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To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:lProj/Aoc&Pres/ClRC/DateDue.indd CHARACTERIZATION OF A DFNBl DELETION ALLELE IN A MICHIGAN KINDRED OF GERMAN DESCENT By Ellen Shields Wilch A DISSERTATION Submitted to Michigan State University in partial fiJIfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics 2010 ABSTRACT CHARACTERIZATION OF A DFNB1 DELETION ALLELE IN A MICHIGAN KINDRED OF GERMAN DESCENT By Ellen Shields Wilch A novel DFNB1 allele identified in a kindred segregating recessively-inherited hearing loss contains a 131.4 kb deletion, upstream of the transcriptional start sites of both GJB2 and GJB6, the genes encoding the gap junction proteins connexin 26 and connexin 30, respectively. The four family members who are compound heterozygotes for this deletion and for the 35delG mutation of GJB2 are all profoundly deaf, and have been since infancy. Heterozygous carriers of this deletion have normal hearing. We demonstrate that this allele segregates with a low-expression phenotype of GJBZ and GJB6 mRNA, assayed by allele-specific PCR of cDNA made from buccal cell RNA of four individuals (three assayed for GJBZ expression, one assayed for GJB6 expression). Considering the evidence of this allele together with that of other DFNB1 deletion alleles, it is likely that the pathogenicity of the DFNB1 deletion alleles is a result of loss of one or more distal GJBZ enhancer elements. One or more critical GJB2 regulatory elements is likely to exist within the genomic interval that is common to the three DFNBl deletions that leave the GJBZ sequence intact; this interval extends from about chr13:19,837,300- 19,932,800 (Mar2006 assembly (NCBI36/hg18)). Assays of candidate enhancer elements identified by cross-species conservation have shown that some sequences within the 131.4 kb deletion interval upregulate or downregulate transcription of a reporter gene in two human epithelial cell lines. Disruption of distal GJBZ regulatory elements may underlie idiopathic hearing loss in persons with monoallelic mutation in the GJBZ coding region. As mutations in GJBZ are the most common cause of congenital hearing loss worldwide, understanding the cis-regulatory landscape of this gene is important in assessing pathogenicity of extragenic sequence variants in suspected DFNB1 alleles, and in interpreting the variation in expressivity of hearing loss in persons with biallelic coding region mutations of 0.182. Copyright by ELLEN SHIELDS WILCH 2010 This dissertation is dedicated to thel9th-century German immigrant settlers of Clinton and Ionia Counties, Michigan, and to their descendants. ACKNOWLEDGEMENTS I thank without ceasing my advisor, Dr. Karen H. Friderici, who is an excellent mentor, a patient teacher, and a valuable friend. My committee members—Dr. Rachel A. Fisher, Dr. Patrick J. Venta, and Dr. Vilma Yuzbasiyan-Gurkan—have provided many, many hours of advice, instruction, and encouragement. Without the mentorship of all four of these people, this dissertation would not exist. I wish to particularly acknowledge Dr. Jill Elfenbein, who died earlier this year. She initiated and contributed significantly to the research described in this dissertation. She was a smart, cheerful, hardworking, and excellent audiologist, research colleague, committee member, teacher, editor, and friend. Her loss is deeply felt. There are too many other people whose support and fi'iendship I wish to acknowledge, but I will do my best to make a list: 0 Dr. Barb Sears, director of the Genetics Program, Dr. Helmut Bertrand, former director, and Jeannine Lee, who takes very good care of all of us. Also, Dr. Richard Miksicek, who served as Genetics Program representative on my committee. 0 Kathy Jernigan, Dr. Meghan Kosarek Drummond, Dr. Soumya Korrapati, Dr. Donna Housley, and Dr. Mei Zhu, who have been the best labmates and friends. 0 Dr. Mei Zhu, Dr. Sainan Wei, Dr. Marty Regier, Kathy Jemigan and others in the Friderici lab past and present who did much of the careful and thoughtful early work on. this project, before I joined the lab. This work included DNA preps, data generation and recording, including the generation and analysis of haplotypes by SNP genotyping and microsatellite genotyping. Dr. Mei Zhu initiated the expression assays detailed in Chapter 2. Dr. Rachel Fisher collected genealogical data, generated an extensive vi analyzed haplotype data, and maintained project data and other records. Dr. Jill Elfenbein coordinated the audiological testing and did much of the testing herself. 0 Dr. John Fyfe, Dr. Brian Schutte, Dr. Deb Schutte, Dr. Donna Koslowsky, Dr. Tejas Kadakia, Erin Wakeling, Dr. Rabeah Al-Temaimi, Dr. Eric Schauberger, Bill Payne, Dr. Tuddow, Dr. Walid F akhouri, Paula, Arianna, Dan, Joe Bonner, Emily, Gabi, Dr. Rachel Spurbeck, Emmy, Ayo, and others on the 5th floor of BPS (or nearby) who (have) contribute(d) to a pleasant and collaborative research environment. 0 Kirk Burkhart, Lauren Broske, Bob Moore, Andrew Reidy, and Sarah Lebeis, who, as undergraduate workers in the Friderici lab, generated useful data included in this dissertation. 0 Dr. Andrea Bréiutigam, Dr. Christopher Vlangos, Dr. Rebecca Slager , Dr. Sandi Clement, Dr. Christina Harzman, Dr. Larissa Reifur, Dr. Laura Yu, and Mary F antacone, my TA and rotation friends. 0 My good friends and neighbors in Albion, who really are too numerous to name without leaving someone important off the list. 0 My parents, Harold and Gloria Shields, who love their kids, and who have waited many years for them all to be out of school. 0 My siblings and siblings-in-law in Maine, Iowa, and elsewhere, and my mother- in-law Jo Wilch. Also, my father-in-law, John Wilch, whom we miss very much. 0 I gratefully acknowledge the many forms of support provided to doctoral students in general, and to me in particular, by Michigan State University. I thank Dr. Walter Esselman of the Dept of Microbiology and Molecular Genetics (MMG), Dr. Richard vii Schwartz of MMG and the College of Natural Science (CNS), Dr. Karen Klomparens of the Graduate School, and Dr. Decolius Johnson. I was the recipient of a University Distinguished Fellowship, a CNS Summer Research Fellowship, and a CNS Dissertation Completion Fellowship. 0 Particular thanks to the MSU-DFS study participants, and to the family members who served on the Research Advisory Committee and the Ethics Committees for this project throughout the years. Human genetics research depends mightily on the generous participation of families, and this family has participated mightily. 0 Love to Thom and the kids, Joe and Miriam. viii TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. xi LIST OF FIGURES .......................................................................................................... xii LIST OF ABBREVIATIONS .......................................................................................... xiii CHAPTER 1: The MSU-DFS Kindred and Hearing loss ................................................... 1 THE MSU-DFS KINDRED AND HEARING LOSS .................................................... 2 THE CELL BIOLOGY OF HEARING .......................................................................... 3 The mammalian hearing and vestibular sensory systems ........................................... 3 The stria vascularis ...................................................................................................... 8 Generation of the endocochlear potential ................................................................. 10 The gap junctional networks of the cochlea and supply of potassium to the endolymph ................................................................................................................. ll CONNEXINS AND CONNEXIN MUTATIONS IN HEARING LOSS .................... 20 The connexin gene and protein families ................................................................... 20 Expression and function of human connexins .......................................................... 22 Distribution of connexins 26 and 30 in the mammalian ear ..................................... 25 Mutations of GJB2 in hearing loss ............................................................................ 26 Connexin 26 mouse models ...................................................................................... 28 GJ B6: GJB2’s mutation-poor, mysterious twin ....................................................... 29 CIS-REGULATION OF TRANSCRIPTION AND CIS-REGULATORY MUTATIONS IN GENETIC DISEASE ...................................................................... 31 Transcriptional enhancers and locus control regions ................................................ 31 Remote enhancers identified in human genetic disease ............................................ 33 Identification of remote cis-regulatory elements ...................................................... 34 REFERENCES .............................................................................................................. 36 CHAPTER 2: Expression of GI B2 and GJ B6 is reduced in a novel DFNB1 allele. ..... 43 ABSTRACT .................................................................................................................. 44 TEXT ............................................................................................................................ 44 ACKNOWLEDGEMENTS .......................................................................................... 65 WEB RESOURCES ...................................................................................................... 66 REFERENCES .............................................................................................................. 67 CHAPTER 3: A novel DFNB1 deletion allele supports the existence of a distant cis- regulatory region that controls GJ B2 and GJB6 expression. ............................................ 69 ABSTRACT .................................................................................................................. 70 INTRODUCTION ........................................................................................................ 71 MATERIALS AND METHODS .................................................................................. 72 Human subjects ......................................................................................................... 72 Array Comparative Genome Hybridization .............................................................. 73 Deletion screening by PCR assay ............................................................................. 73 RESULTS ..................................................................................................................... 74 Identification ofdel(chr13:19,837,344-19,968,698) in an extended family segregating DFNB1 hearing loss .............................................................................. 74 Del(chrl3:19,837,344-19,968,698) segregates as a completely penetrant DFNBl allele and is >300 years old ....................................................................................... 77 Assessment of 35deIG-heterozygotes with SNHL for incidence of pathogenic copy number variation across the DFNB1 locus ............................................................... 82 DISCUSSION ............................................................................................................... 82 ACKNOWLEDGEMENTS .......................................................................................... 91 REFERENCES .............................................................................................................. 93 CHAPTER 4: Enhancer assays of conserved DNA elements in the 131.4 kb MSU-DFS deletion interval ................................................................................................................. 96 INTRODUCTION ........................................................................................................ 97 METHODS ................................................................................................................. 100 Construction of pGL3 luciferase reporter plasmids ................................................ 100 Cell lines and cell culture conditions ...................................................................... 106 Transfections and enhancer assays .......................................................................... 106 RESULTS ................................................................................................................... 107 DISCUSSION ............................................................................................................. 1 15 REFERENCES ............................................................................................................ 120 CHAPTER 5: SUMMARY AND FUTURE DIRECTIONS ......................................... 1.22 REFERENCES ................................................................................. 131 LIST OF TABLES Table 2-1. . Selected haplotype assignments based on genotypes at microsatellites and SNPs within a 620 kb region of chromosome 13 including GJBZ and GJB6... . ...51 Table 3-1. Number of affected individuals with monoallelic GJBZ/GJB6 mutation screened for del(chr1 3 : l 9,837,344-1 9,968,698) ................................................ 83 Table 3-2. List of CNVs in the DFNB1 region .................................................. 83 Table 4-1. Candidate enhancer element locations and lengths, and primers used to amplify elements from genomic DNA .......................................................... 101 Table 4-2. Human-mouse most-conserved sequences (>100 bp) in the region spanned by the three DFNB1 deletions that leave GJZB intact ............................................ 102 Table 4-3. Average expression levels of tested candidate enhancer constructs, and p- values calculated by Student’s t-test, in comparison to the Promoter control... . . . . . ......1 13 xi LIST OF FIGURES Images in this dissertation are presented in color. Figure 1-1. The human ear. ....................................................................... 4 Figure 1-2. Cross-section through the cochlea .................................................... 6 Figure 1-3. Schematic diagram of stria vascularis .............................................. 9 Figure 1-4. Models of potassium movement in cortilymph. ................................ 14 Figure 1-5. Schematic of apposing cell membranes coupled by gap junctions ............ 16 Figure 1-6. Epithelial and connective tissue gap junctional systems of the cochlea ...... 18 Figure 2-1. Map encompassing haplotyped region of 13q11—12 ............................ 47 Figure 2-2. A small portion of the MSU-DFS pedigree ....................................... 50 Figure 2-3. Audiograms of four MSU-DFS family members ................................. 53 Figure 2-4. Southern blot of region around GJBZ .............................................. 56 Figure 2-5. Allele-specific expression assays for GJBZ ....................................... 59 Figure 2-6. Allele-specific expression assay for GJB6 ....................................... 63 Figure 3-1. The MSU-DFS subpedigree ......................................................... 76 Figure 3—2A. Characterization of novel DFNBI deletion allele del(chr13:19,837,344- 19,968,698). ......................................................................................... 79 Figure 3-ZB. Del(chr13:19,837,344-19,968,698) breakpoint junctional sequence ....... 81 Figure 3-2C. Del(chr13:19,837,344-19,968,698) overlaps with DFNB1 deletions deI(GJB6-Dl 381830) and de1(GJB6-D13S I 854) ............................................... 86 Figure 3-2D. Upper VISTA track shows sequence conservation between human (March 2006) and mouse (July 2007) genomes ......................................................... 90 Figure 4-1. Schematic of pGL3-Promoter vector (Promega) ............................... 105 Figure 4-2. Locations of candidate enhancer constructs. ................................... 109 Figure 4-3. Expression assay results ............................................................ 112 xii 35delG array CGH CEPH CNV Cx26 Cx30 dB DFNB1 DFN B4 DSB GJBZ GJB6 K+ kb KCNJI 0 kDa LINE MIM mM mV Na+ nM LIST OF ABBREVIATIONS deletion of a single G nucleotide at c.35 array comparative genome hybridization Centre d’Etude du Polymorphisme Humain copy number variant Connexin 26 Connexin. 30 decibel Deafness locus, autosomal recessive, first identified Deafness locus, autosomal recessive, fourth identified double-strand break gap junction beta 2 gap junction beta 6 potassium kilobase potassium inwardly-rectifying channel, subfamily J, member 10 kflodahon long interspersed nuclear element Mendelian Inheritance in Man [sic] millimolar millivolt sodium nanometer nanomolar xiii NHEJ OMIM PCR SLC 26A 4 SNHL SNP UTR non-homologous end joining Online Mendelian Inheritance in Man [sic] polymerase chain reaction solute carrier family 26, member 4 sensorineural hearing loss single nucleotide polymorphism untranslated region xiv CHAPTER 1 THE MSU-DFS KINDRED AND HEARING LOSS THE MSU-DFS KINDRED AND HEARING LOSS The MSU-DFS kindred is an expanded founder population in central Michigan, centered in Ionia and Clinton counties, and including the towns of Westphalia, Pewamo, and Fowler, among others. Beginning in the 18303 and continuing throughout the remainder of the 19th century, several hundred Catholic German immigrants homesteaded in this part of Michigan. Many, though not all, emigrated from the Eifel region of western Germany. A strong sense of community identity based on a shared German heritage and Catholic faith persists to this day. Marriage between the descendents of the original immigrants has been and remains common. Michigan State University faculty, most notably audiologist Dr. Jill Elfenbein and geneticists Dr. Rachel Fisher and Dr. Karen Friderici, began a long-term collaborative research project with this community in 1996, to investigate the etiology of inherited hearing loss within this population. To date, Dr. Fisher has gathered genealogical records from family and public resources that include 28,256 individuals. Ninety-eight percent of these individuals can be related in a single pedigree (Schutte DL, 2010). Among these, 223 community members have undergone audiological evaluations, given personal and family health histories, and donated DNA. Of the nineteen family members with congenital or prelingual, bilateral, nonsyndromic sensorineural hearing loss, eleven were found to be heterozygous for the 35delG mutation of GJBZ, the gene encoding the gap junction protein connexin 26 (Cx26). Mutations in GJBZ are the most common cause of inherited hearing loss. One person is homozygous for the L445W mutation of SLC 26A 4, the gene encoding the protein pendrin. Mutations in pendrin are also a common cause of inherited hearing loss. Three persons carry monoallelic coding region variants of SLC26A 4; their diagnosis remains under investigation. Four family members, all profoundly deaf, carry the 35delG mutation of GJBZ on one chromosome, and share by descent on the other chromosome a novel deletion mutation that affects the regulation of GJBZ. The identification and characterization of this mutation is the subject of this dissertation. The remainder of this chapter is a literature review, divided into three sections. The first is a description of the mammalian ear, with particular focus on the structures and mechanisms involved in the generation and maintenance of potassium gradients and the endocochlear potential, in which GJBZ and its neighboring gene, GJB6, are highly implicated. The second section concerns the cell biology of the protein products of GJBZ and GJB6, connexins 26 and 30, and the phenotypic consequences of mutations in those genes. The final section is a brief overview of emerging models of cis-regulation of gene transcription, with emphasis on the identification and function of distal regulatory regions. THE CELL BIOLOGY OF HEARING The mammalian hearing and vestibular sensory systems The mammalian ear contains the sensory systems for hearing and for balance, gravity, and acceleration. There are many excellent reviews of the cell and molecular biology of the ear, and the reader is referred to a few of them for more detailed explanation (Dror & Avraham, 2009; Friedman & Griffith, 2003; Petit, 2006; Petit, Levilliers, & Hardelin, 2001; Zdebik, Wangemann, & Jentsch, 2009). The ear is conventionally divided into three regions: outer, middle, and inner (Figure 1-1). The outer ear comprises the pinna and the auditory canal. The geometry of these structures accounts for some degree of amplification of selective frequencies of sound waves Vestibule ' l ' 41,-"); . Semicircular I , , - J 5.2-, Canals "A“. h. .