995) Analysis of clinical variation seen in patients with 18q terminal deletions. Am J Med Genet 59(4), 476-483. Begleiter, ML. and Harris, DJ. (1992) Waardenburg syndrome and meningocele. Am J Med Genet 44(4), 541. Chatkupt, S., Chatkupt, S., and Johnson, W.G. (1993) Waardenburg syndrome and myelomeningocele in a family. J Med Genet 30(1), 83- 84. Moline, ML. and Sandlin, C. (1993) Waardenburg syndrome and meningomyelocele [letter]. Am J Med Genet 47(1), 126. 196. 197. 198. 199. 200. 201. 202. 203. 157 Chatkupt, S., Hol, F.A., Shugart, Y.Y., Geurds, M.P., Stenroos, E.S., Koenigsberger, M.R., Hamel, B.C., Johnson, W.G., and Mariman, EC. (1995) Absence of linkage between familial neural tube defects and PAX3 gene. J Med Genet 32(3), 200-204. Pierpont, J.W., Storm, A.L., Erickson, R.P., Kohn, B.R., Pettijohn, L., and DePaepe, A. (1995) Lack of linkage of apparently dominant cleft lip (palate) to two candidate chromosomal regions. J Craniofac Genet DevBiol 15(2), 66-71. Kawabata, E., Ohba, N.. Nakamura, A., lzumo, S., and Osame, M. (1987) Waardenburg syndrome: a variant with neurological involvement. Ophthalmic Paediatr Genet 8(3), 165-170. Kioussi, 0., Gross, MK, and Gruss, P. (1995) Pax3: a paired domain gene as a regulator in PNS myelination. Neuron 15(3), 553-562. Bankier, A. and Sheffield, L. (1990) Piebaldism, an autosomal dominant trait distinct from Waardenburg syndrome [letter; comment]. Am J MedGenet 37(4), 600-602. Hussels, LE. (1971) Vitiligo versus Waardenburg syndrome. Birth Defects 7(8), 285. Francois, J. (1979) Albinism. Ophthalmologica 178(1-2), 19-31. Pandya, A., Xia-Juan, X., Landa, B.L., Amos, K.S., Israel, J., Lloyd, J., James, A.L., Diehl, S.R., Blanton, SH, and Nance, W.E. (1996) Phenotypic variation in Waardenburg syndrome: mutational heterogeneity, modifier genes or polygenic background? Hum Mol Genet 5(4), 497-502. 204. 205. 206. 207. 208. 209. 210. 158 Reynolds, J.E., Marazita, M.L., Meyer, J.M., Stevens, C.A., Eaves, L.J., Amos, K.S., Ploughman, L.M., MacLean, C., Nance, W.E., and Diehl, SR. (1996) Major-locus contributions to variability of the craniofacial feature dystopia canthorum in Waardenburg syndrome. Am J Hum Genet 58(2), 384-392. Reynolds, J.E., Meyer, J.M., Landa, 8, Stevens, C.A., Arnos, K.S., Israel, J., Marazita, M.L., Bodurtha, J., Nance, W.E., and Diehl, SR. (1995) Analysis of variability of clinical manifestations in Waardenburg syndrome. Am J Med Genet 57(4), 540-547. Tassabehji, M.. Read, A.P., Newton, V.E., Patton, M., Gruss, P., Harris, R., and Strachan, T. (1993) Mutations in the PAX3 gene causing Waardenburg syndrome type 1 and type 2. Nat Genet 3(1), 26-30. Kirkpatrick, S.J. (1992) Waardenburg syndrome type I in a child with deletion (2) (q35q36. 2). Am J Med Genet 44(5), 699-700. de Kok, Y.J., Merkx, G.F., van der Maarel, S.M., Huber, l., Malcolm, 8., Ropers, H.H., and Cremers, PP. (1995) A duplication/paracentric inversion associated with familial X-linked deafness (DFN3) suggests the presence of a regulatory element more than 400 kb upstream of the POU3F4 gene. Hum Mol Genet 4(1 1), 2145-2150. Duttlinger, R., Manova, K., Chu, T.Y., Gyssler, C., Zelenetz, A.D., Bachvarova, RF, and Besmer, P. (1993) W-sash affects positive and negative elements controlling c-kit expression: ectopic c-kit expression at sites of kit-ligand expression affects melanogenesis. Development 118(3), 705-717. Bedell, M.A., Brennan, C.