SPECIFIC BANDENG PATTERNS OF HUMAN CHROMOSOMES BY USE 'OF THE . ’ PROTEOLYTIC ENZYME TRYPS‘IN AND A BUFFERED GIEMSA STAIN Thesis for the Degree of M. s. ‘ mam Sm: umveRsm : .. ‘ ’ GARY L. MARSIGUA - _ 1972‘ ‘ imam av ' HMS & SUNS' 800K BINDERY INC. LIBRARY amoaas grungy. menu; 2%" ABSTRACT SPECIFIC BANDING PATTERNS OF HUMAN CHROMOSOMES BY USE OF THE PROTEOLYTIC ENZYME TRYPSIN AND A BUFFERED GIEMSA STAIN BY Gary L. Marsiglia A modification of the Giemsa banding procedure of Seabright (1971), which employs the enzyme trypsin and a buffered Giemsa stain, was used in a systematic study of 10 controls and 14 patients known to have chromosomal re- arrangements. The patients were selected from the resi- dents at the Lapeer State Home and Training School and from among cases seen in the Genetics Counseling Clinic at Mich- igan State University. Anomalies including sex chromosomal aberrations, autosomal deletions and both balanced and unbalanced auto- somal translocations were found. A detailed discussion of the patients listing clinical findings, routine chromosomal analysis and an interpretation of the Giemsa bands with spe- cific cytogenetic diagnoses are presented. SPECIFIC BANDING PATTERNS OF HUMAN CHROMOSOMES BY USE OF THE PROTEOLYTIC ENZYME TRYPSIN AND A BUFFERED GIEMSA STAIN By Gary L.£Marsiglia A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1972 To Dotti and Tim ii ACKNOWLEDGMENTS I would especially like to thank my professor, Dr. James V. Higgins, for his suggestions, guidance and encouragement during the course of this experimental study. Also, I wish to thank Dr. Herman Slatis and Dr. William Tai for their advice and criticisms. Thanks are likewise due my parents whose love and understanding throughout my life is greatly appreciated. Finally, without the friendship and aid of the fol- lowing individuals this thesis may not have been possible: Sally Cullen, Carola Wilson, Susan Reimer, Lou Betty Richardson, Rachel Rich, Robert Pandolfi, Robert Leider, Michael Madura, Astrid Mack, Frankie Brown and the labora- tory technicians at the Lapeer State Home and Training School. This research was supported by a grant from the Office of the Dean of the College of Natural Science. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . . . . . . Vi INTRODUCTION AND LITERATURE REVIEW . . . . . . . 1 MATERIALS AND METHODS . . . . . . . . . . . . . . 10 RESULTS . . . . . . . . . . . . . . . . . . . . . 12 DISCUSSION . . . . . . . . . . . . . . . . . . . 54 CONCLUDING REMARKS . . . . . . . . . . . . . . . 75 REFERENCES . . . . . . . . . . . . . . . . . . . 76 APPENDIX . . . . . . . . . . . . . . . . . . . . 81 iv LIST OF TABLES Table Page 1. Summary of patients and results by routine karyotype analysis and Giemsa banding . . . . . . . . . . . . . . ZZ Figure 1. (II-FMN 6a. 6b. 8a. 8b. 9a. 9b. 10a. 10b. 11a. 11b. 12. 13a. LIST OF FIGURES Seabright idiogram . Normal male karyotype, 46,XY . Case Case Case' Case Case Case Case Case Case complete Case Case complete Case Case complete Case Case complete Case complete no. no. no. no. no. 110. no. no. no. no. no. no. no. no. no. 110. l, 47,XXX, complete karyotype 2, 47,XYY, complete karyotype . , 48,XXYY, complete karyotype . 46,X iso i, complete karyotype U partial karyotype , 46,XY,Sp-, partial karyotype . , 46,XX,18p-, complete karyotype as O\ 01 h h m V , partial karyotype 7, 46, XX, 21- ,t(21q21q)+, karyotype . 7, partial karyotype 8, 46, KY, 21- ,t(21q21q)+, karyotype . . . . 8, partial karyotype 9, 46, KY, 21- ,t(21q21q)+, karyotype . 9, partial karyotype 10, 46, KY, 14- ,t(l4q21q)+, karyotype . . . . . ll, 46,XX,13q-,t(13q21q)+, karyotype . . . . . . . . vi Page 13 21 25 27 28 30 31 33 35 36 37 38 39 40 41 42 44 46 Figure Page 13b. Cases no. 11 and 12, partial karyotype . . . . 47 14a. Case no. 13, 47, XY ,t(13q21q)+, complete karyotype . . . . . . . . . . . . 48 14b. Case no. 13, partial karyotype . . . . . . . . 49 15a. Case no. 14, 46, XY ,t(8p- q- ,lq+5p+) complete karyotype . . . . . . . . . 52 15b. Case no. 14, partial karyotype . . . . . . . . 53 16. Comparison of banding patterns by 6 different techniques . . . . . . . . . . . . SS 17. Normal segregation in cases no. 11 and 12 . . 7O 18. Idiogram of translocated chromosomes of case no. 14 . . . . . . . . . . . . . . . . . 73 vii INTRODUCTION AND LITERATURE REVIEW Recent advances in human chromosome methodology have made it possible to identify all 22 pairs of autosomes and the X and Y, by their characteristic bands. This present study employs one of the new techniques, Giemsa banding, us- ing 10 controls and 14 patients with chromosomal anomalies. The patients were selected either to confirm a suspected di: agnosis or to point out exactly which chromosomes, on the basis of the bands, were involved in complex structural re- arrangements. Early Methods Lejeune, Gautier and Turpin in 1959, demonstrated that in Down's syndrome the individual possessed 47 instead of 46 chromosomes, being trisomic for a small acrocentric in the C group. This finding was the first aneuploid state de- scribed in man and added much impetus to the field of human cytogenetics. Shortly thereafter, two other trisomies in- volving small autosomes were described, trisomy D (Patau §t_al., 1960) and trisomy 18 (Edwards g£_al., 1960). Iden- tification of other anomalies including translocations, de- letions and sex chromosomal aberrations quickly followed. At first the major difficulty encountered was that most of the chromosomes could not be individually distin- guished from one another, except by group. A search for a method to distinguish pairs within the group was begun.