‘ 'V ‘1 Endolymphatic ' i . canal , 2’ ,f , saCCule External auditory canal V Tympanic . Middle ear Cochlea membrane ossicles 1. f. Eustachian tube Figure 1-1. The human ear. The outer ear comprises the pinna(auric1e) and external auditory canal; the middle ear is the air-filled compartment between the tympanic membrane and the fluid-filled vestibule and cochlea, which comprise the inner ear. Reproduced from (Petit, et al., 2001). conducted through air to the middle car. At the terminus of the auditory canal, sound waves vibrate the tympanic membrane, which is in physical contact with the malleus, one of the ossicles of the middle ear. Sound waves are further modified by the vibration along the ossicles, and are transmitted into the inner ear by vibration of the stapes against the membrane covering the oval window, an opening in the bony covering of the cochlea. The mammalian cochlea is a spiral-shaped structure containing three fluid-filled compartments: the scala tympani, scala vestibuli, and scala media (Figure 1-2). The composition of the fluid within the scala tympani and scala vestibuli, the perilymph, resembles that of typical extracellular fluid. However, the fluid within the scala media, the endolymph, is unusually high in K+ (150 mM) and low in Ca2+ and Na+. Sound waves transmitted across the oval window travel in perilymph along the length of the scala vestibuli. The cochlear hair cells and a variety of surrounding supporting cells are arrayed linearly along the length of the cochlea, comprising the organ of Corti, which rests on the basilar membrane. The shape and mechanical characteristics of the basilar membrane are such that the frequency of a sound wave generated by a pure tone causes vibration of only a specific region of the basilar membrane. The basilar membrane is tonotopically organized, with high frequency sound waves causing vibration at the base of the cochlea, and low frequency sound waves causing vibration at the cochlear apex. Displacement of the basilar membrane with respect to the tectorial membrane, a gel-like structure overlying the organ of Corti, causes deflection of the membrane-bound stereocilia that project from the apical surface of the hair cells. Opening of mechanically- gated channels on some stereocilia is the first in the cascade of events of perilym h ' ,. scala vestl ull . r it; :2 perilymph V scala tympani ‘.'.'.~, . Ifu" I. ' .. . .' ‘ -.. _ . .' A in-é-fizfl"; I V}; r', . ‘ s . . ‘ r . Figure 1-2. Cross-section through the cochlea. ihc, inner hair cell; ohc, outer hair cell; sv, stria vascularis; d, Deiter cell; p, pillar cell; h, Hensen cell; c, Claudius cell; r, root cells; i, interdental cell; is, inner sulcus cell; 05, outer sulcus cell; sp, spiral prominence; tm, tectorial membrane; bm, basement membrane; r, Reissner’s membrane. Reproduced - from Cohen-Salmon et. al. (2002) mechanotransduction, that depend on an lnltlal, rapld 1nflux of K 1nto halr cells Vla these channels, leading to depolarization of the cell with eventual release of neurotransmitter from the basal surfaces of the hair cells. The vestibular labyrinth, the motion- and balance-sensing organ of the inner ear, functions in many ways similarly to that of the cochlea. Endolymph fills the vestibular structures of the saccule and utricle, as well as ducts within the semicircular canals, that together with the endolymphatic sac and duct constitute the membranous labyrinth that is continuous with the scala media of the cochlea. The sensory epithelia of the vestibular system consist of patches of hair cells within the saccule and utricle and at the bases of the three semicircular canals. When motion causes displacement of the otoconium, a granular gel mantling the stereocilia of the vestibular hair cells, deflection of those stereocilia results in a similar mechanotransduction cascade to that of the cochlea, beginning with K+ influx through mechanically-gated channels in the membrane of the stereocilia, and terminating with release of neurotransmitter. There are two populations of hair cells in the mammalian cochlea. A single row of inner hair cells is separated by pillar cells from three rows of outer hair cells. The lateral wall membranes of outer hair cells are enriched in prestin, a motor protein responsible for shortening and stiffening of the outer hair cells in response to membrane depolarization. True hearing is accomplished by the inner hair cells, but the outer hair cells are responsible for amplification (about 50 dB) and frequency refinement, without which hearing is significantly compromised (reviewed in Dallos, 2008). The stria vascularis The stria vascularis (Figures 1-2 and 1-3) is a specialized tissue that forms part of the lateral wall of the scala media, and is integral to two processes critical for proper hearing, the generation of the endocochlear potential and the maintenance of high K+ concentration in the endolymph (Kikuchi, Adams, Paul, & Kimura, 1994; Wangemann, 2002; Zdebik, et al., 2009). It is the most highly vascularized tissue in the human body, indicative of the metabolic energy required for its function. Two layers of epithelial cells form the stria vascularis. Strial marginal cells joined by tight junctions face the scala media. The basolateral membranes of marginal cells face the intrastrial space, which is also bound by strial intermediate cells. The membranes of both marginal and intermediate cells are highly convoluted and in close association with each other, each presenting a large surface area to the narrow (15 nm), highly vascularized intrastrial space. Intermediate cells are connected by gap junctions to an underlying layer of cells, the basal cells, which are also connected by gap junctions to fibrocytes of the spiral ligament. The integrity of the intrastrial space is maintained by the tight junctions of the marginal cells, the basal cells, and the endothelial cells of the capillaries. Endolymph K+ KCNQI -KCN£1 +80 mV Marginal Intermediate Basal Fibrocyte Figure 1-3. Schematic diagram of stria vascularis. Movement of potassium from cochlear lateral wall fibrocytes through the cells of the stria vascularis is responsible for the generation of the endocochlear potential. Reproduced from (Marcus, Wu, Wangemann, & Kofuji, 2002) Generation of the endocochlear potential The endocochlear potential is the large electrical potential of around +80 mV that exists in the endolymph of the scala media. It is this potential that drives the influx of K+ into the mechanically-gated ion channels of the stereocilia at a rate and magnitude required for the appropriate downstream response of the hair cells. Generation of the endocochlear potential within the stria vascularis depends on the two-layer structure of that tissue and on the expression of various ion channels, pumps, and transporters on the membranes of several cell types within and adjacent to the stria. The potassium channel KCNJ 1 0 (Kir4. 1) is expressed on the apical membrane of strial intermediate cells. Potassium concentration within the intrastrial space is low (5 mM K+ ionic activity) compared to that of intermediate cells (60 mM), and diffusion of + . . . . K through these channels IS estimated to be responsrble for generation of most of the resting membrane potential of around +90 mV that exists across this membrane. Low potassium concentration is maintained in the intrastrial space by the action of two ion transport complexes that are highly expressed on the basolateral membrane of strial marginal cells, a Na+,K+ATPase (subunits coded for by ATPIAI and AT P] B] or A TPIBZ) and a Na+,K+,2Cl-cotransporter (N KCC 1 , coded for by SLC 12A 2). Potassium taken up by the marginal cells is then secreted via KCNEl/KCNQI channels expressed on the apical surface of the marginal cells that face the endolymph. 10 The gap junctional networks of the cochlea and supply of potassium to the endolymph The rapid depolarization of hair cells that occurs in response to mechanical deflectlon of stereocrlla depends on a rap1d influx of K into the hair cells, whlch requires a high concentration of potassium (150 mM) in the endolymph, as well as a high positive potential (+80 mV) in the endolymphatic compartment. Both of these physiological necessities are accomplished in coordination, via the movement of potassium and other ions between the various compartments of the cochlea. The basolateralIy-expressed potassium channel KCNQ4 permits the rapid diffusion of potassium out of hair cells following a depolarization event, into perilymph surrounding the basolateral surfaces of hair cells and supporting cells of the Organ of Corti. It has been apparent since the 1980s that perilymph is the source of potassium to the endolymph (reviewed in (Hibino, Nin, Tsuzuki, & Kurachi, 2010)), but there is not yet universal consensus on how that is accomplished. (Zdebik, etal., 2009) discuss three models, all supported by experimental evidence and none mutually exclusive of the others, that are likely to encompass the reality (Figure 1-4). In the first model, K+ may recirculate directly via the perilymph of the scala tympani, which underlies the basilar membrane. The second model suggests that K+ may be taken up from the perilymph adjacent to hair cells by surrounding Deiter’s cells and inner phalangeal cells, through KCC4 and KCC3 K+C1-cotransporters. These channels may operate bi-directionally, allowing movement of potassium into these cells where the external potassium concentration is locally high (presumably in the narrow perilymphatic space adjacent to hair cells), and out of the cells where external potassium concentration is low (in regions of the membrane facing the basilar membrane and scala tympani). Thus, these cells may serve as local buffers of K+ only, rapidly sequestering K+ away from hair cells and releasing it back into perilymph. The third model similarly posits K+ uptake by Deiter’s cells from the perilymph surrounding the hair cells, with subsequent gradient-driven cell-to-cell movement away from the organ of Corti via gap junctions. Figure 1-4. Models of potassium movement in cortilymph. A, K+ diffuses through open cortilymph/perilymph; B, KJr moved into supporting cells is recycled via gap junctions; C, K+ concentration is buffered by sequestration in supporting cells. Reproduced from (Zdebik, et al., 2009). I3 Figure 1-4 14 Gap junctions are channels formed by hexamers of connexin proteins in the membranes of adjacent cells, which provide a means for direct cell-to-cell transfer of ions and small molecules (Figure 1-5). In cells coupled by gap junctions, freeze-fracture electron microscopy reveals plaques, regions of densely packed gap junctional channels. Certain cell populations of the cochlea are extensively coupled by gap junctions (Kikuchi, et al., 1994; Kikuchi, Kimura, Paul, Takasaka, & Adams, 2000); some of the plaques observed in these cell membranes are exceptionally large, having over 100,000 individual channels(Forge, Marziano, Casalotti, Becker, & Jagger, 2003). Gap junctions couple all of the epithelial cells of the organ of Corti, with the exception of the inner and outer hair cells themselves. These supporting cells are coupled on both sides of the organ of Corti to adjacent cells; on the medial side, inner sulcus cells are coupled to interdental cells, and on the lateral side, Claudius cells are coupled to root cells. Notably, gap junctions are not expressed on the apposing cell membranes of root cells and the Type II and Type IV fibrocytes of the spiral ligament. These cell membranes are highly convoluted and extensively interdigitated. Similarly to the basolateral membrane of strial marginal cells, the Na+K+ATPase potassium pump and NKCCl (SLC12A2) potassium transporter are expressed on the membranes of Type II and Type IV fibrocytes. Thus, transfer of K+ from the epithelial cell gap-junctional network to the fibrocytes of the spiral ligament is thought to require its secretion into extracellular perilymph and uptake into adjacent fibrocyte cells by pumps and transporters. Cytoplasmic Transmembrane Extracellular a F'— C - o 2 a "o .0 on a "o choc-000004- o 0.. 00. E E :zf:f:::‘:f:t:t . ' “°"“""“"‘ - :"'::'°°::'.:: -' 32:22::leij . 1' no... no... .... ......n.. un- «ououuuwuol o 8 o a a a '0'. - . ‘I l :2 000.00 - ‘ W . y W ‘1 _ WM utuuoouuu lot-houocuuu; . ,hlolaououuiu ' iddodolouioho ...... IIIQCOIO r I...ll.0lll|.|.oal . ' OI. .0..IO|O. rel ‘ IDOI ntl'bllfiofi‘t Connexon Homomeric Homomeric Heteromeric Heteromeric Channel Homotypic Heterotypic Homotypic Heterotypic Figure 1-5. Schematic of apposing cell membranes coupled by gap junctions. Gap junction channels consisting of interacting hexameric connexons in apposing cell membranes provide channels for the selective transfer of ions and small molecules. Connexins are small, 4-pass, membrane-spanning proteins with cytoplasmic N- and C- terminal domains, one cytoplasmic loop, and two extracellular loops. Connexons and channels with different connexin composition have different selectivity and permeability. Reproduced from (Wei, Xu, & Lo, 2004). 16 All of the fibrocytes of the spiral ligament are coupled by gap junctions, and gap junctions are expressed between spiral ligament fibrocytes and the basal cells of the stria vascularis. Strial basal and intermediate cells are also extensively coupled by gap junctions. Thus, there exists between the organ of Corti and the stria vascularis a veritable highway for the recirculation of potassium from its site of disposal into perilymph from channels in the basolateral membrane of hair cells back to its site of secretion from strial marginal cells into endolymph (Figure 1-6). The model of primarily gap-junctional-mediated movement of K+ is supported by a mouse model of conditional knockout of Connexin 26 in the sensory epithelium ((Cohen-Salmon, et al., 2002), discussed in the following section). connective tissue cell gap junction system stria vascularis spiral ligament K+ endolymph K9 l {x + sc HC Epithelial cell gap junction system _ gap junction Figure 1-6. Schematic showing the circulation of potassium ions through the two gap junctional systems of the mammalian cochlea. FC, fibrocytes; MC, marginal cells, BC, basal cells; 1C, intermediate cells; RC, root cells; SC, supporting cells, HC, hair cells. Reproduced from Zhao et al. (2006). 18 In summary, the intricate cell biology of the mammalian cochlea supports a hearing apparatus that functions over a broad frequency range with high sensitivity and selectivity. This sharpening and amplification is achieved by the function of the outer hair cells, a unique feature of mammalian cochlear anatomy. The hearing process is powered by the stria vascularis, a multi-layered tissue with a complex distribution of ion channels and transporters responsible for achieving the high concentration of potassium in the endolymph. At the same time, movement of potassium through the stria is coupled to an intricate mechanism to generate the required high electrochemical potential (endocochlear potential) of the cochlear endolymph. The metabolic energy required by the stria is provided by extensive vasculature in the intrastrial space. It is suspected that the physical separation of the stria from the organ of Corti is a requirement to minimize vibration from blood flow transmitted to the hair cells. Notably, the movement of potassium through the hair cells and within and through the sensory epithelial supporting cells is accomplished without energy—requiring membrane pumps. It must also be noted that the two cochlear gap-junctional networks, that of the sensory epithelium and that of the spiral ligament connective tissue and strial basal and intermediate cells, can be seen not only as providing routes of transport, but as establishing large reservoirs for the buffering of potassium concentration. A ready sink for potassium may be necessary surrounding the basolateral surfaces of hair cells, and a ready source of potassium, fixed as well with respect to electrochemical potential, has been established as a requirement for proper function of the stria. l9 CONNEXINS AND CONNEXIN MUTATIONS IN HEARING LOSS The connexin gene and protein families Connexins are proteins that hexamerize to form channels in the membranes of most non-circulating cells of vertebrates (Figure 1-5). These channels, called connexons or gap junction hemichannels, associate extracellularly with connexons of adjacent cells to form gap junctions, channels that directly connect the cytosolic spaces of adjacent cells, permitting the transfer of ions and small molecules. Hemichannels may be composed of more than one type of connexin; such connexons are referred to as heteromeric. In forming gap junctions between adjacent cells, homomeric or heteromeric connexons of one cell may associate with connexons on an adjacent cell that are of the same or different composition, thus forming homotypic or heterotypic gap junctions. There are 21 connexin genes in the human connexin gene family, and 20 in the mouse. Connexins are four-pass membrane proteins, with intracellular N- and C-termini, one intracellular loop, and two extracellular loops. Connexin proteins are recognized by some degree of amino acid sequence conservation of some portions of the N-terminus, the membrane-spanning domains, and in the extracellular loops, in paralogs (connexins of the same species) as well as in orthologs (the same connexin across species). Both extracellular loops are characterized by conserved cysteines that make intramolecular disulfide bridges between the two 100p domains. High sequence identity between paralogs within species allows for the assembly of heterotypic gap junctions. Comparative genomics has shown that the large vertebrate connexin gene family is ancient, as there are orthologs to most connexins from mammals to bony fishes 20 (Cruciani & Mikalsen, 2006, 2007). Connexins are subdivided into four classes, a, B, y, and 5, according to amino acid sequence and length. Most of the length variation occurs in the C-terminal domain, which is not highly conserved between orthologs. Connexin proteins are generally named according to their molecular weight, and the corresponding connexin genes are named according to class and order of discovery of the connexin protein or gene. Thus, connexin 26 (Cx26) is a member of the [3 class of connexins which are the shortest, ranging from 25-32 kDa. Human Cx26 is coded for by GJBZ, the second designated gap-junction fl-connexin gene. B-connexins have the shortest C- terminal sequences; a-connexins the longest. Most connexin genes have a similar genomic structure, with one small 5’ non- coding exon, a single intron, and one exon containing a small portion of the 5’ UTR, the entire coding sequence, and the 3’ UTR. However, some connexin genes have been found to have multiple 5’ exons. Alternate promoter use and alternative splicing, associated with tissue-specific expression and/or variation in translation efficiency, has been documented for several connexins, among them Cx30 (Anderson, Zundel, & Werner, 2005), Cx32 (Neuhaus, Dahl, & Werner, 1995; Sohl, Eiberger, Jung, Kozak, & Willecke, 2001; Sohl, et al., 1996), Cx40 (Dupays, etal., 2003), Cx43 (Pfeifer, Anderson, Werner, & Oltra, 2004) and Cx45(Anderson, et al., 2005). Additionally, some connexin genes have more than one coding exon; among them, Cx31.3, Cx36, and Cx40. 