|., Evans, E.P., Copeland, N.G., Jenkins, NA, and Donovan, P.J. (1995) DNA rearrangements located over 100 kb 5' of the Steel (SD-coding region in Steel-panda and Steel- contrasted mice deregulate SI expression and cause female sterility by disrupting ovarian follicle development. Genes Dev 9(4), 455-470. 211. 212. 213. 214. 215. 216. 217. 218. 159 Fantes, J., Redeker, B., Breen, M.. Boyle, S., Brown, J., Fletcher, J., Jones, 8., Bickmore, W., Fukushima, Y., Mannens, M.. et al. (1995) Aniridia-associated cytogenetic rearrangements suggest that a position effect may cause the mutant phenotype. Hum Mol Genet 4(3), 415- 422. Bedell, M.A., Jenkins, NA, and Copeland, MG. (1996) Good genes in bad neighbourhoods [news; comment]. Nat Genet 12(3), 229-232. Epstein, J.A., Glaser, T., Cai, J., Jepeal, L., Walton, 0.8., and Maas, R.L. (1994) Two independent and interactive DNA-binding subdomains of the Pax6 paired domain are regulated by alternative splicing. Genes Dev 8(17), 2022-2034. Ward, T.A., Nebel, A., Reeve, A.E., and Eccles, MR. (1994) Alternative messenger RNA forms and open reading frames within an additional conserved region of the human PAX-2 gene. Cell Growth Differ 5(9), 1015-1021. Hodgkinson, C.A., Moore, K.J., Nakayama, A., Steingrisson, E., Copeland, N.G., Jenkins, N.A. adn Arnheiter, H. (1993) Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein. Cell 74, 395 404. Grompe, M. (1993) The rapid detection of unknown mutations in nucleic acids. Nature Genet 5(2), 111-117. de Morsier, G. (1956) Etudes sur les dysraphies cranioencephaliques. lll. Agenesie du septum lucidum avec malformation du tractus optique: la dysplasie septo-optique. Schweizer Archiv fur Neurologie und Psychiatrie 77, 267. de Morsier, G. (1962) Median Cranioencephalic Dysraphias and Olfactogenital Dysplasia. World Neurology 3, 485-500. 219. 220. 221. 222. 223. 224. 225. 226. 227. 160 Acers, TE. (1981) Optic nerve hypoplasia: septo-optic-pituitary dysplasia syndrome. Trans Am Ophthalmol Soc 79, 425-457. Aicardi, J. and Goutieres, F. (1981) The syndrome of absence of the septum pellucidum with porencephalies and other developmental defects. Neuropediatn'cs 1 2(4), 319-329. ' Brodsky, MC. and Glasier, CM. (1993) Optic nerve hypoplasia. Clinical significance of associated central nervous system abnormalities on magnetic resonance imaging [published erratum appears in Arch Ophthalmol 1993 Apr;111(4):491]. Arch Ophthalmol 111(1), 66-74. Roessmann, U. ( 1989) Septo—optic dysplasia (SOD) or DeMorsier syndrome. J-Clin-Neuroophthalmol 9(3), 1 56-159. Stehr, K., Mayer, U., Pfeiffer, RA, and Reif, R. (1985) Extreme variant of septo-optic dysplasia. Ophthalmic Paediatr Genet 5(3), 159-164. Gendrel, D., Chaussain, J.L., and Job, J.C. (1981) [Congenital hypopituitarism associated with mid-line defects (author's transl)]. Arch Fr Pediatr 38(4), 227-232. Barkovich, A.J., Fram, E.K., and Norman, D. (1989) Septo-optic dysplasia: MR imaging. Radiology 171(1), 189-192. Brook, C.G., Sanders, MD, and Hoare, RD. (1972) Septo-optic dysplasia. Br-Med-J 3(830), 81 1-813. Harris, R.J. and Haas, L. (1972) Septo-optic dysplasia with growth hormone deficiency (De Morsier syndrome). Arch Dis Child 47(256), 973-976. ' 219. 220. 221. 222. 223. 224. 225. 226. 227. 160 Acers, TE. (1981) Optic nerve hypoplasia: septo—optic—pituitary dysplasia syndrome. Trans Am Ophthalmol Soc 79, 425-457. Aicardi, J. and Goutieres, F. (1981) The syndrome of absence of the septum pellucidum with porencephalies and other developmental defects. Neuropediatn'cs 1 2(4), 319-329. ‘ Brodsky, MC. and Glasier, CM. (1993) Optic nerve hypoplasia. Clinical significance of associated central nervous system abnormalities on magnetic resonance imaging [published erratum appears in Arch Ophthalmol 1993 Apr;111(4):491]. Arch Ophthalmol 111(1), 66-74. Roessmann, U. (1989) Septo—optic dysplasia (SOD) or DeMorsier syndrome. J-Clin-Neuroophthalmol 9(3), 156-1 59. Stehr, K., Mayer, U., Pfeiffer, RA, and Reif, R. (1985) Extreme variant of septa-optic dysplasia. Ophthalmic Paediatr Genet 5(3), 159-164. Gendrel, D., Chaussain, J.L., and Job, J.C. (1981) [Congenital hypopituitarism associated with mid-line defects (author's transl)]. Arch Fr Pediatr 38(4), 227-232. Barkovich, A.J., F ram, E.K., and Norman, D. (1989) Septo-optic dysplasia: MR imaging. Radiology 171(1), 189-192. Brook, C.G., Sanders, MD, and Hoare, RD. (1972) Septo-optic dysplasia. Br-Med-J 3(830), 81 1-813. Harris, R.J. and Haas, L. (1972) Septo—optic dysplasia with growth hormone deficiency (De Morsier syndrome). Arch Dis Child 47(256), 973-976. ‘ 228. 229. 230. 231. 232. 233. 234. 235. 236. 161 Hoyt, W.F., Kaplan, S.L., Grumbach, MM, and Glaser, J.S. (1970) Septo-optic dysplasia and pituitary dwarfism. Lancet 1(652), 893-894. lzenberg, N., Rosenblum, M., and Parks, J.S. (1984) The endocrine spectrum of septo—optic dysplasia. Clin Pediatr Phila 23(11), 632-636. Freude, S., Frisch, H., Wrmberger, D., Schober, E., Husler, G., Waldhauser, F., and Aichner, F. (1992) Septo-optic dysplasia and growth hormone deficiency: accelerated pubertal maturation during GH therapy. Acta Paediatr 81 (8), 641 -645. Chen, H.J., Tsai, J.H., Lai, Y.H., Chen, 8.8., and Wang, H1. (1985) [Septo-optic dysplasia with pituitary dwarfismua case report]. Taiwan I Hsueh Hui Tsa Chih 84(9), 1093-1098. Benoit Gonin, J.J., David, M., Felt, J.P., Bourgeois, J., Chopard, A., Kopp, N., and Jeune, M. (1978) [Septo-optic dysplasia with antidiuretic hormone deficiency and central adrenocortical insufficiency. Three cases report in infants (author's transl)]. Nouv Presse Med 7(37), 3327- 3331. Arslanian, S.A., Rothfus, W.E., Foley, T.P., Jr., and Becker, DJ. (1984) Hormonal, metabolic, and neuroradiologic abnormalities associated with septa-optic dysplasia. Acta Endocrinol Copenh 107(2), 282-288. Clark, EA. and Meyer, W.J.d. (1978) Blindness and hypoglycemia: growth hormone deficiency with septo-optic dysplasia. Tex Med 74(2), 47-50. Davis, CV and Shock, JP. (1975) Septo-optic dysplasia associated with see-saw nystagmus. Arch Ophthalmol 93(2), 137-139. Coulter, C.L., Leech, R.W., Schaefer, G.B., Scheithauer, B.W., and Brumback, RA. (1993) Midline cerebral dysgenesis, dysfunction of the hypothalamic-pituitary axis, and fetal alcohol effects. Arch Neurol 50(7), 771-775. 