1. Connexin genes are found on eight human chromosomes, both singly and in clusters. The genes coding for connexins 26, 30, and 46 (GJBZ, GJB6, and GJA3) are 21 tandemly arranged within less than 100 kb at 13q12.11. Two other clusters of connexin genes occur on human chromosome 1. Expression and function of human connexins Connexins are expressed in the cell membranes of nearly all non-circulating cells of vertebrates. Most cell types express more than one connexin, and most connexins are expressed in more than one cell type; for instance, keratinocytes express at least six different connexins, and human Cx43 is expressed in at least 35 different tissues(Laird, 2006). Gap junction channels and hemichannels are known to permit the selective transfer of ions and molecules of less than about 1 kDa, including metabolites and signaling molecules such as cyclic adenosine monophosphate (CAMP) and inositol 1,4,5- trisphosphate (1P3) (Beltramello, Piazza, Bukauskas, Pozzan, & Mammano, 2005). Passage of ions and molecules through gap junction channels is not a passive process, dependent solely on channel pore size, but is in fact highly regulated. The gating status as well as the selective permeability of gap junction channels is determined by interactions between amino acid residues in the cytoplasmic domains of the connexon, within and between connexins. Changes in membrane voltage, transjunctional voltage, + pH, Ca2 concentration, and phosphorylation status are all known to affect the opening and closing of gap-junctional channels. A high-resolution (3.5 A) structure of human Cx26 recently solved has yielded insights into the functionality of a number of the residues of the Cx26 protein (Maeda, et al., 2009). Orientation of the four transmembrane helices of each connexin creates of the hexameric connexon a broad, shallow bowl at the cytoplasmic entrance to the channel. Two of the four transmembrane 22 helices of each connexin extend significantly into the cytoplasm to create this broad surface. Hydrogen bonding between residues of the N-terminal helix and the first transmembrane helix of the adjacent connexin form a narrow-diameter ring of the six N- terminal helices of the connexon at the channel entrance. These hydrogen bonds may be disrupted by conformational changes in one or more of the connexins as a result of changes in the cytoplasmic chemical or electrical milieu, favoring interactions between residues of the N-terminal helices and consequent plugging of the channel. Certain features of the general structure of a Cx26 gap junction, as well as the gating model proposed by (Maeda, et al., 2009) are generalizable from Cx26 connexons to many others, including heteromeric connexons, as there is extensive conservation of many residues in the modeled domains, including nearly universal conservation across a and B connexins of the D2 and W3 residues of the N-terminal helix, the two residues most implicated in the gating function. (Maeda, et al., 2009) do not model the C-terminus or cytoplasmic loop of Cx26; these domains are highly variable across connexins. It is believed that the variability of these domains contributes to the specificity in permeability of connexons of differing composition. It is known that the C-terminus of many connexins (although not Cx26) is a target of phosphorylation, and that many kinases act on connexins, and that such interactions modulate gap junction function. Connexins, like other membrane proteins, are translated on rough endoplasmic reticulum, and oligomerized into connexons before delivery to the plasma membrane. Hemichannels diffuse within the plasma membrane to the periphery of gap junction plaques, sites of many closely packed gap junctions, where newly arriving hemichannels dock to hemichannels in adjacent cell membranes. Although it was believed that all 23 hemichannels are constitutively closed until successful docking with hemichannels on apposing cells, it is now known that hemichannels may function independently of gap junction formation. Connexins turn over rapidly, having half-lives of a few to several hours. Degradation of aging gap junctions occurs by invagination and budding off of unique double-membraned vesicles, called connexisomes, from the center of plaques(Laird, 2006). There is a growing body of evidence that connexin gap junction formation and function is tied to the formation and function of adherens junctions and tight junctions, that connexins interact with proteins of adherens junctions and tight junctions, and that connexins interact with cytoskeletal proteins and other cytoplasmic proteins (Dbouk, Mroue, El-Sabban, & Talhouk, 2009). Thus, regulation of gap junction function may occur, not solely through mechanisms that modulate the opening and closing of individual channels, but also by mechanisms that govern the rate of synthesis, assembly, and degradation of gap junctions. The formation of gap junction plaques between and among sets of cells within tissues makes of those cells a functional, though nontraditional, syncitium. With respect to the ions and molecules that pass through particular configurations of gap junctions, diffusion can occur across large numbers of cells quickly and with little or no investment of metabolic energy. This has three main functional consequences for cells connected by gap junctions. Firstly, cells coupled by gap junctions may simply benefit from a long- distance, low-energy import and/0r export mechanism. In the model of gap-junctional + + movement of K away from the sensory epithelia of the cochlea, energy-requiring K pumps can be positioned in the spiral ligament, minimizing ‘noisy’ vascularization near the organ of Corti. Secondly, cell-to-cell signals can be easily propagated and amplified. 24 (Beltramello, et al., 2005) provided evidence for gap-junction mediated diffusion of 1P3 in supporting cells of the organ of Corti, coupled to the propagation of intercellular Ca2 waves. Gap junctions are known to be selectively permeable to other second messengers, including CAMP. In addition to coordinating physiological responses across populations of cells, gap junctional communication is important in development. Thirdly, extensive coupling of cells provides large sources and sinks. For example, the K+ diffusion potential across the membrane of strial intermediate cells depends on an enormous reservorr of K , supplied by the extensrve gap-junctional network connecting spiral ligament fibrocytes to the strial basal cells, which are coupled to the intermediate cells. Distribution of connexins 26 and 30 in the mammalian ear Connexins 26, 30, 31, 36, and 43 are known to be expressed in the mammalian ear. A number of studies have determined connexin expression by immunoprecipitation, freeze fracture, immunogold labeling, and immunohistochemistry, in cochlear preparations from mouse, guinea pig, human, and other mammals ((Forge, Becker, et al., 2003; Forge, Marziano, et al., 2003; Jagger, Nevill, & Forge, 2010; Liu, Bostrom, Kinnefors, & Rask-Andersen, 2009; Zhao & Yu, 2006). Connexins 26 and 30 are the most abundant connexins in the cochlea, with high levels of expression in all of the supporting cells of the sensory epithelium, and in several cell types comprising the lateral wall of the cochlea. Expression of Cx26 and Cx30 overlaps in several cell types, although there are regions of both gap-junctional networks where expression of one connexin predominates, or is expressed exclusively; co-labeling of both connexins in organ of Corti supporting cells suggests the existence of heteromeric and/or heterotypic 25 gap junctions (Liu, et al., 2009). Co-assembly of Cx26/Cx30 connexons or gap junctions was demonstrated in mouse cochlear preparations by co-immunoprecipitation (Ahmad, Chen, Sun, & Lin, 2003). Connexins 26 and 30 are known to make functional heteromeric gap junctions in vitro (Marziano, Casalotti, Portelli, Becker, & Forge, 2003; Yum, et al., 2007). Mutations of GJB2 in hearing loss Over 220 mutations of GJB2 have been reported to cause recessively inherited nonsyndromic hearing loss (Hilgert, Smith, & Van Camp, 2009). The great majority of these are missense mutations. About one-quarter to one-third of the total are nonsense mutations or small deletions which cause a frameshift and premature stop codon. Many of these mutations result in translation of severely truncated, non-functional protein that is rapidly turned over. For instance, the 35delG mutation results in a premature stop codon after a single frameshift substitution in the 12th codon (Denoyelle, et al., 1997). Cx26 protein is not detected in skin biopsies of persons homozygous for this mutation. One splice site mutation (IVSl+1G—>A; (Denoyelle, et al., 1999)) and one promoter mutation (-44C—>T; (Matos, et al., 2007)) have also been reported. Mutations associated with AR hearing loss (DFNBIA) are distributed across the protein, in every domain. Fewer than a dozen mutations of GJBZ associated with AD hearing loss (DFNA3A) have been reported; these mutations also occur throughout the protein. Functional studies of a number of recessive and dominant mutations indicate a range of defects, including impaired trafficking to the plasma membrane, inability of hemichannels to form gap junctions with adjacent cells, and varying degrees of channel dysfunction of assembled gap junctions (variously summarized in (Bicego, et al., 2006; Mese, Londin, Mui, Brink, 26 & White, 2004; White, 2000). Differences between recessive and dominant mutations have been shown to be due to presence or absence of dominant-negative effects when mutant Cx26 constructs are expressed with wild-type Cx26 or Cx30, including mistrafficking of assembled connexons as well as channel dysfunction. The DFNBlA phenotype is characterized by congenital, bilateral, sensorineural hearing loss that is not generally progressive, with a flat or sloping audiogram. There is considerable variation in the degree of hearing loss, which is very rarely nonpenetrant. The severity of hearing loss correlates with category of mutation; greater hearing loss is associated with a genotype of biallelic known or expected null or protein-truncating mutations, and milder hearing loss is associated with biallelic non-truncating (missense) mutations (Azaiez, et al., 2004; Cryns, et al., 2004; Snoeckx, et al., 2005). Of note, there is considerable variation in degree of hearing loss among affected persons with the same genotype, indicating genetic and/or environmental effects beyond that conferred by any particular combination of mutations. However, no modifier loci have been identified to date. Although the vast majority of mutations of GJBZ cause AR NSHL, several are responsible for dominantly inherited conditions and syndromes that include hearing loss along with skin disorders related to defects of keratinization. These mutations tend to occur in the N-terrninal domain or in the first extracellular loop of the Cx26 protein. GJBZ mutation may cause PPK (palmar-plantar keratoderma, hyperkeratosis of the palmar and plantar surfaces of the hands and feet), KID (keratitis-ichthyosis-deafness) syndrome (MIM 148210, vascularizing keratitis with progressive EKV 27 (erthrokeratoderma yariabilis)), Vohwinkel syndrome (MIM 124500, mutilating keratoderma), or Bart-Pumphrey syndrome (MIM 149200, knuckle pads and leukonychia (white nails)). Connexin 26 mouse models Contrary to the human phenotype, where complete loss of Cx26 protein leads only to NSHL and no other apparent pathology, knockout of connexin 26 in mouse is embryonic lethal (Gabriel, et al., 1998). (Cohen-Salmon, et al., 2002) generated a transgenic, conditional mouse knockout of Gij in the sensory epithelium of the organ of Corti. Development of the cochlea was normal, and endocochlear potential (EP) in P12- P13 conditional knockout mice was comparable to that of wild-type mice. However, death of border cells and inner phalangeal cells, supporting cells in proximity to inner hair cells, was observed starting at P14. Cell death progressed to include other supporting cells, as well as the outer hair cells themselves. By 3 weeks of age, significant hearing loss was detected by ABR (auditory brainstem response). In adult mice, both EP and K+ concentration of endolymph were reduced. The authors postulate that cell death results initially from failure of supporting cells around the inner hair cells to adequately transport K+ out of the cortilymph (the perilymph in the extracellular spaces of the organ ofConD. A mouse model expressing a human GJBZ transgene bearing the R75W mutation associated with dominantly inherited hearing loss (Inoshita, et al., 2008; Kudo, et al., 2003; Minekawa, et al., 2009) confirms earlier evidence from expression of human R75W GJBZ in Xenopus oocytes, that R75W connexins exert a dominant-negative effect 28 on wild-type Cx26 (Richard, et al., 1998). Defects in supporting cells in the organ of Corti in the R75W transgenic mouse were observed, and felt to underlie the dysmorphology of the organ of Corti and to explain the hearing loss phenotype, as the outer hair cells themselves maintained electromotility (Minekawa, et al., 2009). No vestibular phenotype is apparent in this mouse, even though Cx26 is expressed in vestibular supporting cells, and morphology and function of the stria vascularis appears normal (Kudo, et al., 2003). GJB6: GJBZ’s mutation-poor, mysterious twin Evidence for co-assembly of Cx26/Cx30 heteromeric connexons and gap junctions is now reasonably well established (see above). GJB6 shares the DFNB1 locus with GJBZ and is located about 30kb telomeric to GJBZ. Connexins 26 and 30 show high amino acid sequence identity. Synteny at this locus is preserved in mammals, birds, and reptiles, but the duplication that gave rise to GJB2 and GJB6 in tandem is not observed in fish genomes (Cruciani & Mikalsen, 2006). While over 200 mutations of GJB2 are now reported to cause nonsyndromic recessively inherited hearing loss (Hilgert, et al., 2009), no coding region mutations of GJB6 at all are associated with recessive hearing loss, and only a single missense mutation is associated with dominant hearing loss. However, there are new four identified DFNB1 deletion mutations. Two are deletions in which GJB6 is truncated, with abrogation of GJBZ expression shown for one (F. J. del Castillo, et al., 2005; I. del Castillo, et al., 2002; Feldmann, et al., 2009; Lerer, et al., 2001; Pallares-Ruiz, Blanchet, Mondain, Claustres, & Roux, 2002; Rodriguez-Paris & Schrijver, 2009). The third is a large (>920 kb) deletion in which the entire gene sequences of both GJBZ and GJB6 are missing (Feldmann, et al., 2009). The fourth is the 29 deletion identified in our lab, and that is the subject of this dissertation. This deletion segregates with reduced expression of both GJBZ and GJB6 (Wilch, et al., 2006) [Chapter 2 of this dissertation]; (Wilch, et al., 2010)[Chapter 3 of this dissertation). The GJB6 coding region is closely scrutinized in individuals with suspected DFNB1 hearing loss; curiously, the only null alleles of GJB6 that have been identified are those in which GJBZ firnction is compromised as well. The Cx30 knockout mouse lacks the endocochlear potential and has significant hearing impairment that worsens in concert with degeneration of the sensory epithelium that begins at P18 (Teubner, et al., 2003). Although this mouse does not display an overt vestibular phenotype, loss of saccular hair cells has been documented (Qu, et al., 2007). Overexpression of Cx26 in this mouse rescues both hearing (Ahmad, et al., 2007) and saccular hair cells (Qu, et al., 2007). I discuss GJB6 and its relationship to GJBZ further in Chapter 4 of this document. To summarize: 1. The pathogenicity of the three DFNB1 deletions so far identified that leave GJBZ intact very likely results from loss of expression of GJB2. 2. The weight of evidence to date does not support a model of inheritance of monoallelic mutation of both GJB2 and GJB6 (digenic inheritance) as a cause of recessively inherited hearing loss. 3. One or more cis-regulatory elements controlling the expression of GJBZ and GJB6 exist somewhere within the genomic interval common to the three DFNBI deletions discussed. This interval extends from about 150 kb to 250 kb upstream of the 30 transcriptional start site (TSS) of GJB6 (~ 180 kb to 280 kb upstream of the T88 of GJBZ). 4. Other mutations occurring in these yet-to-be identified GJB2-regulatory elements may explain the hearing loss in some persons with monoallelic mutation of GJBZ. Sequence variation within these elements may underlie some fraction of the variable expressivity of hearing loss in persons with biallelic GJBZ mutation. 5. We have begun to characterize sequence elements in this region that are capable of enhancing expression of a reporter gene in a cell culture assay system. CIS-REGULATION OF TRANSCRIPTION AND CIS-REGULATORY MUTATIONS IN GENETIC DISEASE Transcriptional enhancers and locus control regions Investigations in the last twenty years have gone a long way toward building a rich conceptualization of the eukaryotic cis-regulatory landscape. Arrays of enhancer and silencer elements may be associated with transcriptional regulation of target genes, controlling transcription rates as well as temporal- and tissue-specificity of transcriptional activity. It is now known that such elements may exist at vast distances from the genes they regulate. These elements are thought to interact with basal and proximal promoters via looping of intervening DNA. Enhancer-bound transcription factors many interact directly with protein components of the basal transcriptional machinery, or with other proteins bound to the proximal promoter, to stabilize the pre-initiation complex and increase the rate at which the RNA Polymerase II complex is recruited to the transcription start site. Enhancer elements are also associated with the recruitment of 31 chromatin-modifying complexes. Some chromatin modifiers propagate covalent histone modifications along the chromatin, establishing transcriptional]y-permissive regions of ‘open’ chromatin that spread in both directions from an enhancer element. Enhancers may provide more transient points of recruitment for other types of chromatin- remodeling proteins, such as SWI/SNF complexes, that function to ‘nudge’ nucleosomes with respect to their associated DNA, increasing accessibility of basal promoters to the transcriptional machinery. Enhancer elements may also up-regulate transcription by mediating the movement of target genes into ‘transcription factories’ or other nuclear subcompartments permissive to transcription. A requirement for preventing promiscuous interactions between enhancers and non-target genes is fulfilled, at least in part, by insulator elements. One such element that is widespread across mammalian genomes and is relatively cell-type-invariant is defined by a number of binding sites for CTCF (CCCTC-binding factor). Insulators are thought to have two general activities. Enhancer-blocking is the selective prevention of association of a non-target gene with an enhancer in cis; this may involve CTCF/chromatin/protein interactions that define boundaries of active chromatin loops permitting interactions only between enhancers and genes in the same loop domain. Insulators are also found at the boundaries of active (euchromatic) and silenced (heterochromatic) chromatin domains, indicating a barrier function with respect to the spread of chromatin-modifying complexes. 32 Remote enhancers identified in human genetic disease A number of genetic diseases can be caused by disruption of distal cis-regulatory elements. Among the most dramatic and best-characterized examples is preaxial polydactyly resulting from gain-of—function mutations of a conserved non-coding sequence element that drives expression of sonic hedgehog (Shh) in vertebrate limb development. Among humans, mice, and cats, twelve different single-nucleotide mutations have been identified within an 800 bp conserved region located le from the T88 of Shh. In transgenic mice, this sequence element is sufficient to drive expression of a reporter gene in a manner that capitulates the wild-type expression pattern of Shh in limb buds; replacement of the wild-type enhancer sequence with mutant constructs results in ectopic expression of Shh in a pattern that correlates to the polydactyly subphenotypes in humans and cats (Lettice, et al., 2003; Lettice, Hill, Devenney, & Hill, 2008; Lettice, et al., 2002). Many human diseases are associated with cytogenetically-diagnosed translocations, deletions, duplications, and inversions. Some of these phenotypes have now been shown in some cases to be attributable to disruption of cis-regulatory elements whose target genes are located at a considerable distance from these regulatory sequences. For instance, aniridia (MIM 106210) is commonly caused by haploinsufficiency of PAX 6 ; chromosomal rearrangements associated with aniridia have breakpoints that occur up to 125 kb away from PAX6 itself. Analyses of this distant downstream region have now identified several sequence elements that have been shown in transgenic mouse studies to drive relevant, tissue-specific expression of a reporter (Kleinjan, et al., 2006; Kleinjan, et al., 2001). 