237. 238. 239. 240. 241. 242. 243. 244. 245. 246. 162 Elster, AB. and McAnamey, ER. (1979) Maternal age re septo-optic dysplasia [letter]. J Pediatr 94(1), 162-163. Dominguez, R., Vila Coro, A.A., Slopis, J.M., and Bohan, T.P. (1991) Brain and ocular abnormalities in infants with in utero exposure to cocaine and other street drugs. Am J Dis Child 145, 688-695. Donat, J.F. (1981) Septo—optic dysplasia in an infant of a diabetic mother. Arch Neurol 38(9), 590-591. Landrieu, P. and Evrard, P. (1979) [Septo-optic dysplasia: clinical study and elements of genetic counseling]. J Genet Hum 27(4), 329-341. Benner, J.D., Preslan, M.W., Gratz, E., Joslyn, J., Schwartz, M., and Kelman, S. (1990) Septo-optic dysplasia in two siblings. Am J Ophthalmol 109(6), 632-637. Ellenberger, C., Jr. (1972) Septo-optic dysplasia. Br Med J 4(839), 552. Huseman, C.A., Kelch, R.P., Hopwood, NJ, and Zipf, W.B. (1978) Sexual precocity in association with septo-optic dysplasia and hypothalamic hypopituitarism. J Pediatr 92(5), 748-753. Fitz, CR. (1983) Holoprosencephaly and related entities. Neuroradiology 25(4), 225-238. Fitz, CR. (1994) Holoprosencephaly and septa-optic dysplasia. Neuroimaging Clin N Am 4(2), 263-281. Leech, R.W. and Shuman, RM. (1986) Holoprosencephaly and related midline cerebral anomalies: a review. J Child Neurol1(1), 3-18. 247. 248. 249. 250. 251. 252. 253. 254. 163 Patel, H., Tze, W.J., Crichton, J.U., McCormick, A.Q., Robinson, G.C., and Dolman, CL (1975) Optic nerve hypoplasia with hypopituitarism. Septa-aptic dysplasia with hypopituitarism. Am J Dis Child 129(2), 175- 1 80. St. John, J.R.a.R., BL. (1957) Congenital Absence of the Septum Pellucidum. Am J Surgery 94, 974-980. Vlfilliams, J., Brodsky, M.C., Griebel, M., Glasier, C.M., Caldwell, D., and Thomas, P. (1993) Septa-aptic dysplasia: the clinical insignificance of an absent septum pellucidum. Dev Med Child Neural 35(6), 490- 501. Pagan, RA. and Stephan, M.J. (1984) Septo-aptic dysplasia with digital anomalies. J Pediatr 105(6), 966-968. LaRoche, GR. (1984) Septa-aptic dysplasia and median cleft face syndrome [letter]. Am J Dis Child 138(8), 795-796. Stewart, G., Castro Magana, M.. Sherman, J., Angulo, M.. and Collipp, P.J. (1983) Septa-aptic dysplasia and median cleft face syndrome in a patient with isolated growth hormone deficiency and hyperprolactinemia. Am J Dis Child 137(5), 484-487. Teng, R.J., Wang, P.J., Wang, TR, and Shen, Y.Z. (1989) Apert syndrome associated with septa-optic dysplasia. Pediatr Neural 5(6), 384-388. Michaud, J., Mizrahi, EM, and Urich, H. (1982) Agenesis of the vemlis with fusion of the cerebellar hemispheres, septa-optic dysplasia and associated anomalies. Report of a case. Acta Neurapathal Bert 56(3), 161-166. 255. 256. 257. 258. 259. 260. 261. 262. 263. 164 Kaufman, L.M., Miller, MT, and Mafee, MP (1989) Magnetic resonance imaging of pituitary stalk hypoplasia. A discrete midline anomaly associated with endocrine abnormalities in septa-optic dysplasia. Arch Ophthalmal107(10), 1485-1489. Byrd, SE. (1989) Magnetic resonance imaging of supratentarial congenital brain malformations. J Natl Med Assoc 81(8), 873—881. Shapiro, MB. and Senapathy, P. (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nuc Acids Res 15(17), 7155-7174. Ragan, PK. and Schneider, TD. (1995) Using information content and base frequencies to distinguish mutations from genetic polymorphisms in splice junction recognition sites. Hum Mutat 6(1), 74-76. Declerck, A., Casteels, l., Demaerel, P., and Dralands, L. (1994) Septa-aptic dysplasia. Bull Soc Beige Ophtalmol 254, 157-161. Kumura, D., Miller, J.H., and Sinatra, FR. (1987) Septa-aptic dysplasia: recognition of causes of false-positive hepatabiliary scintigraphy in neonatal jaundice. J Nucl Med 28(6), 966-972. Sherlock, DA. and McNical, LR. (1987) Anaesthesia and septa-optic dysplasia. Implications of missed diagnosis in the peri-operative period. Anaesthesia 42(12), 1302-1305. Preston, R.A., Past, J.C., Keats, B.J.B., Aston, C.E., Ferrell, R.E., Priest, J., Nauri, N., Lasken, H.W., Morris, C.A., Hurtt, M.R., et al. (1994) A gene for Crouzon craniofacial dysostosis maps to the long arm of chromosome 10. Nat Genet 7(2), 149-153. Bellus, G.A., Hefferan, T.W., Ortiz de Luna, R.l., Hecht, J.T., Hartan, W.A., Machada, M., Kaitila, l., McIntosh, l., and Francamana, CA. (1995) Achandraplasia is defined by recurrent GSBOR mutations of FGFR3. Am J Hum Genet 56(2), 368-373. 165 264. Stailav, l., Kilpatrick, MW, and Tsipauras, P. (1995) A common FGF R3 gene mutation is present in achandraplasia but not in hypachandraplasia. Am J Med Genet 55(1), 127-133. 265. Rousseau, F.. Saugier, P., Le Merrer, M., Munnich, A., Delezoide, A.L., Marateaux, P., Bonaventure, J., Narcy, F., and Sanak, M. (1995) Stop cadan FGFR3 mutations in thanatophoric dwarfism type 1 [letter]. Nat Genet 10(1), 11-12. 266. Tavonnina, P.L., Shiang, R., Thompson, L.M., Zhu, Y.Z., Vlfilkin, D.J., Lachman, R.S., ercax, W.R., Rimain, D.L., Cahn, DH, and Wasmuth, J.J. (1995) Thanatapharic dysplasia (types I and II) caused by distinct mutations in fibroblast growth factor receptor 3. Nat Genet 9(3), 321- 328. 267. Bellus, G.A., McIntosh, l., Smith, E.A., Aylswarth, A.S., Kaitila, |., Horton, W.A., Greenhaw, G.A., Hecht, J.T., and Francamano, CA. (1995) A recurrent mutation in the tyrosine kinase domain of fibroblast growth factor receptor 3 causes hypachandraplasia. Nat Genet 10(3), 357-359. 268. Park, W.J., Meyers, G.A., Li, X., Theda, 0., Day, D., Orlaw, S.J., Jones, MC, and Jabs, E.W. (1995) Novel FGFR2 mutations in Crouzon and Jackson-Weiss syndromes show allelic heterogeneity and phenotypic variability. Hum Mal Genet 4(7), 1229-1233. 269. Ades, L.C., Mulley, J.C., Senga, I.P., Morris, L.L., David, D.J., and Haan, EA. (1994) Jackson-Weiss syndrome: clinical and radiological findings in a large kindred and exclusion of the gene from 7p21 and 5qter. Am J Med Genet 51(2), 121-130. 270. Schaumann, B. (1979) Comparative den'nataglyphic analysis in two types of acracephalasyndactyly: Saethre-Chatzen syndrome and Pfeiffer syndrome. Birth Defects 15(6), 661-667. 