33 Identification of remote cis-regulatory elements Modularity of enhancer elements is an emerging theme, and is perhaps to be expected in genes with important developmental roles, with highly specific patterns of expression in a wide variety of cell types, and with constraints on timing of expression during development (Visel, et al., 2009). Highly investigated regulatory regions of developmentally important genes, in particular, are suggestive of cis-regulatory landscapes with certain general features. First, regulatory sequence may occupy a large span of genomic space, from several 105 of kilobases to more than a megabase. These large regions may appear as ‘gene deserts’ (Nobrega, Ovcharenko, Afzal, & Rubin, 2003); large spans of nongenic DNA are in fact associated with some developmentally important genes. Transposon exclusion zones may also mark extensive regions of cis- regulatory DNA. Second, cross—species conservation is, not surprisingly, associated with function in intergenic and intronic sequence (Pennacchio, et al., 2006; Prabhakar, et al., 2006; Woolfe & Elgar, 2008; Woolfe, et al., 2005). As discussed above, quantitative variation associated with genetic disease, related to ectopic expression or to loss of expression, has been clearly localized to highly conserved cis-regulatory sequence. Thirty-five years ago, King and Wilson (King & Wilson, 1975)presciently suspected that regulatory rather than coding-sequence variation would be shown to be responsible for a great deal of phenotypic variation between closely related species. Because changes to amino acid sequence of a protein expressed in a variety of tissues may have negative pleiotropic effects, phenotypic variation resulting from mutation in a cis-regulatory region affecting timing or amount of expression in only a single tissue may be more likely to be tolerated than variation that is introduced by alteration of amino acid 34 sequence (Wray, 2007). In a meta-analysis of several surveys assessing evidence for positive selection in both protein-coding and noncoding sequence, Haygood et al. (Haygood, Babbitt, Fedrigo, & Wray, 2010) detected such a trend, finding less adaptive change in the coding regions of genes with patterns of broad expression, relative to those genes with expression limited to a single tissue. Thus, while conservation remains the most straightforward means by which to identify candidate enhancer elements, those studies which return the most striking results are perhaps cherry-picking. That is, key developmental regulators expressed early in development such as SHH, PAX genes, and HOX genes, are those most expected to have regulatory sequences intolerant of variation, and to preserve ancient, shared homology across many phyla. The likely story for many other genes whose diversity of expression across phyla may be responsible for phyletic diversity of phenotype characteristics may perhaps be less simple to predict. Functional studies such as that detailed in Chapter 4 of this dissertation are absolutely critical to understanding complicated patterns of cis-regulation. 35 REFERENCES Ahmad, S., Chen, 8., Sun, J., & Lin, X. (2003). Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice. Biochem Biophys Res Commun, 307(2), 362-368. Ahmad, S., Tang, W., Chang, Q., Qu, Y., Hibshman, J ., Li, Y., et al. (2007). Restoration of connexin26 protein level in the cochlea completely rescues hearing in a mouse model of human connexin30-linked deafness. 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American Journal of Human Genetics 79(1):]74-9. 43 ABSTRACT In a large kindred of German descent, we found a novel allele that segregates with deafness when present in trans with the 35delG allele of GJBZ. Qualitative polymerase chain reaction-based allele-specific expression assays show that expression of both GJB2 and GJB6 from the novel allele is dramatically reduced. This is the first evidence of a deafness-associated regulatory mutation of GJBZ, and of potential coregulation of GJB2 and GJB6. TEXT Mutations in GJB2, the gene encoding gap junction protein connexin 26 (Cx26), are the most common cause of prelingual-onset, recessively inherited, nonsyndromic, sensorineural hearing loss (SNHL) in humans. GJBZ and GJB6, which encodes connexin 3O (Cx30), comprise the DFNBI/A3 locus at 13q12 (Figure 2-1). In the vertebrate cochlea, Cx26 and Cx30 are coexpressed as heteromeric connexons in nonsensory cells of the organ of Corti as well as in subsets of cells of the spiral ligament and stria vascularis (Ahmad, Chen, Sun, & Lin, 2003; Forge, Becker, et al., 2003; Forge, Marziano, Casalotti, Becker, & Jagger, 2003), and are implicated in the maintenance of cochlear K+ gradients necessary for proper hair-cell function (Kikuchi, Kimura, Paul, Takasaka, & Adams, 2000; Wangemann, 2002). More than 80 recessive mutations of GJB2, nearly all affecting proper translation of the Cx26 protein, and several dominant missense mutations are implicated in DFNB1/A3 hearing loss. Others cause skin disease with accompanying SNHL, including Vohwinkel syndrome (MIM 124500), Bart-Pumphrey syndrome (MIM 149200), palmoplantar keratoderma (PPK [MIM 148350]), and keratitis-ichthyosis-deafness (KID 44 [MIM 148210]). Three dominant missense mutations of GJB6 have been shown to cause the skin disorder Clouston syndrome (hidrotic ectodermal dysplasia [MIM 129500]). In contrast to the abundance and diversity of GJBZ hearing-loss mutations, only a single dominant mutation of GJB6 causing nonsyndromic hearing loss had been published (Grifa, et al., 1999) before the identification of two large deletions- of 309 kb and 232 kb: del(GJB6-D1381830) and del(GJB6-D1381854), respectively-truncating the 5’ end of GJB6 (Figure 2-1). These deletions segregate with hearing loss when present either homozygously or heterozygously with each other or in trans with a recessive GJBZ mutation (F. J. del Castillo, et al., 2005; 1. Del Castillo, et al., 2003; 1. del Castillo, et al., 2002; Lerer, et al., 2001; Pallares-Ruiz, Blanchet, Mondain, Claustres, & Roux, 2002). Investigators have suggested, but have not demonstrated, that loss of appropriate regulation of GJBZ from chromosomes bearing these deletions may underlie the hearing loss in these individuals which may be exacerbated by loss of one GJB6 allele as well. Common et al. (Common, et al., 2005) showed immunohistochemical evidence that Cx26 expression is disrupted in certain skin-cell types in an individual bearing the larger deletion in trans with GJBZ 35delG, suggesting disruption of a GJB2 cis-regulatory element located within this deleted interval. Mutation screening of GJBZ in individuals whose hearing loss and family history are consistent with GJB2 deafness reveals a significant number of subjects with only one identified mutation. Additional screening for GJB6 deletions explains only some of these heterozygotes. 45 Figure 2-1. Map encompassing haplotyped region of 13q1 1-12 showing locations of GJBZ, GJB6, genotyped microsatellite and SNP markers, and breakpoints of del(GJB6- D1 3S1830) and del(GJB6-D13S1854), all shown approximately to scale. The locations of variants used for allele-specific expression assays--GJB2 35delG, GJBZ +94, and rs 7333214, are also indicated. Boxes indicate exons, and the hatched boxes indicate coding regions of the two genes, Vertical lines show approximate locations of genotyped microsatellites (in bold) and SNPs. D13SZ32 is the most proximal microsatellite showing recombination of the novel haplotype in affected individuals. The transcriptional start sites of GJBZ and GJB6 are indicated by right-angled arrows. 46 —§ telomere del(GJB6-Dl381830,-D13S1854) GJBG (0x30) GJBZ (Cx26) ‘— centromere 35deIG ~90kb ~50kb ~50kb rs7333214 +94 ~80k.b' l j ..... 4 47 ...... VQZQLQL LSJ 681609631 1v9919z181 1968188151 818198131 9006188131 17 LOVVGSJ 911-8210 ~5kb 1788918691 .LLLZB IV 170 1.39963J 191717sz 30 LZS‘QGSJ 980609631 9172 IdNS 91711766151 86039963J 988 LSLSSJ 91438610 Figure 2-1 We report here evidence for a novel pathogenic DFNB1 allele for which we demonstrate reduction in or loss of detectable expression of message from both GJBZ and GJB6. Figure 2-2 shows a small portion of the pedigree of MSU-DFS, a large American kindred of mainly German descent. After obtaining written informed consent, we collected DNA and performed audiological testing on >200 family members. We screened all samples for GJBZ 35delG and for SLC26A4 L445W. Ten MSU-DFS family members are hearing impaired due to homozygosity of 35delG (one, DF5-68, is shown in Figure 2-2); one deaf family member has received a diagnosis of Pendred syndrome (MIM 274600) and is homozygous for SLC26A4 L445W (not shown in pedigree). Microsatellite and SNP genotyping across the GJBZ/GJB6 genomic region (Figure 2-1 and Table 2-1) allowed us to define a number of haplotypes. Three distinct haplotypes bearing 35delG exist within the family, indicating more than one founder for 35delG in this community (two 35delG haplotypes appear in Figure 2-2). Notably, four deaf family members, DF5-20, -70, -122, and -l 94, are heterozygous for 35delG and share a common haplotype (indicated in Figure 2-2 by a black bar with a star) that is longer than 600 kb and shorter than 3.1 Mb on their non—35delG chromosome. These individuals are profoundly hearing impaired, in contrast to DF5-68, who is homozygous for 35delG and is the father of DF5-70 (Figure 2-3). Of the 14 family members who carry the novel allele, all those with 35delG on their other chromosome (4 individuals) have hearing loss, whereas all those with any other allele (10 individuals) have normal hearing (P<.001, by Fisher’s exact test). We also performed a linkage analysis (easyLINKAGE Plus v.3.01RC1, SuperLink v1.4) at the DFNB1 locus, using 28 individuals (shown in boxed areas in Figure 2-2). We assigned the same mutant status to both the novel haplotype and 48 Figure 2-2. A small portion of the MSU-DF 5 pedigree, with DFNBI haplotypes indicated for selected individuals. Five individuals with congenital SNHL are represented by blackened symbols. One of these, DF5-68, is hearing impaired because of homozygosity of GJBZ 35delG (AG). The other four are heterozygous for GJBZ 35delG and bear the same haplotype on their non-35delG chromosome (black bar with star). Individuals contained within the boxed areas were included in the linkage analysis. 49 c) ”mu”. )1»— D \\\\\\.\\\‘ D 35AG alleles novel DFNB1 allele AW 05:.5 194 DF5-69 _- o DF5-122 DF5- 72 \\\\\\\\\\ O ’IIIIIIIIIa DF5-65 DPS-66 DF5-67 AG * a) * Figure 2-2 El 'IIIIIIIII AG 0 lll * I I [III] I AG* 50 *[l 0.83% $— \\\\\\\\\‘ *AG EAG AG DF5-70 DF5-71 VIII/I’ll, 11 AG F 5-68 III/III” m D133 F Effie DF5-48 * Table 2-1. Selected haplotype assignments based on genotypes at microsatellites and SNPs within a 620 kb region of chromosome 13 including GJB2 and GJB6. Novel Location DFNBI Marker (bp) Allele A B C D E D13Sl316 19,580,089-442 6 6 6 5 5.5 6 rs3751385 19,660,956 C T T C C C 35delG 19,661,685 + AG AG + + + rs9552098 19,662,895 C T T C C C rs7994748 19,664,130 T C C T C T GJBZ +94 19,664,944 C C C A C C SNP1245 19,665,308 T C C T C T rs9509086 19,665,957 C A ND C A ND rs9552102 19,668,529 T A ND T T ND A21445T 19,695,038 A A A A T A rs9552104 19,695,038 C G ND C C C A19277T 19,695,038 A ND A ND T A rs93 1 53 84 19,695,038 A G G A G A rs7333214 19,694,497 C C C C A C D13Sl75 19,746,380-734 4 3 3 2 4 2 rsl 3379006 19,795 ,790 T C C T C C rs7337318 19,795,91 1 C G G C G G rsl3378957 19,795,932 A C C A C C r512873647 19,796,279 T C C T C C rs9509139 19,796,316 A G G A G G rsl 1616264 19,796,330 A T T A T T rs944014 19,837,040 G A G G G G rs9552241 20,025,569 ND ND G C G G r32772175 20,198,712 T T ND A A A Note—The “A” and “B: columns indicate delG (AG) alleles carried by DF5-20 and DF5-70, respectively. The “C” column corresponds to the haplotype shown by a box with stippling, “D” to the haplotype shown by a box with horizontal lines, and “E” to the haplotype shown by a white box in figures 2, 5, and 6. Locations are based on UCSC Genome Browser, May 2004 assembly. + = wild type; ND = not determined. 51 Figure 2-3. Audiograms of four MSU-DF 5 family members (identified by ID/sex/age). dB HL = decibels hearing level; ANSI = American Standards Institute. . DF5-20, -70, and -122 are 35de1G (35AG) heterozygotes bearing the novel pathogenic allele. Note that all three profound SNHL at all tested frequencies. By contrast, DF5-68, homozygous for GJB2 35delG and the father of DF5-70, has significantly more residual hearing, particularly in his left ear. Although only pure-tone air- conduction thresholds are shown (circle = right ear; X = left ear, arrows = threshold beyond tested limit), bone conduction thresholds immittance measures (not shown) indicate that the hearing loss in all four family members is sensorineural. Frequency (Hz) Frequency (Hz) 125 250 500 1000 2000 4000 8000 10 125 250 500 1000 2000 4000 8000 .10 g 18 DF5-20/M/62yrs g DF5-70/F/9yrs *' 20 35AG/novel ‘— 35AG/novel g 30 «723 _. 50 _, I 60 I g 70 03 80 90 100 110 Frequency (Hz) Frequency (Hz) 10 125 250 500 1000 2000 4000 8000 125 250 500 1000 2000 4000 8000 0 o g 10 DF5-122/F/53yrs 10 DF5-68/M/~35yrs 93 20 35AG/novel § 20 35AG/35AG <73 30 92 30 Z 40 _. 40 :5 50 ‘2 50 ..I I 50 3 60 '0 I 80 ca 80 100 100 “0 110 Figure 2-3 53 the 35delG allele and all other haplotypes were considered nonmutant. The resulting LOD score of 2.08 indicates that there is a good likelihood that the novel haplotype harbors a DFNB1 mutation. We sequenced the entire coding regions and 5' and 3' UTRs of both GJBZ and GJB6 in three of our affected probands, including splice sites, alternative exons and promoter regions, the entire single intron of GJB2, and several kilobases of sequence around each gene, and found no sequence variants unique to the novel haplotype. Heterozygosities found in sequencing also showed that GJBZ and GJB6 are intact on both chromosomes and that the affected individuals do not bear either del(GJB6-D1381830) or del(GJB6-DI3SI854). Southern blotting indicated no rearrangements or unusual methylation around GJBZ (Figure 2-4). We hypothesized that an unidentified cis-regulatory element of GJBZ exists within the DFNBI locus and is disrupted on our novel pathogenic allele. Given the impracticality of searching for candidate variants across the entire locus, we first sought to acquire evidence of loss of GJB2 expression from this allele. We developed three allele-specific PCR assays to assess the relative abundance of transcript from each allele, using tissue both easily available and expected to express Cx26. To isolate RNA, buccal cells from several family members were collected on Cytosoft brushes, and were immediately stored in lml Trizol (Gibco/Life Technologies) on ice. Some samples were stored at —20°C for one to several days. RNA was isolated following the Trizol protocol, was resuspended in 20 ul H20, and was stored at —80°C. We followed a modified 54 Figure 2-4. Southern blot of region around GJBZ (with transcriptional start site indicated by right-angled arrow, and exons by black boxes), for DF5-20 and an unrelated control. 32P-labeled probe indicated by wavy-lined box. Detected probe is associated with expected digestion product sizes, and results are similar for both DPS-20 and the control. Since Nru I is a methylation-sensitive restriction enzyme, these results also preclude unusual methylation on either allele of DF5-20. Five ug aliquots of DNA from DF5-20 and an unrelated control were digested with 1 U of Bgl II, Nru I, or Sca I (all New England Biolabs) in single digests, or with l U of Bgl II or Sca II in combination with IU of Nru I for double digests. Each digestion was incubated for 20 hours at 37°C in the appropriate concentration of NEB Buffer 3 (New England Biolabs) in 50 [11 reactions. 45 ul of each digested sample (after addition of 5 ul loading dye to each tube) was run on a 0.7% agarose gel with Markers II and III (Roche), and transferred to a nylon membrane following a standard protocol. A 618 bp PCR product amplified from genomic sequence 500 bp- 1 kbp upstream of the transcriptional start site of GJBZ was radioactively labeled with ~50 uCi [0-32P]dATP following the protocol provided with the Invitrogen Random Primers DNA Labeling System, and purified by Chroma Spin column (BD Biosciences). The probe was hybridized to the membrane overnight at 65 0C following a standard protocol; the membrane was washed and exposed on a Phospholmager overnight and then on X-ray film for 4 days (film image shown in figure). 55 Bglll Bglll/Nrul Nrul Scal/Nrul control DF5-20 control DF5-20 12508 bp 2067 bp 865 bp 747 bp 865 hp 3931 bp H ‘ 747 bp {-5 control DF5-20 control DF5-20 A - Bgl l| digest 41339 bp Bgl Il/Nru ldouble digest 2067 b Nru I digest g 12508 bp V Wh- Sca | Bgl ll Nru l Bgl ll Nru l 56 Sea lggest Bgl n control Scal o ‘1‘ in u. D Sca I —— Superscript II (Invitrogen) reverse/transcription protocol for cDNA synthesis; 5-8 ul template RNA, 1 ul of 0.5 ug/ul oligo(dT) primer, 1 ul of lO-mM dNTP, and 2 ul H20 were heated to 70°C for 10 min, were chilled on ice, and then were mixed and incubated at 42°C for 2 min after 4 ul of 5X first-strand buffer and 2 ul of 0.1M dithiothreitol was added. Addition of 1 ul Superscript II reverse transcriptase was followed by incubation at 42°C for 50 min, then incubation at 70°C for 15 min. A final incubation at 37°C for 20 min followed addition of 1 ul RN aseH. A negative control to test for genomic DNA contamination was generated for each sample by omitting the Superscript II and replacing it with 1 ul H20. These negative controls were carried through the assays side by side with the corresponding sample. In no instance was product observed for these controls (data not shown). We designed a cDNA-specific PCR assay to assess the relative abundance of 35delG and non-35delG product amplified from GJB2 cDNA synthesized from 35delG heterozygotes (Figure 2-5A). A standard PCR-based assay for the identification of 35delG in genomic DNA (Wilcox, Osborn, & Dahl, 2000) is based on the introduction of a restriction site for BstNl by a 3' mismatch primer (5'- GCTGGTGGAGTGTTTGTTCACACCCGC-3') that overlaps the run of Gs between nt30 and nt35. The BstNl restriction site is present in PCR product amplified from wild- type (non-35delG) alleles only and is absent in PCR product amplified from 35delG alleles. We paired a 5' primer located in exon 1 (5'- CGCAGAGACCCCAACGCCGAGA-3') with the 3' mismatch primer to generate a 139 57 Figure 2-5. Allele-specific expression assays for GJBZ. A, Schematic showing design of (F) located in the first, noncoding exon of GJB2 is separated on genomic DNA from the reverse mismatch primer (R), located in the coding region of GJB2, by a >3 kb intron (F ' = forward primer for genomic 35delG assay). The BstNl site is introduced into product amplified from wild-type (non-35delG [indicated by “+”]) template. B and D, Pedigrees including DF5-70 and DF5-20, heterozygous for both GJBZ 35delG and the novel pathogenic allele (black bar with star). Also indicated are the 35delG and +94 genotypes are also indicated. C and E, Results of cDNA-specific assay for GJBZ expression based on 35delG genotype. Products of 139 bp were amplified from all tested family members (ud = undigested, no enzyme added, d = addition of restriction enzyme). DF5-20, -70, and -71 (normal-hearing sibling of DF5-70) are all heterozygous for 35delG; however, after digestion with BstNl, only DF5-71 clearly shows both 139-bp and 110-bp products, indicating representation of two different alleles at the cDNA level. DF5-20 and DF5-70, who both bear the novel pathogenic non-35delG allele, show either no or barely detectable 110-bp product, indicating underrepresentation of this allele in the pool of amplified product (M = Marker V [Roche]). F, Results of cDNA-specific assay for GJBZ expression based on genotype at GJB2 +94. Products of 139 bp (ud=undigested) were amplified from both tested family members, who are both heterozygous (A/C) at +94. Although theldigested product (d) amplified from DF5-72 cDNA clearly indicates heterozygosity at the cDNA level, lack of any detectable 139-bp product in the digested product of DF5-67 indicates underrepresentation of the novel (C) allele among the amplified product. 58 A F R F' «- -fj - a\\\\\\\\\\\\\s- BseY1+site 139 bp PCR product from cDNA: L f BstN1 site i BseY1 site BstN1 site (+94 assay) (35delG assay) B DF5-68 DF5.59 35AG E E I+ [1+ M DF5-68 DPS-69 DF5- 70 DF5- 71 +AGAG ”uddudduddud --- --‘afi139bp(AG) DF5~70 DF5—71 =5“ - 4' 110 bp (...) 124 bp ‘ ' 104 bp * ‘* AG + AG + D DF5-72 DF5-20 M DF5-20 +/+ +94+ICEA bIC EC 124b wild 6&3 "an 3510 104 hp: ...... l 110bp(+) .,.. . W} DF5—65 DPS—666) DPS-67 C A C A C ICI a E E I F DPS-67 DF5-72 + + + AG + + * * M ud d ud d 124hp.,,_, I- Q. *139bp(C) 104 ”9"“ up - 4109 bp (A) Figure 2-5 59 bp PCR product from cDNA template only, which, when incubated with BstN1 will yield digestion products of 110 bp and 29 bp only if the 35delG mutation is not present. For each individual we assayed, we amplified from cDNA template and from the corresponding negative control. A 20-ul PCR (1-5 [1] template cDNA, 2 pl 10X Qiagen buffer, 4 ul Q solution (Qiagen), 0.16 ul 25-mM dNTP (Invitrogen), 1 ul of each 20-uM primer, 5 U Invitrogen T aq DNA polymerase, and remainder H20) was denatured for 3 min at 94°C, followed by 40 cycles at 94°C for 303, 66°C for 305, and 72°C for 303, with a final extension at 72°C for 5 min. 0f each PCR, 10 ul was digested with ~10 U BstN1 (New England Biolabs) for >2h, and 3 ul of each reaction was run on a 3.5% NuSieve 3:1 agarose gel containing 0.3 jig/ml ethidium bromide. The results (Figure 2-5, B-E) for the two deaf 35delG heterozygotes that we assayed, DF5-20 and DF5-70, indicate that, although they are heterozygous for 35delG at the genomic DNA level, the barely detectable or absent 110 bp product indicates underrepresentation of GJBZ transcript from the novel (non-35delG) allele. Results from the parents and sibling of DPS-70 are consistent with their genotypes and provide appropriate controls. Unbiased amplification and digestion of both 35delG and non- 35delG alleles is shown by the result for DF5-7l, the sibling of DF5-70, who is also heterozygous for 35delG but carries a normal non-35delG chromosome. Sequencing of genomic DNA across the 139-bp target sequence (not shown) confirmed that there are no differences between these two individuals that would give rise to a bias in either amplification or digestion. To demonstrate that loss of expression of this allele is not unique to individuals carrying the 35delG mutation on their other chromosome 13, we developed a second 60 assay to determine allele-specific GJB2 expression in a normal-hearing adult offspring of DF5-20 bearing the novel haplotype (Figures 2-5A and 2-5D). DF5-67 carries a previously unreported exon 1 SNP at position +94. BseY1 digestion of the 139-bp product amplified with the same primers as described above will yield 102-bp and 27-bp products only if an A is present at position +94 (10 ul of each PCR product was digested with 5 U BseY1 enzyme [New England Biolabs] for >2 h, 5 pl 1% SDS was added, and electrophoresis was performed as above). Although both DF5-67 and ~72 are heterozygous for +94 at the genomic level, all of the product amplified from DF5-67 cDNA was digested (Figure 2-5F), indicating that no or very little of the PCR product had been amplified from template derived from the novel allele which bears the common variant, C. Again, sequencing of genomic DNA across the target sequence shows no differences between DF5-67 and DF5-72 that would yield biased amplification or digestion of the resulting amplimers. We hypothesized that loss of expression of GJB2 might be accompanied by loss of expression of GJB6, since these two genes lie within 30 kb of each other, and their products are coexpressed in the cochlea. DF5-65, a normal-hearing adult child of DF5- 20, bears the novel haplotype and is heterozygous for rs7333214 in the 3'UTR of GJB6, allowing us to test this hypothesis (Figures 2-6A and 2-6B). Using a forward primer located in the fourth of the noncoding exons (5'-CACCATTGGCTTCTAGGCAC-3') and a reverse primer located close to the 3' end of the 3'UTR (5'- CCACACTGTTCCGTCTACAT-3'), we amplified a 1,550-bp product from cDNA template only, in a 20-ul PCR under the following conditions: 2 ul of 10X Qiagen 61 Figure 2-6. Allele-specific expression assay for GJB6. A, Schematic showing design of prCH4 IV digestion assay for allele-specific expression of GJB6 mRNA using l-550- bp PCR product amplified from cDNA only. A forward primer (F) located in exon 5 of GJB6 is separated on genomic DNA from the reverse primer (R), located in the GJB6 3'UTR, by a >6 kb intron in addition to ~1,500 bp of the final exon. B, Pedigree showing rs 7333214 genotypes of assayed subjects. DF5-65 bears the novel pathogenic allele (black bar with star) and is heterozygous for rs 7333214. C, Results of cDNA- specific assay for GJB6 expression. Digestion of product amplified from DF5-72 yields 460-bp as well as 292-bp and 168-bp products, indicating that both GJB6 alleles are expressed. Digestion of product amplified from DPS-65 yields a robust 460-bp band, but the band at 292 bp is only barely visible. This indicates underrepresentation of the novel allele among the amplified product. 62 F R ‘ ‘ —El-Cl-l:l—D W- prCiH4/*V sites prCH4lV site rs7333214 . . If rs7333214=C 1550 bp PCR product from cDNA: _ . _ 460 bp + 292 bp + 168 bp /' '7' ' t K 537 bp 214 bp 338 hp 460 hp B C M DF5-65 DF5-72 DF5-72 DF5-20 rs7333214 -> A c c c 500 bp _’ - 5 '4—460 hp OT. 400 bP " “I- , ,. rs7333214=A DF565 . '\ 292 & 168 hp ...- — -/ rs7333214=C * Figure 2-6 63 buffer, 4 [.11 Q solution (Qiagen), 0.16 ul of 25-mM dNTP (Invitrogen), 1 ul of each 20- uM primer, 5 U Taq DNA polymerase (Invitrogen), and remainder H20 were denatured for 5 min at 94°C, followed by 50 cycles at 94°C for 305, 57°C for 305, and 72°C for 2 min, with a final extension at 72°C for 5 min. Digestion of this product for ~2h at 37°C with ~10 U prCH4 IV (New England Biolabs) yields digestion products of 537, 460, 338, and 214 bp if an A (the rare variant) is present at rs7333214; the 460-bp product is digested to 292- and 168-bp products if a C is present at rs7333214 (the common variant, carried, in this case, on the novel allele). Two microliters of each reaction was run on a 1.5% agarose gel (10 ug ethidium bromide/30ml) in 0.5X Tris-borate-EDTA buffer. Comparison of the result for DF5-65 with that for a control (DF5-72, his mother) who is also heterozygous for rs7333214 (Figure 2-6B) and whose DNA is identical across the target sequence, shows that the 292- and 168- bp products, derived from amplification of the novel allele, are significantly underrepresented in DF5-65 (Figure 2-6C). This is consistent with the interpretation that expression of GJB6 as well as that of GJBZ is diminished from the novel allele. Additionally, since the cDNA-specificity of each assay depends on the amplification of PCR product from only cDNA from which a long intron has been properly spliced out, failure to accumulate PCR product might also be expected from an allele that is mutant for proper splicing. It is highly unlikely that GJBZ and GJB6 on this chromosome both contain unidentified splice mutations. This study provides evidence that the MSU-DFS family is segregating a novel DFNB1 allele that is characterized by significant reduction in expression of both GJBZ and GJB6. This loss of expression is heritable, cis-acting, and not due to a simple parent—of-origin epigenetic modification. We have sought and found no coding-region 64 mutations, splice-site mutations, or any other sequence variant unique to the novel allele within or close to GJBZ or GJB6, suggesting that loss of function of an as-yet- unidentified cis-regulatory element(s) is responsible for our observations. It is possible that a locus-control region (LCR) regulates the coexpression of GJBZ and GJB6. An LCR was initially described in the B-globin locus, where it is absolutely required for expression of any of the genes in this cluster. Other examples include the growth hormone and TH2 cytokine loci (Dean, 2006). Such an element(s) may be located within the common interval deleted on chromosomes bearing del(GJB6-D13S1830) or del(GJB6-D13Sl 854). The severity of the hearing loss in the individuals in our kindred who are heterozygous for 35delG and this novel allele is similar to that of individuals who are del(GJB6-D13S1830)/35delG, who as a group have been shown to have more severe hearing loss than 35delG homozygotes (Snoeckx, et al., 2005). Beyond the identification of the basal promoters of both genes (Essenfelder, Larderet, Waksman, & Lamartine, 2005; Kiang, Jin, Tu, & Lin, 1997; Tu & Kiang, 1998), nothing is known of cis-acting elements that regulate GJB2 and GJB6. The common observation of an excess of deaf individuals bearing only a single identified GJB2 mutation strongly suggests that GJB2 regulatory mutations remain to be identified. ACKNOWLEDGEMENTS We thank the members of the MSU-DFS family for their generous participation, and Dr. Thomas Friedman, Dr. Robert Morel], and Dr. Patrick Venta for helpful discussion and critical reading of the manuscript. This research was supported by the Michigan State University Foundation and by National Institute on Deafness and Other Communication Disorders grant DC004568 (to K.H.F.). 65 WEB RESOURCES Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/0mim/ for Vohwinkel syndrome, Bart-Pumphrey syndrome, PPK, KID, hidrotic ectodermal dysplasia, and Pendred syndrome. 66 REFERENCES Ahmad, S., Chen, 8., Sun, J ., & Lin, X. (2003). Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice. Biochem Biophys Res Commun, 307(2), 362-368. Common, J. E. A., Bitner-Glindzicz, M., O'Toole, E. A., Barnes, M. R., Jenkins, L., Forge, A., et al. (2005). 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J ., Alvarez, A., Telleria, D., et al. (2002). A deletion involving the connexin 30 gene in nonsyndromic hearing impairment. N Engl J Med, 346(4), 243-249. Essenfelder, G. M., Larderet, G., Waksman, G., & Lamartine, J. (2005). Gene structure and promoter analysis of the human GJ B6 gene encoding connexin 30. Gene, 350(1), 33-40. Forge, A., Becker, D., Casalotti, S., Edwards, J., Marziano, N., & Nevill, G. (2003). Gap junctions in the inner ear: comparison of distribution patterns in different vertebrates and assessement of connexin composition in mammals. J Comp Neural, 46 7(2), 207-231. Forge, A., Marziano, N. K., Casalotti, S. 0., Becker, D. L., & Jagger, D. (2003). The inner ear contains heteromeric channels composed of cx26 and 0x30 and deafness-related mutations in cx26 have a dominant negative effect on cx30. Cell Commun Adhes, 10(4-6), 341-346. Grifa, A., Wagner, C. A., D'Ambrosio, L., Melchionda, S., Bemardi, F., Lopez-Bigas, N., et al. (1999). 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GJ B2 mutations and degree of hearing loss: a multicenter study. Am J Hum Genet, 77(6), 945-957. Tu, Z. J., & Kiang, D. T. (1998). Mapping and characterization of the basal promoter of the human connexin26 gene. Biachim Biophys Acta, 1443(1-2), 169-181. Wangemann, P. (2002). K+ cycling and the endocochlear potential. Hear Res, 165(1-2), 1-9. Wilcox, S. A., Osborn, A. H., & Dahl, H. H. (2000). A simple PCR test to detect the common 35delG mutation in the connexin 26 gene. Mal Diagn, 5(1), 75-78. 68 CHAPTER 3 Wilch, E., Azaiez, H., Fisher, R. A., Elfenbein, J., Murgia, A., Birkenhager, R., et al. (2010). A novel DFNB1 deletion allele supports the existence of a distant cis- regulatory region that controls GJB2 and GJB6 expression. Clin Genet, 78(3), 267- 274. 69 ABSTRACT Eleven affected members of a large German-American family segregating recessively inherited, congenital, non-syndromic sensorineural hearing loss (SNHL) were found to be homozygous for the common 35delG mutation of GJB2, the gene encoding the gap junction protein Connexin 26. Surprisingly, four additional family members with bilateral profound SNHL carried only a single 35delG mutation. Previously, we demonstrated reduced expression of both GJBZ and GJB6 mRNA from the allele carried in trans with that hearing the 35delG mutation in these four persons. Using array comparative genome hybridization (array CGH), we have now identified on this allele a deletion of 131.4 kb whose proximal breakpoint lies more than 100 kb upstream of the transcriptional start sites of GJBZ and GJB6. This deletion, del(chrl3:l9,837,344- 19,968,698), segregates as a completely penetrant DFNB1 allele in this family. It is not present in 528 persons with SNHL and monoallelic mutation of GJBZ or GJB6, nor have we identified any other candidate pathogenic copy number variation by arrayCGH in a subset of 10 such persons. Characterization of distant GJB2/GJB6 cis-regulatory regions evidenced by this allele may be required to find the ‘missing’ DFNBI mutations that are believed to exist. KEYWORDS connexin 26, connexin 30, DFNB1, gene expression regulation, GJBZ, GJB6, sensorineural hearing loss, sequence deletion 7O INTRODUCTION Mutation in the Connexin 26 (Cx26) gap junction protein-encoding gene GJB2 (MIM 121011) is the most commonly identified cause of congenital, recessively inherited, sensorineural nonsyndromic hearing loss (DFN BIA [MIM 220290]) (Hilgert, Smith, & Van Camp, 2009; Kenneson, Van Naarden Braun, & Boyle, 2002). In the inner ear, Cx26 and Cx30 (encoded by GJB6 [MIM 604418], which lies 30 kb telomeric to GJBZ on human chromosome 13) are expressed in non-sensory cells of the organ of Corti and in cells of the spiral ligament and stria vascularis. Cx26/Cx30 cochlear gap junctions have been implicated in maintenance of K homeostasrs 1n the inner ear (Kikuchi, Kimura, Paul, Takasaka, & Adams, 2000; Wangemann, 2002). However, more recent studies point to a complex suite of functions for gap-j unctional networks throughout the cochlea involving participation of second messengers and including generation of the endocochlear potential (Beltramello, Piazza, Bukauskas, Pozzan, & Mammano, 2005; Teubner, et al., 2003; Zhao, Yu, & Fleming, 2005). This intricate set of roles depends on a varied distribution and composition of connexon hemichannels and gap junctions throughout the epithelial supporting cell and connective tissue gap-junctional networks of the cochlea (Ahmad, Chen, Sun, & Lin, 2003; Forge, Becker, et al., 2003; Forge, Marziano, Casalotti, Becker, & Jagger, 2003; Jagger & Forge, 2006; Zhao & Yu, 2006), therefore requiring tight regulation of expression of GJBZ and GJB6. Although more than 200 mutations of GJBZ, two deletions involving GJB6 (DFNBI B [MIM 612645]), and one deletion encompassing both GJBZ and GJB6, constitute the reported set of DFNB1 mutations (F. J. del Castillo, et al., 2005; 1. del Castillo, et al., 2002; Feldmann, et al., 2009; Hilgert, Smith, et al., 2009), population 71 screening commonly yields an excess of individuals with hearing loss who carry only a single identified GJBZ mutation or DFNB1 deletion (Azaiez, et al., 2004). This finding strongly suggests that additional mutations that lie outside of the proximal promoter and transcribed regions of GJBZ remain to be identified. Here we identify a novel 131.4-kb deletion, distant from the transcriptional start sites of both GJBZ and GJB6, that segregates as a DFNB1 allele in the extended family in which it is found, and that also segregates with reduced expression of either GJBZ or GJB6 message in four family members assayed. This finding has relevance for the identification of distant GJBZ and GJB6 cis-regulatory elements, as sequence variations within these elements may prove to. constitute the bulk of ‘missing’ GJB2 mutations in DFNBI hearing loss. MATERIALS AND METHODS Human subjects Participating MSU-DF5 subjects were ascertained through the Oyer Speech- Language-Hearing Clinic within the Department of Communicative Sciences and Disorders at Michigan State University. Audiologic examination of family members included otoscopy, tympanometry, and pure-tone and air- and bone-conduction thresholds. DNA was isolated from blood, saliva, or cheek swabs. Genealogical information was collated from family histories and from publicly available records. Additional subjects were ascertained from hearing loss referrals to the Molecular Otolaryngology Research Laboratories at the University of Iowa. Written informed consent for genetic and audiological testing was obtained from all participants and from the parents of minors. The Michigan State University Institutional Review Board and the University of Iowa Human Subjects Committee approved all procedures. 72 Array Comparative Genome Hybridization To examine potential cis-regulatory regions for mutation, we looked first for copy number variants (CNVs) by array comparative genome hybridization (array CGH) using a finely tiled CGH microarray designed and manufactured by Nimblegen Systems. Included on the array were 384,000 overlapping oligomeric probes of 45-80 nucleotides in length from regions of non-repetitive sequence on human chromosome 13q11-12, spanning ~6.5 Mb from the most centromeric annotated sequence at chr13:18,000,000 to chr13:24,500,000 (Build 36.1). We provided Nimblegen with DNA from MSU-DF5-20, one of the four deaf family members with monoallelic mutation of GJB2. Nimblegen carried out the labeling and hybridizations on MSU-DF5-20 as well as on DNA from 10 additional unrelated GJB2 heterozygotes with severe-to-profound non-syndromic sensorineural hearing loss (SNHL). Identification of the breakpoints of del(chrl 3:19,837,344-19,968,698) was done by bidirectional Sanger sequencing of the 628 bp polymerase chain reaction (PCR) product amplified with primers flanking the region of copy number loss (see the following section). Confirmation of regions of deletion and duplication in the additional GJBZ heterozygotes and screening in 160 Centre d’Etude du Polymorphisme Humain (CEPH) controls was done by PCR with flanking primers; these products were not sequenced for breakpoint identification. Deletion screening by PCR assay To screen for del(chrl3:19,837,344-19,968,698), we designed a two-product multiplex PCR assay. Two primers (5'-TGGGACCAGGTCTGTTGTT-3' and 5'- ATTGCGACTTGCTTTTCGTT-3') flank the deletion breakpoint and amplify a 628-bp 73 product from genomic DNA bearing the deletion; the second primer pair (5'- GCAGCCATCTCATGGTCTCT-3' and 5'-CCAACACAATTGGGTCACTCT-3') amplifies an 836-bp product from sequence within the deleted interval. In a 20-pl reaction containing 1.5 mM MgCl2, 25 uM primer, 0.2 mM dNTP, and 0.5 U Taq polymerase, genomic DNA (10-40 ng) was denatured for 5 min at 94°C, followed by 35 cycles of 94°C for 30 s, 66°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 7 min. Products were resolved on a 1% agarose gel containing 0.3 ug/ml ethidium bromide. DNA from 220 MSU-DFS family members, 160 CEPH controls, and 528 patients with hearing loss who are heterozygous for a known GJBZ/GJB6 mutation was assayed. RESULTS Identification of del(chrl3:19,837,344-19,968,698) in an extended family segregating DFNB1 hearing loss Figure 3-1 shows a small portion of the pedigree of MSU-DF 5, an extended family descended from a founder community of 500 German-Catholic emigrant families who settled in Michigan in the 19th century. Eleven living family members with hearing loss are known to be homozygous for the common European founder mutation of GJBZ, 35delG (only one of those individuals is included in Figure 3-1). Unexpectedly, four other family members with bilateral profound sensorineural hearing loss (SNHL) carry only a single 35delG mutation but share the same haplotype on their non-35delG chromosome. Previously, we documented allele-specific reduction of GJBZ and GJB6 expression from this allele in buccal cells from four heterozygous individuals (two deaf, 74 Figure 3-1. The MSU-DF5 subpedigree shown here includes the four profoundly deaf family members who are compound heterozygotes for del(chrl3:l9,837,344-19,968,698) (green shading) and for the 35delG mutation of GJBZ (blue shading) which segregates within this family. 0f the 220 family members screened, 27 unaffected persons are heterozygous for the deletion and are negative for 35delG; they are shown here, although a number of unaffected siblings who are negative for both mutations are not. The most recent common ancestors of the known carriers of del(chrl3: 19,83 7,344-19,968,698) were born in the early 18th century in northern Europe. We previously documented reduced expression of GJBZ in three persons (red arrows) and GJB6 in one person (yellow arrow) from the allele now known to bear del(chrl3:19,837,344-19,968,698). 