271. 272. 273. 274. 275. 276. 277. 278. 166 Tsukahara, M.. Hagiwara, K., and Kajii, T. (1985) Pfeiffer syndrome or Saethre-Chatzen syndrome? Jinrui Idengaku Zasshi 30(2), 51-56. Pfeiffer, R.A., Ratt, H.D., and Angerstein, W. (1990) An autosomal dominant facia-audia symphalangism syndrome with Klippel-Feil anomaly: a new variant of multiple synastases. Genet Cauns 1(2), 133- 140. Pfeiffer, R.A., Junemann, G., Palster, J., and Bauer, H. (1973) Epiphyseal dysplasia of the femoral head, severe myopia and perceptive hearing loss in three brothers. Clin Genet 4(2), 141-144. . van Lupke, A. (1989) [Evaluation of impulse noise]. Laryngarhinaatalagie 68(1 0), 561 -562. Pfeiffer, RA. (1987) New syndrome: mixed hearing loss, mental deficiency, growth retardation, short clubbed digits, and EEG abnormalities in manazygaus female twins. Am J Med Genet 27(3), 639-644. Schell, U., Hehr, A., Feldman, G.J., Rabin, N.H., Zackai, E.H., de Die Smulders, C., Viskachil, D.H., Stewart, J.M., Wolff, G., Ohashi, H., et al. (1995) Mutations in FGFR1 and FGFR2 cause familial and sporadic Pfeiffer syndrome. Hum Mal Genet 4(3), 323-328. Rutland, P., Pulleyn, L.J., Reardon, W., Baraitser, M., Hayward, R., Jones, B., Malcolm, 8., Winter, R.M., Oldridge, M., Slaney, SF, and et al. (1995) Identical mutations in the FGFR2 gene cause both Pfeiffer and Crouzon syndrome phenotypes [see comments]. Nat Genet 9(2), 173-176. Rahatgi, M. (1991) Cloverleaf skull—a severe form of Crauzan's syndrome: a new concept in aetiology. Acta Neurachir Wren 108(1-2), 45-52. 279. 280. 281. 282. 283. 284. 285. 286. 167 Siratnak, J., Brodsky, L., and Pizzuta, M. (1995) Airway obstruction in the Crouzon syndrome: case report and review of the literature. Int J Pediatr Otarhinalaryngal 31 (2-3), 235-246. Fehlaw, P. (1988) [Pseudo-Crouzon syndrome with defective mental development]. Pediatr Grenzgeb 27(4), 327-330. Oldridge, M.. erkie, A.O., Slaney, S.F., Paale, M.D., Pulleyn, L.J., Rutland, P., Hackley, A.D., Wake, M.J., Galdin, J.H., Winter, R.M., et al. (1995) Mutations in the third immunaglabulin domain of the fibroblast growth factor receptor-2 gene in Crouzon syndrome. Hum Mal Genet 4(6), 1 077-1082. Reardon, W., Wrnter, R.M., Rutland, P., Pulleyn, L.J., Jones, B.M., and Malcolm, S. (1994) Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome. Nat Genet 8(1), 98-103. Neilsan, KM. and Friesel, RE. (1995) Constitutive activation of fibroblast growth factor receptor-2 by a point mutation associated with Crouzon syndrome. J Biol Chem 270(44), 26037-26040. Del Gatta, F. and Breathnach, R. (1995) A Crouzon syndrome synonymous mutation activates a 5' splice site within the lllc exon of the FGF R2 gene. Genomics 27(3), 558-559. Garry, M.C., Preston, R.A., White, G.J., Zhang, Y., Singhal, V.K., Lasken, H.W., Parker, M.G., Nwakara, N.A., Past, J.C., and Ehrlich, GD. (1995) Crouzon syndrome: mutations in two spliceafarms of FGFR2 and a common point mutation shared with Jackson-Weiss syndrome. Hum MaIGenet 4(8), 1387-1390. Steinberger, D., Mulliken, J.B., and Muller, U. (1995) Predispasitian far cysteine substitutions in the immunaglabulin-like chain of FGFR2 in Crouzon syndrome. Hum Genet 96(1), 113-115. 