75 .- cum-e on- $0 w 2w E 2:5 _ = $-me $-me we. oLIn. w mate O._.D 2:8 2:8 Oru NEE ... m w% i csotm 26:03 .3229: F E um>mmmm .o_o__m 5:220 Eot nausea": commoaxo 09.0 a 3388 no: 2992?: m E uo>mmmm .o_o__m 8:23 So... nausea. commocaxo mmsw - o_o__m c3226 .2 026mm: ..Izw o_o__m 90.6mm --*at e_e__e 8:28 76 with 35delG in trans; two hearing, with normal GJBZ and GJB6 alleles in trans) (WILCH et al. 2006). Sequencing of the coding regions, 5' and 3' UTRs, proximal promoter regions, and splice sites of both GJB2 and GJB6, as well as several kilobases of sequence upstream of GJB2 failed to identify any other sequence variant unique to this low- expression allele. A single copy region identified by array CGH (Figure 3-2A) was confirmed by PCR and sequencing (Figure 3-2B) to be a deletion of 131.4 kb that is carried in trans with the 35delG mutation in the four deaf 35delG heterozygotes. The proximal breakpoint of this deletion is in intergenic sequence at chr13: 19,837,344 within a simple repeat of (TG)2I, leaving 9 TG dinucleotides intact on the deleted chromosome. The distal breakpoint occurs at chr13:19,968,698 within a long interspersed nuclear element located in the second intron of CR YLI . The sequence flanking the breakpoints does not clearly indicate a mechanism by which the deletion was generated. No homology exists between the sequences including and immediately adjacent to the breakpoints. The existence of only a single base pair of microhomology at the breakpoint junction is consistent with non- homologous end joining and double-strand break repair. Del(chr13:19,837,344-19,968,698) segregates as a completely penetrant DFNB1 allele and is >300 years old 0f the 220 MSU-DPS family members tested, 27 are heterozygous and none are homozygous for the deletion. The deletion is present on the chromosomes from which reduced expression of GJBZ and GJB6 message was documented (in MSU-DF5-20, -65, -67, and -70), and in all four deaf 35delG heterozygotes, indicating that the deletion- 77 Figure 3-2A. Characterization of novel DFNBI deletion allele del(chrl3:l9,837,344- l9,968,698). Custom array comparative genome hybridization (array CGH) across a 6.5 Mb region of human chromosome 13 indicates a region of single copy number loss in MSU-DF5-20. 78 ooodooéw 898on _ ooodoodm - firm 25E 0868.? _ 832% 802522 e. e. E 9 Z L 0 1' Z' Olinl OO'I 79 Figure 3-2B. Del(chr13: 19,83 7,344-19,968,698) breakpoint junctional sequence amplified from MSU-DF5-20 DNA and aligned with human genome reference sequence (Build 36.1) shows a single basepair of microhomology at the breakpoint. 80 m3. Beam. 500400000500an.n.0.3goo.5295o985m.oooaooaoaooaoooaoaoodogaoogaBananao mvumommw _______________________:____________________________________________________ 500500005009990550ooafiaaoooaogaoooaooaoaooa00055054950535.5990 omega 2... ____:_.__________.__ __ .22: _ _ __ _ _ .2... _ 40044090004000933350omagmaooosavannaBogoaooaooofimafiaaoaogoaggoa 8:me www.mmmdw adamafiadaaooodoogaogaoogfion.oooaooaon.owmaaaaaoafifiogaaofiaoaofifia $8me _______:2________________________________________ _ _ ___ 9400955490098ogaogeoogmooaoooaooa09090.55aoaoadaoaoaoaoaaooaoo0040 ombuo :. _ __ _ _ __________________._______________ 00009809969BUBBUBBOBBOBBOBUBOBOBUBOSURGE1090908090909.0909080809090998900005 m thmmmw a a a 328.2 02 $8888 586: 81 bearing chromosome segregates in this pedigree as a completely penetrant recessive DFNBI allele (Figure 3-1). This deletion was not identified in 160 CEPH controls. Under the assumption of identity-by-descent, and by choosing the least number of meioses to relate carriers, the deletion chromosome can be traced back to four individuals born in Germany between 1702 and 1723, all equally likely to have contributed this allele to the MSU-DPS pedigree. Although the deletion allele is thus at least 300 years old and may exist outside of this pedigree, it has not been identified among 528 persons with hearing loss, who are heterozygous for a single GJBZ/GJB6 mutation, from the United States, Brazil, Iran, and several European countries (Table 3-1). Assessment of 3SdelG-heterozygotes with SNHL for incidence of pathogenic copy number variation across the DFNBI locus To determine if deletions may constitute a frequent mechanism for DFNB1 deafness, we performed array CGH on DNA from 10 unrelated 35delG-heterozygous patients with severe-to-profound SNHL. We identified copy number variants (CNVs) across the region (Table 3-2), including 5 deletions ranging in size from 130 bp to 450 bp, and 3 duplications between 65 bp and 960 bp in size. Three regions were associated with either duplication or deletion. CNVs were confirmed by PCR with flanking primers, although the breakpoints were not precisely identified. All of these CNVs were present in 160 CEPH controls at frequencies higher than 20%. DISCUSSION Of the nearly 100 loci mapped for human inherited non-syndromic hearing loss since the early 19905, 46 genes have been identified with sufficient evidence to establish 82 Laboratory (Investigators) N Reported ethnicity or nationality USA/Iowa (Smith/Azaiez) 55 USA/Virginia (Pandya) 132 96 caucasian, 4 black, 25 hispanic, 7 other Spain (del Castillo) 50 Brazil (Sartorato/da Silva Costa) 40 France (Le Marechal) 41 Netherlands (Kremer/Hoefsloot) 27 Germany/Freiburg (Birkenhéiger) 62 Germany/Koln (Bolz/Kubisch) 10 Germany/Mainz 31 23 German, 5 Turkish, 2 Moroccan, l (Haaf/ Schneider) Spanish Italy (Murgia) 22 Belgium (Van Camp/Wgyts) 58 40 Belgian, 18 Iranian Total 528 Table 3-1. Number of affected individuals with monoallelic GJBZ/GJB6 mutation screened for del(chrl 3:19,837,344-19,968,698). Position* Variation Length* (bp) Chr13: 19686728-19686792 dup 65 Chr13: 19691 120-19691940 dup 820 Chrl3:19722020-l 9722070 del/dup 50 Chrl3:19749870-19750200 del 340 Chr13:19768000-19768130 del 130 Chr13219818780-19819200 del 420 Chr13: 19835210-19835420 del/dup 200 Chr13:19863060-19864020 del/dup 960 Chr13: 19934100-19934550 del 450 Chr13:19969740-19970700 dup 960 Chr13:19974330-19974480 del 150 CNV, copy number variant; del, deletion; dup, duplication. * Length and position are approximate. Table 3-2. List of CNVs in the DFNB1 region. 83 causation. Mutations in two of these genes, GJBZ and SLC26A 4, cause a significant proportion of hearing loss globally, with loss-of-function mutations of GJBZ estimated to be responsible for more than half of all recessively inherited SNHL in developed countries (Hilgert, Smith, et al., 2009). This knowledge has changed the medical evaluation of families segregating presumed autosomal recessive SNHL and has made accurate genetic counseling, recurrence chance estimation, phenotype-genotype correlations and prognosis for progression of hearing loss possible when two mutant alleles of GJBZ are identified. It is widely accepted that unidentified mutations of GJB2 exist that are cis-regulatory in nature. Deletion alleles that abrogate expression of GJB2 without disrupting the gene sequence are important for identifying candidate cis- regulatory regions for mutation analysis. The deletion we have described, del(chrl3:19,837,344-19,968,698), represents the fourth DFNB1 deletion allele, and the third in which GJBZ is left intact. Del(GJB6- D13SI830) (1. del Castillo, et al., 2002; Lerer, et al., 2001; Pallares-Ruiz, Blanchet, Mondain, Claustres, & Roux, 2002) and del(GJB6-D13S1854) (F. J. del Castillo, et al., 2005), deletions of 309 kb and 232 kb, respectively, truncate GJB6 and extend telomerically (Figure 3-2C). Lerer et a1. (Lerer, et al., 2001) showed GJB2 expression from both alleles in lymphocytes from a patient heterozygous for del(GJB6-D13S1830) and on this basis suggested that a digenic interaction between GJBZ and GJB6 may be responsible for the hearing loss in these individuals. Our allele-specific expression assay results on del(chrl 3:19,837,344-19,968,698), which suggest otherwise, were consistently replicated using stratified epithelial cell-derived cDNA from buccal swabs, whereas assays of lymphocyte-derived cDNA from the same individuals gave inconsistent results 84 Figure 3-2C. Del(chr13:19,837,344-19,968,698) overlaps with DFNBI deletions del(GJB6-Dl3Sl830) and del(GJB6-D1381854) over a 95.4 kb interval upstream of the transcriptional start sites (indicated by right-angled arrows) of GJB2 and GJB6. CR YLI is also shown. 85 N rodmmd Froowdhwdw .mEEE coEEoo 9. 3 hrtrmo A. .......... ... .......... . ............ .........V _.9_ _ __ _= 8035242689 amxov emso s. e __ ___ .7 I 9. «8 :8 5m 588:8 ox Fm? AmmodmmdwrmvmfimmdfimEozov e. mom 685206938 03 Bear... E 38.8 3868.2 amxov mmso flrl 86 (data not shown). This discrepancy may indicate that regulation of GJBZ and GJB6 differs with tissue type, and that expression assayed in lymphocytes may not be relevant to expression in cochlear cells. Common et al. (2005 ) also demonstrated loss of normal Cx26 expression from the deletion-bearing allele in stratified epithelial skin cells in an individual with hearing loss and a 35delG/del(GJB6-D13SI830) genotype. These results are consistent with hypotheses of complex cis-regulation of GJBZ and loss of an important GJBZ regulatory element as a result of these deletions. Qualitative loss of GJBZ expression from alleles bearing del(GJB6-D13S1830) in compound heterozygosity with GJBZ mutations/variants E47X, M34T, and V27I/E114G has since been demonstrated in three individuals (Rodriguez-Paris & Schrijver, 2009), further supporting this mechanism of action. Feldmann et al. (2009) have provided additional evidence against the GJB2/GJB6 digenic hypothesis. They have documented a >920 kb deletion encompassing GJBZ, GJB6, GJA3 and several other genes that is present in trans with the V84M mutation of GJBZ in a deaf individual. Three other persons with normal hearing in this pedigree carry this contiguous gene deletion, indicating that loss of single copies of GJB2 and GJB6, per se, cannot explain the pathogenicity of the two GJB6-truncating deletion alleles, del(GJB6-D13Sl830) and del (GJB6-D13S l 854). Del(chr13:19,837,344-1 9,968,698) is of particular interest as it leaves both GJB2 and GJB6 completely intact, has a proximal breakpoint substantially farther from GJB2 than the GJB6-truncating DFNB1 deletion alleles, and segregates with reduced expression of both GJBZ and GJB6. These findings suggest that time- and tissue-specific enhancer elements for both genes may lie a considerable distance upstream, and/or that a 87 locus control region exists for these two genes. We hypothesize a similar mechanism of pathogenesis for all three deletion alleles that involves, in addition to loss or reduction in expression of GJB6, the loss of a critical cis-regulatory element for GJB2 located within the common 95.4 kb genomic interval that is deleted in all three alleles. Sequence conservation between species is one metric by which candidate non-coding regulatory DNA may be identified. Human-mouse sequence conservation and rank VISTA (rVISTA) calculations of potential regulatory function (Brudno, et al., 2003; Loots, Ovcharenko, Pachter, Dubchak, & Rubin, 2002) (Figure 3-2D), reproduced from the VISTA browser) show numerous candidate regulatory elements within this interval. Because we found del(chrl3:19,837,344-19,968,698) in only one extended family, as an alternate, less parsimonious hypothesis, del(chrl3:l9,837,344-l9,968,698) could be in linkage disequilibrium with a pathogenic single nucleotide substitution or other undetected DNA lesion closer to both GJBZ and GJB6 that disrupts cis-regulatory function of both genes coordinately. This possibility also permits a common mechanism of pathogenesis of the three deletion alleles which leaves GJB2 intact, and accommodates the evidence of loss of GJBZ/Cx26 expression observed from the del(GJB6/D13S1830) allele by Rodriguez-Paris et a1. (2009) and by Common et al. (2005). The identification of additional deletions is possible with the increasing accessibility of array CGH and other techniques to assess CNV. Although patients with severe-to-profound hearing loss and monoallelic GJBZ mutation are candidates for 88 Figure 3-2D. Upper VISTA track shows sequence conservation between human (March 2006) and mouse (July 2007) genomes over the 95.4-kb common deletion interval indicated in Fig. 2C; pink-colored peaks represent regions of 100+ bp of non-coding sequences conserved at 270% identity. Lower track shows ranleSTA (rVISTA) predictions (-logi0[P-value]) of potential regulatory function based on multispecies sequence conservation combined with identification of transcription factor binding sites (http://genome.lbl.gov/vista/index.shtml). 89 :3” 2:5 $58.2. $63.3 xmdvmdw xm.mmm.m_. xmdwmdr 0.02.de ed 4 — _ _ _ . . om - $8 .. _ __ _. . ., _ _. _ ;__ jliljalgiwdjlni $03 ul.I-IIEI- éégHflHmflliiwlfl .. ...Iiniwiri.n§a§.: I F 1 - EEG t xmxmmdr xmkmmdr xwfinmdr xmsowdr xwkmwdv xmfivmdw 0.0 fl «.— u__ d “J? ~‘L . 90 Figure 3-2D. Upper VISTA track shows sequence conservation between human (March 2006) and mouse (July 2007) genomes over the 95.4-kb common deletion interval indicated in Fig. 2C; pink-colored peaks represent regions of 100+ bp of non-coding sequences conserved at 270% identity. Lower track shows rankVISTA (rVISTA) predictions (-log.0[P-value]) of potential regulatory function based on multispecies sequence conservation combined with identification of transcription factor binding sites (http://genome.lbl.gov/vista/index.shtml). 89 od o.m e\eom $00? EA sear.— xmdomdr xmdmmdv 0593.9 xmdmmdr xm.m~o.m_. xmarmdr _ fl - — _r u .. H _. _ _. _ ....W. _ ...fi. w j‘qaltjl L J_ xm.3m.9 xmsmmdr xmfibdr cascade stmmdw 05.5%.? . . _ J . n n. .1 . _. a... ._._.2_ _ _ .1 .1 ; 74114134111. .. 11. Elli -l l l 11 1111.41.-- 1 ll 90 pathogenic CNV, our findings from 10 such patients suggest that while CNV is common, identifying the regulatory regions may require an alternative approach such as enhancer- reporter assays. The CNVs we found among our 10 patients were all found among CEPH controls at frequencies higher than 20%. Sequence variation within regulatory elements can give rise to allele-specific quantitative differences in expression of message that may yield phenotypic variation (Cowles, Hirschhorn, Altshuler, & Lander, 2002; Knight, 2004). Variability in degree of hearing loss is a well-documented feature of GJBZ deafiress, even among persons with biallelic null (protein-truncating) mutations that completely abolish Cx26 protein expression (Azaiez, et al., 2004; Snoeckx, et al., 2005). No modifier genes have yet been identified to explain this phenotypic variability (Hilgert, Huentelman, et al., 2009). We hypothesize that sequence variation in cis that regulates GJB6 expression from a distance may be partially responsible for some of this variability. In summary, our studies of del(chrl3:19,837,344-19,968,698) support the presence of distant cis-regulation of GJB2 and substantially reduce the span of chromosome 13 that is most strongly implicated in this function. We also provide the first evidence of distant cis-regulation of GJB6. Additional efforts to elucidate and characterize distant GJBZ and GJB6 cis-regulatory regions are clearly warranted. ACKNOWLEDGEMENTS We are grateful to the many MSU-DF5 family members for their participation. This work was supported by NIDCD grant DC004568 to KAF, an MSU Foundation grant to RAF and a Families and Communities Together Coalition grant to J E . RJHS is the Sterba Hearing Research Professor, University of Iowa College of Medicine, who supported the project in part 91 with National Institutes of Health (N IH)-National Institute on Deafness and Other Communication Disorders (N IDCD) grant DC02842. EW thanks Dr. Patrick J. Venta for critical reading of the manuscript. ABBREVIATIONS Array CGH array comparative genome hybridization CEPH Centre d’Etude du Polymorphisme Humain CNV copy number variant CX26 Connexin 26 CX30 Connexin 30 DSB double-strand break LINE long interspersed nuclear element NHEJ non-homologous end joining PCR polymerase chain reaction SNHL sensorineural hearing loss UTR untranslated region 92 REFERENCES Ahmad, S., Chen, S., Sun, J., & Lin, X. (2003). Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice. Biochem Biophys Res Commun, 307(2), 362-368. Azaiez, H., Chamberlin, G. P., Fischer, S. M., Welp, C. L., Prasad, S. D., Taggart, R. T., et al. (2004). GJB2: the spectrum of deafness-causing allele variants and their phenotype. Hum Mutat, 24(4), 305-311. Beltramello, M., Piazza, V., Bukauskas, F. F., Pozzan, T., & Mammano, F. (2005). Impaired permeability to Ins(1,4,5)P3 in a mutant connexin underlies recessive hereditary deafness. Nat Cell Biol, 7(1), 63-69. Brudno, M., Do, C. 8., Cooper, G. M., Kim, M. 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J ., Alvarez, A., Telleria, D., et al. (2002). A deletion involving the connexin 30 gene in nonsyndromic hearing impairment. N Engl J Med, 346(4), 243-249. F eldmann, D., Le Marechal, C., Jonard, L., Thierry, P., Czajka, C., Couderc, R., et al. (2009). A new large deletion in the DFNB1 locus causes nonsyndromic hearing loss. Eur J Med Genet, 52(4), 195-200. Forge, A., Becker, D., Casalotti, S., Edwards, J ., Marziano, N., & Nevill, G. (2003). Gap junctions in the inner ear: comparison of distribution patterns in different vertebrates and assessement of connexin composition in mammals. J Comp Neural, 46 7(2), 207-231. 93 Forge, A., Marziano, N. K., Casalotti, S. 0., Becker, D. L., & Jagger, D. (2003). The inner ear contains heteromeric channels composed of cx26 and cx30 and deafness-related mutations in cx26 have a dominant negative effect on cx30. Cell Commun Adhes, 10(4-6), 341-346. Hilgert, N., Huentelman, M. J., Thorburn, A. 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Proc Natl Acad Sci U S A, * 102(51), 18724-18729. 