287. 288. 289. 290. 291. 292. 293. 294. 168 Meyers, G.A., Orlaw, S.J., Munro, l.R., Przylepa, K.A., and Jabs, E.W. (1995) Fibroblast growth factor receptor 3 (FGF R3) transmembrane mutation in Crouzon syndrome with acanthasis nigricans. Nat Genet 1 1 (4), 462-464. Richtsmeier, J.T. (1987) Comparative study of normal, Crouzon, and Apert craniofacial morphology using finite element scaling analysis. Am J Phys Anthrcpal 74(4), 473-493. Carter, C.O., Till, K., Fraser, V., and Coffey, R. (1982) A family study of craniosynostosis, with probable recognition of a distinct syndrome. J Med Genet 19(4), 28028-5. de Leon, G.A., de Leon, G., Graver, W.D., Zaeri, N., and Alburger, PD. (1987) Agenesis of the corpus callosum and Iimbic malformation in Apert syndrome (type I acracephalasyndactyly). Arch Neural 44(9), 979-982. de Leon, G.A., de Leon, G., Graver, W.D., Zaeri, N., and Alburger, P. (1989) Agenesis of the corpus callosum in Apert syndrome? [letter]. ArchNeuraI 46(5), 479. Beligere, M, Harris, V., and Pruzansky, S. (1981) Progressive bany dysplasia in Apert syndrome. Radiology 139(3), 593-597. F leraw, W. and Szamak, F. (1979) [The Apert syndrome (acrocephalosyndactylia). A case study]. Fartschr Med 97(10), 438- 439. lzumikawa, Y., Naritami, K., lkema, S., Goya, Y., Shirama, N., Yashida, K., Yara, A., and Hirayama, K. (1988) Apert syndrome with partial palysyndactyly: a proposal on the classification of acracephalasyndactyly. Jinrui Idengaku Zasshi 33(4), 487-492. J 295. 296. 297. 298. 299. 300. 301. 302. 303. 304. 169 Marateaux, P. and Fanfria, MC. (1987) Apparent Apert syndrome with palydactyly: rare pleiotropic manifestation or new syndrome? Am J Med Genet 28(1), 153-158. Nicholas, KC. and Jargensan, R.J. (1975) Apert syndrome. Birth Defects 11(2), 396. Pyrkasz, A., Grzywna, W., and Grzybawski, A. (1988) [Apert syndrome]. Pediatr Pal 63(9), 590-592. Ruppert, F., Schultz, K., Weisenbach, J., and Bazzay, L. (1974) [Apert's syndrome (acrocephalosyndactyliaj Orv Hetil 1 15(36), 2127- 21 30. Salomon, L.M., Medenica, M., Pruzansky, S., and Kreibarg, S. (1973) Apert syndrome and palatal mucapalysaccharides. Teratology 8(3), 287-291. Saw, D., Camara, B., Kuakuvi, N., Traare, A., Diagne, l., Sall, M.G., Moreira, C., and Senghar, G. (1988) [Apert syndrome or type I acracephalosyndactylia. Apropos of a case]. Dakar Med 33(1-4), 11 - 13. Szumera, B., Mazurek, A., and Jarczyk, K. (1972) [Wheatan-Apert syndrome]. Pol Tyg Lek 27(6), 231-232. Watersan, J.R., DiPietra, MA, and Barr, M. (1985) Apert syndrome with frantanasal encephalacele. Am J Med Genet 21(4), 777-783. Phillips, 8.6. and Miyamata, RT. (1986) Congenital conductive hearing loss in Apert syndrome. Otalaryngal Head Neck Surg 95(4), 429-433. Gould, H.J. and Caldarelli, DD. (1982) Hearing and atapathalagy in Apert syndrome. Arch Otalaryngal108(6), 347-349. 305. 306. 307. 308. 309. 310. 311. 312. 313. 170 V\filkie, A.O., Slaney, S.F., Oldridge, M., Poole, M.D., Ashwarth, G.J., Hackley, A.D., Hayward, R.D., David, D.J., Pulleyn, L.J., Rutland, P., et al. (1995) Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome [see comments]. Nat Genet 9(2), 165-172. Barballa, L. and Menendez, l. (1983) Derrnataglyphics in Saethre- Chatzen syndrome: a family study. Acta Paediatr Hung 24(3), 269-279. Bianchi, E., Arica, M., Padesta, A.F., Grana, M., Fiari, P., and Beluffi, G. (1985) A family with the Saethre-Chatzen syndrome. Am J Med Genet 22(4), 649-658. Gabrielli, O., Marani, E., Barbata, M.. Pierleani, C., and Felici, L. (1989) [Type-Ill acracephalasyndactylia (Saethre-Chatzen syndrome). Description of 2 cases]. Pathologica 81(1073), 295-300. Galluzzi, F., Salti, R., Marianelli, L., and La Cauza, C. (1980) [The Saethre-Chatzen syndrome. Clinical case]. Minerva Pediatr 32(5), 325- 328. Jimenez Garcia, M. and Gonzalez Cortes, MC. (1977) [The Saethre- Chatzen syndrome (acrocephalosyndactylia type lll)]. BaI Med Hosp Infant Mex 34(4), 903-908. Kreibarg, S., Pruzansky, S., and Pashayan, H. (1972) The Saethre- Chatzen syndrome. Teratology 6(3), 287-294. Pantke, O.A., Cohen, M.M., Jr., Wrtkap, C.J., Jr., Feingald, M.. Schaumann, B., Pantke, HO, and Garlin, R.J. (1975) The Saethre- Chatzen syndrome. Birth Defects 11(2), 190-225. Reardon, W.. McManus, S.P., Summers, D., and Winter, RM. (1993) Cytogenetic evidence that the Saethre-Chatzen gene maps to 7p21.2. Am J Med Genet 47(5), 633-636. 314. 315. 316. 317. 318. 319. 320. 321. 171 Shidayama, R., Hirana, A., lio, Y., and Fujii, T. (1995) Familial Saethre- Chotzen syndrome with or without palydactyly of the toe. Ann Plast Surg 34(4), 435-440. Friedman, J.M., Hanson, J.W., Graham, CB, and Smith, D.W. (1977) Saethre-Chatzen syndrome: a broad and variable pattern of skeletal malformations. J Pediatr 91 (6), 929-923. erkie, A.O., Yang, S.P., Summers, D., Poole, M.D., Reardon, W.. and Winter, RM. (1995) Saethre-Chatzen syndrome associated with balanced translacatians involving 7p21: three further families. J Med Genet 32(3), 174-180. Reid, C.S., McMarraw, L.E., McDonald McGinn, D.M., Grace, K.J., Ramos, F.J., Zackai, E.H., Cohen, M.M., Jr., and Jabs, E.W. (1993) Saethre-Chatzen syndrome with familial translocation at chromosome 7p22. Am J Med Genet 47(5), 637-639. Evans, CA. and Christiansen, R.L. (1976) Cephalic malformations in Saethre-Chatzen syndrome. Acrocephalosyndactyly type III. Radiology 121(2), 399-403. Morell, R., Pierpont, J., Gua, W., Friedman, T.B., Ha, L., Spritz, R.A., Erickson, RP. and Asher, J.H., Jr. (1996) Possible digenic inheritance of Waardenburg Syndrome type 2 (W82) and ocular albinism (OA) phenotypes. [in press]. Morell, R., Carey, M.L., Lalwani, A.K., Friedman, TB. and Asher, J.H., Jr. (1996) Three mutations in the paired homeodomain of PAX3 that cause Waardenburg Syndrome type 1. [in press]. Asher, J.H., Jr., Harrison, R.W., Morell, R., Carey, ML. and Friedman, T.B. (1996) Effects of PAX3 modifier genes on craniofacial morphology, pigmentation, and viability: a murine model of Waardenburg Syndrome variation. Genomics 34, 285-298. 172 322. Sadler, T.W. (1990) Langman's medical embryology, sixth edition. Williams and Wilkins, Baltimore. 323. Heimer, L. (1983) The human brain and spinal cord. Springer-Verlag New York Inc. "Illllllll'lllllllll“