1:- 95 CHAPTER 4 Enhancer assays of conserved DNA elements in the 131.4 kb MSU-DFS deletion interval 96 INTRODUCTION Mutations in GJBZ, the gene encoding the gap junction protein connexin 26 (Cx26), are the most frequent cause of congenital deafness globally. GJBZ is a small gene that is easy to screen for coding region, splicing, and basal and proximal promoter mutations by direct sequencing. Occupying about 5 kb of genomic sequence on human chromosome 13q12, the gene consists of a 116 bp noncoding exon, a 3.2 kb intron, and a 5 second exon containing 22 bp of 5' UTR sequence, the entire 678 bp coding sequence, and 1.4 kb of 3' UTR sequence. Over 200 mutations causing recessively inherited “1r hearing loss have been identified in GJBZ (Hilgert, Smith, & Van Camp, 2009); fewer than two dozen other mutations are reported to cause dominantly inherited hearing loss or syndromic forms of hearing loss that include various symptoms of skin dysfunction. The DFNBI locus also includes two other connexin genes, GJA3 (Cx46) and GJB6 (Cx30). Cx46 is expressed in lens only (Gerido & White, 2004) and several mutations of GJA3 have been associated with dominantly inherited cataract. Like Cx26, Cx30 is also expressed in the cochlea and in stratified skin epithelial cells. In the cochlea, the two connexins have overlapping, although non-identical, expression patterns, and are known to co-assemble into heteromeric connexons (Ahmad, Chen, Sun, & Lin, 2003; Zhao & Yu, 2006). Hemichannels of heteromeric Cx26/Cx30 connexons have been shown to have gating and transport properties that differ from homomeric connexons of either Cx26 or Cx30. In spite of this intimate co-expression in the cochlea, the mutation spectrum of GJB6 differs considerably from that of GJB2. A single missense mutation of GJB6 is associated with dominantly inherited hearing loss; three 97 missense mutations cause Clouston syndrome (MIM129500), a hair-nail dystrophy without hearing loss. With the notable exception of the two deletions discussed below, del(GJB6-D1381830) and del(GJB6-Dl3Sl854), no mutations of GJB6 are associated with recessively inherited nonsyndromic hearing loss. It has been apparent for some time that many people with hearing loss have only monoallelic mutation of GJBZ, more than can be expected to be incidental carriers of a GJBZ mutation with hearing loss of a different etiology. The carrier frequency of GJBZ mutations varies with population, but can be as high as 3-4%; the proportion of individuals with hearing loss who are found to have monoallelic GJBZ mutation is not uncommonly 10% or higher (Kenneson, Van Naarden Braun, & Boyle, 2002). As it is relatively straightforward to thoroughly screen the GJB2 gene itself, it is generally believed that additional, regulatory mutations of GJBZ must exist within the DFNB1 locus, but at some distance from the gene itself; that is, beyond the one to several kilobases of upstream sequence that is typically interrogated when sequencing efforts fail to turn up a coding region mutation. For other genes, the catalog of such mutations has been growing substantially over the last several years. Four large deletions have been identified that segregate as DFNB1 mutations. Two of these, del(GJB6-Dl 3S1830) and del(GJB6-Dl3Sl824), are deletions of 309 kb and 232 kb, respectively, that both truncate the 5' end of GJB6 and extend telomerically to include several 3' exons of CR YLI, and leave GJBZ sequence intact (F. J. del Castillo, et al., 2005; 1. del Castillo, et al., 2002; Lerer, et al., 2001; Pallares-Ruiz, Blanchet, Mondain, Claustres, & Roux, 2002). A hypothesis of digenic inheritance with GJB2 was 98 made on the basis of results of allele-specific expression assayed in peripheral leukocytes (Lerer, et al., 2001). Those authors detected expression of GJBZ from the del(GJB6- D1381830) allele, contradicting the hypothesis that this allele leads to loss of function of GJBZ. The characterization of two additional deletions at this locus (F eldmann, et al., 2009; Wilch, et al., 2010) along with two studies of the del(GJB6-D13Sl830) mutation in particular (Common, et al., 2005; Rodriguez-Paris & Schrijver, 2009) together contradict the hypothesis of digenic inheritance of GJBZ and GJB6. Firstly, the 131.4 kb deletion characterized by our lab (Wilch, et al., 2010; Wilch, et al., 2006) overlaps with the two deletions previously described, but has a proximal breakpoint more than 140 kb distant from the transcriptional start site of GJB6, thus leaving both GJB6 and GJB2 sequences intact. We demonstrated that this deletion segregates with low expression of both GJBZ and GJB6 mRNA, by qualitative allele-specific expression assays on buccal cell cDNA. This finding suggests that one or more regulatory elements for GJB2 are disrupted within this deleted interval, at least 170 kb upstream of the transcriptional start site of GJBZ. Similar assays on persons bearing the del(GJB6-D13S1830) allele heterozygously with coding-region variants of GJBZ also indicated reduced expression of GJBZ mRNA from the deletion allele (Rodriguez-Paris & Schrijver, 2009). This result is consistent with that of Common et al. (2005), who showed by immunohistochemistry lack of Cx26 protein expression in certain stratified epithelial skin cells in a person with hearing loss who is a compound heterozygote for del(GJB6-D13S1830) and the 35delG null mutation of GJBZ. Finally, 8 >920 kb deletion that removes all three of the connexin genes at this locus was recently reported (F eldmann, et al., 2009). In the pedigree in which this deletion was 99 identified, the proband carried the deletion in trans with the known pathogenic V84M mutation of GJBZ, and had been profoundly deaf since infancy. Three family members with normal hearing were also found to be carriers of the deletion. This finding contradicts a model of digenic inheritance, as these carriers have lost one allele of GJBZ and one allele of GJB6, albeit in cis, but maintain normal hearing. Expression in leukocytes assayed by Lerer et al. (2001) may not be relevant to expression in cochlear or skin cells, and may explain this single experimental result supportive of digenic inheritance. Thus, it is appropriate to identify and test candidate enhancer elements within the deleted intervals of the three deletions that leave GJBZ intact. Here, I report on the results of luciferase enhancer assays on several conserved elements within the 131.4 kb deletion region. Among the elements assayed, two were found to have enhancer activity, driving expression of the luciferase reporter under the control of an SV40 promoter. Three elements showed silencer activity. Two constructs, differing only by one nucleotide within the 702 bp sequence, yielded allele-specific differences in expression. These two constructs also showed cell-specific differences in expression. METHODS Construction of pGL3 luciferase reporter plasmids Six sets of primers were designed to amplify 700-1200 bp candidate sequence elements (Table 4-1). Candidate sequences were chosen from among a set of human- mouse conserved sequences (at least 100 hp at 68% or greater identity, over a 100bp sliding window) tabled from Vista (http://pipeline.lbl.gov) (Table 4-2). Ideally, all 100 .100 bp) in the region spanned by the three DFNB1 deletions that leave GJ2B intact. Location 1 Lenth, hp I % identity, human-mouse Conserved elements common to del(GJB6—D13S1830) and del(GJB6-D1331854) 19730166-19730257 100bp 70.00% 19730790-19730905 120bp 71.70% 19737446-19737741 297bp 75.10% 19740981-19741092 1 14bp 72.80% 19741260-19741361 103bp 71.80% 19742396-19742479 85bp 75.30% 19779910-19780063 158bp 74.10% 19780200-19780292 94bp 75.50% 19828527-19828667 141bp 76.60% Conserved elements common to all three deletions, intergenic 1983 8242-1 983 8339 98bL 70.40% 19855245-19855410 166bp 68.70% 19858147-19858257 lllbp 73.90% 19875532-19875648 1 17bp 69.20% Conserved elements common to all three deletions, intronic (CR YLI) 19883338-19883590 (included in A) 254bp 81.10% 19886680-19886878 201bp 74.60% 19892138-19892264 (included in B) 127bp 71.70% 19898889-19899008 (included in C) 124M) 69.40% 19899273-19899387 (included in C) 116bp 71.60% 19910910-19911007 98bp 75.50% 19912961-19913076 117bp 70.10% 19914056-19914459 (included in D) 405bp 76.80% 19916684-19916796 1 14bp 75.40% 19919824-19919988 1656p 70.30% Conserved elements common to MSU-DFS deletion and del(GJB6—D13S1830), intronic (CR YLI) 19934455-19934749 (included in E) 2981);) 73.20% 19935030-19935118 (included in E) 89bp 80.90% 1 993 72 1 6-1 993 73 99 189bp 69.80% 19937556-19937663 1 13bp 70.80% 19941365-19941585 225bp 70.20% 19941620-19941709 97bp 71 .10% 19948667-19948762 98bp 71.40% 19954587-19954745 (included in F) 159bp 74.80% 19966012-199661 15 1059p 71.40% 19966281-19966370 100bp 70.00% 102 conserved sequences would have been included in candidate enhancer constructs; however, difficulty with both amplification and cloning of candidate elements redefined this as a pilot project. Primers were designed with XhoI (each forward primer) or MluI (each reverse primer) recognition sequence added to the 5' ends, in order to clone the amplified product, in genomic orientation, into the multiple cloning site of the pGL3- Promoter vector (Promega Corporation). This vector (Figure 4-1) contains the SV40 promoter and modified coding sequence of firefly luciferase, as well as the E. coli ampicillin-resistance gene. Inserts were amplified with Pfi: polymerase with template DNA from MSU-DFS family members. Amplified DNA was digested overnight with XhoI and MluI, purified and concentrated by QiaQuick (Qiagen), ligated with T4 DNA ligase to XhoI/MluI-digested and QiaQuick-purified pGL3-Promoter at room temperature for 2 hours (NEB), and transformed into competent E. coli DHSa cells. Colonies grown on LB agar with ampicillin were screened by whole colony PCR for insert. Correct sequence was confirmed, and plasmid DNA for all transfections was purified by a single Promega Pure-Yield midiprep. The pGL3-Basic vector lacks the SV promoter, and was used in these experiments as a negative control. The pGL3-Control vector contains an SV40 enhancer element upstream of the SV40 promoter, and was used in these experiments as a positive control. The pRL-TK vector contains the coding sequence of Renilla luciferase. Cotransfection of pRL-TK with the pGL3 plasmid allows expression from the pGL3 vector to be corrected for transfection efficiency. 103 Figure 4-1. Schematic of pGL3-Promoter vector (Promega) 104 E. 950:. «m0? Bax qmom_maz mnm_ooz mvm EUEI +03 :9500. +02 no: .05.... 258 000_ov>m Ecumm 005 006an 00> _ m momm . a m 885$ 0m ___mm .225 mm sex I 56505.30: .5 mm :36 . E :22 9 3:2 C 606 to C m Bax 30:03.00. hE< 0:30.903 .0: 0:0 00:00 {11‘ acozqcomcmz \ B86 2:8 80550 105 Cell lines and cell culture conditions Human vaginal stratified epithelial cell line VK2/E6E7 (ATCC Catalog No. CRL- 2616) was obtained from ATCC. Cells were grown in keratinocyte serum-free medium (Gibco-BRL 17005-042) with 0.1 ng/ml human recombinant epidermal growth factor, 0.05 mg/ml bovine pituitary extract, and 44.1 mg/L calcium chloride. HaCaT cells, human skin keratinocytes, were obtained from a neighboring lab. Cells were grown in DMEM (Gibco 11965) with 10% fetal bovine serum and 100 U/ml penicillin- streptomycin at 37°C and 5% C02. Transfections and enhancer assays Cells were grown to 50-80% confluency in 40 ml medium in 96-well half-area tissue culture plates (Coming 3885). Each transfection mix consisted of 900 ng of the appropriate pGL3 plasmid, 90 ng pRL-TK plasmid, and 40 ul OptiMEM, in a final volume of 50 ul. This yielded a final concentration of 18 ng/ul of pGL3 plasmid. FuGENE6 transfection reagent (Roche) was added in a 6:1 ratio to each transfection mix, and each well transfected with 2.5 ul mix (~50 ng plasmid total). Transfections of all constructs were done at the same time, and all wells contained cells plated from the same cell preparation. Transfections of the pGL3-Basic and pGL3-Control vectors, as well as empty pGL3 -Promoter vector were also made at the same time. Cells were incubated for 24 hours, and firefly and Renilla luciferase expression assayed by DualGlo luciferase assay system (Promega), according to the manufacturer’s protocol. Six to nine replicate wells were transfected with each construct. Background luminescence was assayed from three to six untransfected wells. 106 The results given and discussed below are based on a single experiment, so do not account for variation introduced by error introduced in pipetting small volumes of plasmid into the transfection mix. The concentrations of plasmid stocks were determined by Nanodrop, and volumes of between 1.6 ul and 4.7 ul pipetted into each transfection mix to yield 18 ng pGL3 plasmid per ul of mix. Following one set of experiments, the same volumes of plasmid stocks were pipetted into water to make a final volume of 50 ul, and 5 ul of this run on a 5% agarose gel. The mass of DNA in each well was assessed on the Storm Imager (GE). Results are calculated and shown using both sets of mass calculations. RESULTS Over the genomic interval of our deletion, chr13:19,837,344-19,968,698, there are 24 intergenic or intronic, non-exonic, non-UTR conserved sequences (Table 4-2). Twenty of those sequences are located in CR YLI introns; four are intergenic. Fourteen sequences are common to the three DFNB1 deletion alleles that leave GJBZ sequence intact. Our deletion defines the proximal boundary of the ~94 kb common interval, at chr13:19,837,344, and the del(GJB6-D13Sl854) deletion defines the distal boundary of the common interval, around chr13:19,935,000 (Figure 4-2). There are nine conserved sequences in the 130 kb between GJB6 and the proximal boundary of the common deleted interval. Nine candidate enhancer constructs were made by cloning PCR-amplified sequence into the multiple cloning site of the pGL3-Promoter vector. Two constructs were made for each of three of the six genomic regions investigated. Two identical 107 Figure 4-2. Locations of candidate enhancer constructs. 108 N-.. 0.50:. r w r .. w r .r .. .. PJ}EU 00:00 .0092". $8. K $8.8 $8.8 $8.8 $3.8 $8. K $8.2 $8.8 $8.8 $8.: $8.8 $8.8 $0 :8 $3.8 $0 F. 8 $8. E $3. I $8.8 $8.8 $8. E $8.8 988332288905va 828. 828:8 £03 _mha 8%. :3 -80_ —80_ 82. 22050.0 30:05.0 0820ch _oooommmF _ooooommF “ n 8. 8 $8.8 $8.8 _ $90.8 Booomwmp ”m 2:0 0.00m 109 constructs were cloned separately for regions C and F; these two pairs of constructs (C1 and C2; F 1 and F2) serve as technical replicates. The two constructs B1 and B2 are an allelic pair; the 702 bp insert sequence is identical except for the nucleotide at SNP rs7320760. This is a highly polymorphic SNP that lies within the 127 bp human-mouse conserved sequence element. Expression assay results are shown in Figure 4-3, and p values are shown in Table 4-3. Student’s t-test was used for all statistical comparisons, and starred comparisons are significant at p<0.001. Stars above columns in Figure 4-3 indicate significant difference between the mean expression observed for that construct compared to mean expression from the empty pGL3-Promoter plasmid (“promoter control”). The region D element is 1168 bp in length. This element contains the longest human-mouse conserved sequence in 250 kb of sequence upstream of GJB6, 405 bp conserved at 76.8%. This is the only element that contains >100 bp of sequence conserved at >70% in chicken; about 250 bp are conserved above 70%. This element is located in the third intron of CR YLI, about 2 kb upstream of intron 4. In both cell lines assayed, this element displayed a repressor effect, with about a five-fold reduction in luciferase expression relative to the promoter-only construct in HaCaT cells, and a two- fold reduction in VK2/E6E7 cells. The region C construct is 979 bp in length, and its sequence is located in the fifth intron of CR YLI . This construct contains about 240 bp of conserved sequence. This element also displayed a repressor effect in both cell lines, a greater than two-fold reduction relative to the promoter-only constructs. Plasmid preps from two separate llO Figure 4-3. Expression assay results. 111 8'. m m n a m .I: e I e c * c T 2 a K a m v H u .m .m m n n o .m 0 C S .q. u a a G r r p m. D. E x E .....o. .m.............. 33.250: .mucmumoEEE >=2E 111.16 um~=mEEc .wucoummEEE >52: Figure 4-3 112 .8280 88:55 05 8 acmgafioo E .82-. 9.5me .3 38.3.8 mos—91. Ea £63850 52858 882980 388 mo £96. 3.82me 0353.. 5-9 93$. 3.: mode No.3 3.3 .928 .oucmccw mod m~.m NNé 3.2 .958 .BoEoE ~o.o .56 :6 mad 8mm Hooodv BA «Nd mvaod and Neda N”. Hooodv omd cod 800.0 mmd 3.3 E Hmmvd omd wwé Hnmwd mv.~ 3.3 m Hooodv nmd SA Soodv mma «md n. Hooodv mmd ofim Seed :4 cod 8 Hooodv «Nd mmé Hooodv SA mm: C Hooodv woa $6 806 m~.~ Ned N mv.33 m Hooodv mma moda Humod and 3.: H m..m..m m mommd Rd «.mfi 836 cm...“ 3.3 < 58:85 c2838. . 82.859. .3058; 5.83% . um~_.mE._oc .m> 8583.83. .m> 8588.25. 828:8 .m:_m> a 22.5% ammum>< .m:.m> a Emccmum omega. 2.8 Bmm\~x> c. cofimmaxm 2.8 ...mUm... 5 5.8296 ll3 region C clones were assayed in this experiment, and t-tests indicate that their means are not significantly different from each other. T-tests comparing expression mean of each region C construct against that of the promoter-only construct all yielded highly significant p values. The region C and region D constructs were the only constructs to show repressor activity consistently in both cell lines. The 791 bp region F construct contains conserved sequence that is located beyond the distal breakpoint of del(GJB6-D1 3S1854), so is deleted in only two of the three DFNBI deletion alleles. Two region F constructs were cloned, prepped and the sequences of the inserts confirmed to be identical. Both constructs showed enhancer activity. A nearly two-fold increase in luciferase expression was significant for both constructs in VK2 cells. Both constructs also showed enhancer activity in HaCaT cells, although the mean of expression of one construct, at about 1.5 times the level of the promoter-only construct, was only significant at p<0.03, which is not a sufficiently small p value for multiple testing. The most interesting pattern of activity was that of the region B constructs. These two constructs are an allelic pair, differing by a single nucleotide out of the 702 bp sequence. rs7320760 is a transition single nucleotide polymorphism located within the 127 bp of human-mouse conserved sequence. The B1 construct displays the major C allele, which is calculated to have a population allele frequency of 0.6-0.7, based on a set of European, Chinese, and Japanese genotypes. In HaCaT cells, the B1 construct had no effect on luciferase expression. However, the BZ construct had a significant repressor effect, with a 2 to 3-fold reduction relative to the promoter-only construct. Conversely, ll4 in VK2/E6E7 cells, the B] construct showed enhancer activity, with a 2-fold increase over the promoter-only mean. The B2 construct also showed enhancer activity, significant but perhaps modest; the p-value for one set of calculations (ImageQuant quantified plasmid amount) is 0.0035, above the cutoff for significance for multiple comparisons. In sum, these data indicate a real difference in expression between the two constructs, in each cell type. The data also indicate, for each of these two constructs, a real difference in expression between cell types. Two constructs, those of regions A and E, demonstrated no effect in either cell line. The conserved sequences represented in these constructs are among the longest and most conserved in the MSU-DPS deletion interval. The region A conserved sequence, 254 bp conserved at 81.1%, is common to all three deletions, and is located in intron 6 of CRYLI . The region B sequence is just telomeric to the distal breakpoint of del(GJB6- D1381 854), so is only included in two of the three deletion regions. The region E construct contains two conserved sequences, 298 hp at 73.2% and 89 hp at 80.9%, separated by about 250 bp of intervening sequence. DISCUSSION Hearing loss has complex etiology, and much congenital or prelingual hearing loss cannot be diagnosed based on any distinguishable audiological or anatomical characteristic. Determining the genetic or environmental etiology of hearing loss is important to decisions regarding therapies and to the early detection and management of other conditions that may present later than hearing loss in some syndromes. ll5 Mutations in GJBZ as the cause of hearing loss must be reliably ruled out in order to justify searching for less prevalent mutations in other genes associated with deafness. However, the inheritance and pathogenesis of DFNBI hearing loss is not completely understood. Many people with hearing loss carry monoallelic mutation of GJBZ at a significantly higher rate than the population allele frequency of those mutations. Two hypotheses can be made to explain this observation. The first is that unidentified mutations exist in cis-regulatory DNA, that some of these cis-regulatory elements may exist at a considerable distance from GJB2 itself, and disruption of one or more general or ear-specific enhancer elements by wholesale deletion or by small deletion or substitution of one or more critical nucleotides results in lost or reduced transcription of GJBZ. Very little of the regulatory landscape of GJB2 is known, beyond the proximal and basal promoter regions (Matos, et al., 2007; Tu & Kiang, 1998). The evidence of several large deletions that segregate as DFNB1 mutations now clearly shows that a cis- regulatory region exists, at a considerable distance from both GJBZ and GJB6. The alternative hypothesis is that hearing loss due to mutations in GJBZ may rarely be inherited as a dominant trait with incomplete penetrance. Since the 35delG null mutation is commonly the monoallelic mutation identified in this circumstance, this hypothesis requires that GJB2 is occasionally haploinsufficient in these heterozygous individuals. This hypothesis is conceivable, given the number and sophistication of molecular interactions involving Cx26 in hearing that are coming to be understood. These two hypotheses are not necessarily mutually exclusive. In fact, allelic variation in expression of GJBZ resulting from distant functional cis-regulatory variants on a non-mutant allele may contribute to this hypothesized occasional haploinsufficiency, in combination with ll6 variation at other loci that encode proteins that interact with Cx26, or that function in the same cell biological pathways. We have demonstrated sequence-specific and cell-type-specific enhancer and repressor activity in genomic sequences distal to GJBZ and GJB6, utilizing a luciferase reporter system and two epithelial cell lines that are confirmed to express endogenous Cx26 and Cx30. These experiments provide three important findings. First, there are sequences within the interval common to the three DFNB1 deletions leaving GJBZ intact that are capable of enhancer activity, and whose loss might therefore explain the pathogenicity of these deletions. Second, cross-species conservation is an appropriate criterion to use to identify candidate cis-regulatory elements. Four of six 700-1200 bp regions assayed demonstrated statistically significant up- or down-regulation of the luciferase reporter in one or both cell lines. Third, the experimental assay system is able to reproducibly detect enhancer or repressor activity in our hands. The pGL3 family of vectors includes three vectors for control transfections. The pGL3-Control vector includes SV40 enhancer and promoter elements in front of the luciferase coding region. Expression from this vector was on the order of 6-fold (in HaCaT cells) to 14-fold (in VK2/E6E7 cells) that of the promoter-only construct (pGL3-Promoter). Expression from the pGL3 —promoter vector was robust compared to that of the pGL3 -Basic vector, which lacks both enhancer and promoter sequences. Expression from this vector was always reliably close to zero. Assaying six to nine technical replicates transfected with each experimental construct (same transfection mix and same cell suspension in each of the six to nine “7 replicate wells) yielded reasonable standard deviations. T-tests comparing the means of expression of each construct against the mean of expression of the promoter-only construct were generally highly significant (p<0.0001). The expression means were sometimes statistically different between duplicate constructs (C1 and C2 are identical constructs that were cloned separately, as are F 1 and F2); however, the direction (up— vs. down-regulation) was never different, and significance of effects relative to the promoter- only construct was maintained in three of four comparisons. This experiment leaves some important questions unanswered, but suggests means of redress. First, of the sequences with identified transcriptional regulatory effect, do any interact with the GJB2 or GJB6 promoter in vivo? Our lab has been supplied with the PGL3-Basic vector with the GJB2 promoter cloned in (gifi of Dr. Hela Azaiez and Dr. Richard Smith, University of Iowa), so that these and other candidate regulatory elements can be assessed on their ability to drive expression of the luciferase gene under the control of the GJB2 promoter. Ultimately, an experimental approach such as 3C (chromosome conformation capture) may be best suited to answer this question. This technique preserves and visualizes interactions that bring regions of chromatin into close contact. First, DNA and proteins are cross-linked in cells expressing the protein of interest, and the DNA is then fragmented by restriction digest and religated; PCR products that result from amplification across ligation junctions represent close interactions between those genomic regions, such as looping and contact via transcription factors between enhancer elements and basal or proximal promoter elements. 118 Connexins 26 and 30 are each expressed in a number of tissues. In both inner ear and skin, where each of these proteins has been most closely investigated, expression patterns are intricate and very cell-type specific. An important question is whether any sequence shown to have transcriptional regulatory activity drives expression of either protein in any cells of the cochlea itself. As no human cochlear-derived immortalized cell line has yet been developed, this is a question that cannot be answered in cell culture experiments. One approach is that of recombineering of BAC transgenes containing putative regulatory elements, such as that used to localize tissue-specific enhancers of GDF 6 (Mortlock, Guenther, & Kingsley, 2003; Portnoy, et al., 2005). In summary, these experiments suggest that some elements within the 131.4 kb deletion interval may have regulatory fimction. We have not yet demonstrated specific, in vivo interactions between these elements and the GJBZ and GJB6 promoters. ll9 REFERENCES Ahmad, S., Chen, S., Sun, J ., & Lin, X. (2003). Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice. Biochem Biophys Res Commun, 307(2), 362-368. Common, J. E., Bitner-Glindzicz, M., O'Toole, E. A., Barnes, M. R., Jenkins, L., Forge, A., et al. (2005). Specific loss of connexin 26 expression in ductal sweat gland epithelium associated with the deletion mutation del(GJB6-D13S1830). Clin Exp Dermatol, 3 0(6), 688-693. del Castillo, F. J ., Rodriguez-Ballesteros, M., Alvarez, A., Hutchin, T., Leonardi, E., de Oliveira, C. A., et al. (2005). A novel deletion involving the connexin-3O gene, del(GJB6-d1331854), found in trans with mutations in the OJ B2 gene (connexin- 26) in subjects with DFNBI non-syndromic hearing impairment. J Med Genet, 42(7), 588-594. del Castillo, I., Villamar, M., Moreno-Pelayo, M. A., del Castillo, F. J ., Alvarez, A., Telleria, D., et al. (2002). A deletion involving the connexin 30 gene in nonsyndromic hearing impairment. N Engl J Med, 346(4), 243-249. Feldmann, D., Le Marechal, C., Jonard, L., Thierry, P., Czajka, C., Couderc, R., et al. (2009). A new large deletion in the DFNB1 locus causes nonsyndromic hearing loss. Eur J Med Genet, 52(4), 195-200. Gerido, D. A., & White, T. W. (2004). Connexin disorders of the ear, skin, and lens. Biochim Biophys Acta, 1662(1-2), 159-170. Hilgert, N., Smith, R. J ., & Van Camp, G. (2009). Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics? Mutat Res, 681(2-3), 189-196. Kenneson, A., Van Naarden Braun, K., & Boyle, C. (2002). GJB2 (connexin 26) variants and nonsyndromic sensorineural hearing loss: a HuGE review. Genet Med, 4(4), 258-274. Lerer, I., Sagi, M., Ben-Neriah, Z., Wang, T., Levi, H., & Abeliovich, D. (2001). A deletion mutation in GJB6 cooperating with a GJ B2 mutation in trans in non- syndromic deafness: A novel founder mutation in Ashkenazi Jews. Hum Mutat, 18(5), 460. Matos, T. D., Caria, H., Simoes-Teixeira, H., Aasen, T., Nickel, R., Jagger, D. J ., et a1. (2007). 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J ., & Kiang, D. T. (1998). Mapping and characterization of the basal promoter of the human connexin26 gene. Biochim Biophys Acta, 1443(1-2), 169-181. Wilch, E., Azaiez, H., Fisher, R. A., Elfenbein, J ., Murgia, A., Birkenhager, R., et al. (2010). A novel DFNBI deletion allele supports the existence of a distant cis- regulatory region that controls GJ B2 and GJ B6 expression. Clin Genet, 78(3), 267-274. Wilch, E., Zhu, M., Burkhart, K. B., Regier, M., Elfenbein, J. L., Fisher, R. A., et al. (2006). Expression of GJB2 and GJB6 is reduced in a novel DFNB1 allele. Am J Hum Genet, 79(1), 174-179. Zhao, H. B., & Yu, N. (2006). Distinct and gradient distributions of connexin26 and connexin30 in the cochlear sensory epithelium of guinea pigs. J Comp Neurol, 499(3), 506-518. 121 CHAPTER 5 SUMMARY AND FUTURE DIRECTIONS 122 Mutations in GJBZ are the most common cause of hearing loss globally. More than 200 recessively-acting hearing-loss mutations of GJB2 have been identified since GJB2 was found to be the causative gene in DFNB1 hearing loss in 1997. The discovery of a novel deletion mutation that segregates as a DFNBl allele in a large Michigan kindred is the major finding of this dissertation. Although this mutation has so far been identified only in this extended family, its characterization strongly supports the existence of a previously-unknown, distant cis-regulatory region for GJBZ, and for GJB6. Our enhancer assays of candidate cross-species conserved sequences within the deleted interval show that some non-exonic sequences up- or down-regulate expression of a reporter gene driven by a generic promoter. Globally, mutations within the DFNB1 locus have not been fully elucidated. In many populations, many persons with hearing loss are found to bear monoallelic mutation of GJBZ. This phenomenon has two explanations. The first is that unidentified, fully penetrant DFNB1 mutations exist. Since the coding-, splice site-, promoter-, and untranslated-regions of GJB2 are easily sequenced, this argues for distant, cis-regulatory mutations that abrogate the expression of GJB2, such as the deletion that we have found in the MSU-DFS kindred. Although this particular deletion has so far not been identified outside of this kindred, it is important because it narrows the interval within which variation in the DNA of other persons with presumed DFNB1 hearing loss may be evaluated for pathogenicity. The second explanation is that some pathogenic alleles of GJBZ may occasionally be dominantly-acting, with reduced penetrance. Penetrance is a measure of the sufficiency of a mutation, at a single locus, to confer a phenotype. Fully-penetrant alleles 123 confer phenotype in perfect accordance with the rules of single-gene inheritance. So, under the assumption of full penetrance, a DFNB1 allele segregates with hearing loss only when present in homozygosity or in compound heterozygosity with another DFNBI allele. Most DFNB1 alleles result in loss of function of the connexin 26 protein; as the phenotype is recessively inherited, these alleles are thus usually haplosufficient with respect to the trait of hearing—the protein product that is the translated result of transcription from only one active locus is sufficient to maintain normal hearing. Conceptualize a continuum, with one end corresponding to no expressed Cx26 protein at all and a hearing-loss phenotype. The other end of the continuum is set a bit arbitrarily, but corresponds to a ‘usual’ amount of Cx26 protein expressed from two active, ‘normal’ loci, and a normal-hearing phenotype. Somewhere along that continuum— between 50% and 0% of the ‘usual’ dose of Cx26 protein—will be a point at which the transition between the normal-hearing phenotype and the hearing-loss phenotype occurs. Where does this point exist? For Cx26 and hearing loss, this will depend not only on some minimal, sufficient level of the Cx26-specific biological tasks accomplished by some minimal, sufficient number of Cx26 molecules expressed in the relevant cells in the cochlea, but also on all sorts of other tasks being accomplished by other molecules in those cells that will determine what the minimal, sufficient number of Cx26 molecules must be. One can imagine a cochlea where dysfunction of one (in a digenic model of inheritance), a few (in an oligogenic model of inheritance) or several (in complex disease) other molecules is such that even 50% of the usual dose of Cx26 is not sufficient to accomplish the Cx26-specific tasks. In this manner, a null, hypofunctional, or 124 hypomorphic allele, which normally confers no hearing-loss phenotype in trans with a normal GJB2 allele, may, on some geneticbackgrounds, be haploinsufficient. Thus, concepts that are applied to describing inheritance of phenotype in human pedigrees, particularly those of penetrance, expressivity, dominance, and recessivity, are perhaps most appropriately thought of, not as properties inherent to any particular allele, but as properties emerging from the interactions between alleles and their genetic/physiological/environmental contexts. Further, the body of literature and the accumulated understanding of the DFNB1 locus—the number of DFNB1 alleles, their nature with respect to the above concepts, the population genetics of DFNB1 hearing loss, the difficulty of the tasks of sorting out pathogenic mutations and benign variants, in particular, the difficulty of interpreting monoallelic mutation segregating with a phenotype that is traditionally understood as recessive—illustrates not only the fluidity of these genetic concepts, but also the interrelationship of the concepts with each other. Thus, penetrance is directly implicated in the assignment of dominance/recessivity. As well, all of this impinges on the traditional distinctions between single-gene and complex disease (Scriver & Waters, 1999). All of the above is, for me, the most important genetic lesson of the DFNB1 locus. With respect to the future directions of this project, this thinking is relevant to the formulation of hypotheses to test in the MSU-DFS kindred with regard to two phenomena: 1) unexplained congenital and/or prelingual hearing loss in four MSU-DFS study participants (none of whom segregate any known DFNB1 alleles), and 2) the complex phenotype of age-related hearing loss (presbycusis). As of the date of this dissertation, there are no MSU-DFS study participants who have hearing loss and monoallelic mutation of GJBZ. However, four study participants 125 have a history of prelingual hearing loss, but no genetic diagnosis at this time. For three of these individuals, we recently sequenced SLC26A 4, the gene encoding the ion- transporter protein pendrin, mutations of which cause nonsyndromic hearing loss (DFNB4), hearing loss with EVA (enlarged vestibular aqueduct, a finding that can only be discerned radiologically), and Pendred syndrome (hearing loss with thyroid dysfunction and EVA) (Pryor, et al., 2005). Two of our MSU-DF 5 study participants were found to harbor the D724G mutation monoallelically. This mutation is thought to be a fully penetrant, recessive mutation causing hearing loss with or without thyroid involvement (Pera, et al., 2008). Monoallelic D7240 mutation with hearing loss in these two persons suggests unidentified mutation on each chromosome in trans; however, our sequencing efforts have ruled out SLC26A4 coding region mutation. One MSU-DFS study participant bears the L597S mutation of SLC26A 4, also monoallelically. This mutation is associated with some degree of altered protein function in vitro, and is found in excess as monoallelic mutation among persons with hearing loss with EVA (Choi, et al., 2009). Although the weight of evidence suggests that this variant is probably not pathogenic, Choi et al. (2009) suggest that it may contribute to hearing loss under some circumstances. Sequencing of SLC26A4 in the fourth MSU-DFS subject is not yet complete, but has so far failed to yield any candidate pathogenic mutation. None of the four subjects harbors the 131.4 kb DFNBI deletion, or the 35delG or any other coding- region mutation of GJBZ. As in any recessively-inherited phenotype with locus heterogeneity, some people may carry a single mutant allele at an interrogated locus, but display the relevant phenotype due to mutation at a different unknown or uninvestigated locus. Alternatively, the phenotype may have an environmental etiology; this is 126 sometimes the case in congenital hearing loss, as certain prenatal infections may go unrecognized. In these three cases, fithher investigation of SLC26A4 is appropriate, for the following reasons: First, in three of the four subjects, one mutation (or debated variant) of SLC26A4 has already been identified. Second, two of the four subjects are children with one parent who does not share MSU-DFS ancestry. Because GJBZ and SLC26A4 are the two genes that are by a large margin most commonly responsible for recessively-inherited or spontaneous congenital hearing loss, mutation in one of these two genes should be suspected first. A more specific lesson of the DFNB1 locus, elucidated in this dissertation, is that some pathogenic alleles may harbor distant cis-regulatory mutations. For the four subjects, and any additional subjects with hearing loss and no GJBZ mutation that may in future be recruited into the MSU-DFS study, haplotyping of the SLC26A4 locus may identify a novel DFNB4 allele, shared between some of these subjects, and only in compound heterozygosity with an identified SLC26A4 mutation in persons who also have hearing loss. This finding would justify looking hard for candidate regulatory mutation, and for working up an expression assay that might yield functional evidence that a regulatory mutation of SLC26A4 exists on this allele. Cataloguing and evaluation of all variants within a haplotype shared between family members may not be practical; however, array CGH is a means by which potential candidate deletions, at least, may be identified. Chromosomal rearrangements that do not involve copy number variation are more difficult to look for. Paired-end massively-parallel sequencing permits discovery of such rearrangements at the same time that SNP genotyping is accomplished. 127 Although the existence of additional mutation at the DFNB1 locus is generally accepted, the literature is muddier with respect to SLC26A4 and the DFNB4 locus, in particular, with respect to the presumed dominance/recessivity of the many missense alleles of SLC26A 4. It seems that dominance with reduced penetrance may be an assignment of convenience in those situations where a second mutation in trans cannot be identified. With respect to MSU-DPS and monoallelic mutation of SLC26A4, it may be important to investigate haplotypes and/or coding-region variants of other cochlear- expressed genes, to assess potential contributions of other candidate loci to digenic or oligogenic inheritance of hearing loss. Evidence exists suggesting digenic interactions between SLC26A4 and F 0X11 (Yang, etal., 2007) or KCNJ] 0 (Yang, et al., 2009) in some persons with hearing loss and monoallelic mutation of SLC26A4. We have largely ruled out this possibility in one of our study subjects, but currently do not have sufficient heterozygous markers typed at these two loci to rule this out in the other two subjects. Expression studies, such as those described for GJBZ and GJB6 in this dissertation, are not so easily accomplished for SLC26A 4, as pendrin is not expressed in tissues that are easily obtained from living subjects. However, pendrin has been found to be expressed in placenta, as well as kidney, thyroid, and cochlea; expression studies in placental tissue may be possible with members of this kindred. A lesson of the MSU-DFS kindred is that large-family studies may be particularly useful in guiding investigation into phenotypes with locus- and allelic-heterogeneity. One person with monoallelic mutation of GJB2 is not remarkable; four family members with monoallelic mutation of GJB2 is. Elucidation of the shared haplotype on their common ‘non-mutant’ chromosome justified time- and labor-intensive efforts to search 128 for a pathogenic variation. Further, had this kindred not also been segregating the 35delG mutation, the deletion allele would never have been associated with a phenotype. To date, we have identified four (perhaps five) hearing-loss alleles segregating in the MSU- DF 5 kindred: 35delG and the 131.4 kb deletion for DFNB1/GJBZ, and L445W and D724G (and L597S) for DFNB4/SLC26A 4. Of our >200 study participants, more than 40% carry one or more of these five alleles. The potential for identification of significant associations of variation in cochlear-expressed genes with presbycusis and/or noise- induced hearing loss is significant, as is the potential to clarify the status of the L597S allele. Work with this kindred is moving forward under Drs. Brian Schutte and Debra Schutte of Michigan State University; they seek to expand the number of study participants by more than lO-fold, and to investigate complex disease genetics. This kindred is a population of a size and degree of relatedness that fills an important gap between highly-consanguineous, isolated extended families, that have been particularly valuable for the discovery of mutations in recessive, single-gene disease, and large populations of unrelated persons, that provide cases and controls for association studies, that have yielded some insight into complex disease. Where risk alleles in complex disease become less-penetrant alleles in single-gene disease, study populations such as the MSU-DFS kindred may be particularly valuable in elucidating pathogenic combinations of alleles of several genes, and in clarifying genotype-phenotype relationships. This particular community has also been generous with their long-term involvement and dedication to achieving the goals of our research. Our critical 129 experiments could not have been made without the long—standing, productive working relationships that we have enjoyed with individual participants as well as with community members who have been part of our advisory and ethics committees over the last dozen or so years. We have gone back to participants many times for more cheek swabs, more blood, and more information. We have gained additional participants from among interested friends and relatives who have been put in touch with us by our dedicated committee members. I sincerely thank them for making this dissertation possible, and for making it better. 130 REFERENCES Choi, B. Y., Stewart, A. K., Madeo, A. C., Pryor, S. P., Lenhard, S., Kittles, R., et al. (2009). 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