- Autoradiography was first applied to human chromosomes by Morishima g£_al., in 1962. Somewhat earlier, the basic fea- tures of chromosome organization and duplication had been revealed when Taylor §E_Elx (1957) introduced tritiated thymidine into their study of Vicia faba chromosomes. Many of the first investigations on human chromosomes focused at— tention on the asynchronous pattern of DNA replication of the X chromosomes in females. ”They also suggested that some of the autosomes exhibited replication patterns that could be useful in characterizing chromosomes not distinguishable by morphology (Gilbert, gt_al., 1962). Modifications of techniques made it possible to autoradiographically identify the chromosomes of groups B, D and E (Schmid, 1963). Pairs l, 2, 3 and 16 and frequently the Y were easily recognized by morphology and measurement. Chromosomes in groups P, G and C, except for the late replicating X, however, remained indistinguishable from one another. Banding Patterns In 1968, T. Caspersson, at the Institute for Medical Cell Research and Genetics, in Stockholm, Sweden, began ex- perimenting with a fluorescent alkylating agent. It was hypothesized that an alkylating agent might interact and ac- cumulate in guanine-rich segments of DNA, specifically at- tacking the N-7 atom of guanine. A fluorescent alkylating agent whose presence on the chromosome could be detected by ultramicrofluorescence, would specifically allow one to vis- ualize chromosomal loci with a high guanine content. Since both quinicrine and quinicrine mustard were highly fluores- cent in the visible range, they were chosen to test this hy- pothesis. Both gave a rather diffuse fluorescence, but the mustard additionally demonstrated a clear pattern of cross striations which extended across both sister chromatids. By applying this stain to Vicia faba and Trillium, charac- teristic banding patterns for each chromosome were demon- strated (Caspersson gt_al., 1969). The next step was to attempt this technique with hu- man chromosomes. In 1970, Caspersson §£_a1,, showed that in man, each pair also produced distinct bands when stained with the quinicrine mustard. By this method each individual chrom- osome pair could be distinguished. Originally, Caspersson had thought that the alkyla- ting group in the quinicrine mustard was reacting with the DNA in two ways: (1) preferentially acting on the guanine moieties and (2) by intercalation in the double helix. He believed that this was why the bands appeared distinct for each chromosome pair. This was found not to be true when it was observed that quinicrine dihydrochloride, which lacks the alkylating group and therefore should not bind to the guanine- rich areas, produced bands at the same sites as the mustard (O'Riordan g£_al,, 1971). Proflavine and acriflavine, which are also fluorescent agents without alkylating groups, like- wise produced the same bands. Britten and Kohne in 1968, showed that a large frac- tion of the DNA of higher organisms reassociated faster than would be predicted from the DNA content of the cell. Another fraction of the DNA was observed to reassociate at the ex- pected rate. These findings led them to conclude that cer- tain areas of the DNA are redundant. Their survey further pointed out that the repeated DNA occurs widely in higher or- ganisms and is ubiquitous among eucaryotes. Hybridization of the DNA with radioactive nucleic acid, detectable by autoradiography, allowed an investigation of the distribution of the repeated sequences within the genome (Jones, 1970; Pardue and Gall, 1970). By using the technique of in situ hybridization these investigators showed that mouse satellite DNA, hybridized with the DNA in the cen- tromeric regions of all the metaphase chromosomes except the Y. This was also observed for the RNA complementary to the satellite DNA. The centromeric regions were not only labeled, but also intensely stained with Giemsa. Because it is known that constitutive heterochromatin contains DNA that is pri- marily of the satellite type (Corneo e£_al,, 1970), they con- cluded that the centromeric areas of the mouse were likewise heterochromatic. [While many definitions of the term het- erochromatin exist, whenever it is mentioned in this review, it will refer to the constitutive heterochromatin that con- tains most of the satellite DNA (i.e., the highly repetitive DNA), unless otherwise indicated.] Arrighi and Hsu (1971) and Yunis, Roldan, Yasmineh and Lee (1971), working independently, demonstrated that the centromere regions in man could be selectively stained with Giemsa after denaturation followed by renaturation of the DNA in chromosome preparations. Since it had been shown that the centromeric regions in the mouse chromosomes are composed of repetitive DNA and since these regions are heave ily stained by the Giemsa, it was suggested that the heavily "blocked areas” on human chromosomes also represented redun- dant DNA. The denaturation step consisted of NaOH treatment followed by the renaturation which was an incubation in sa- line sodium citrate (SSC). This renaturation procedure is similar to the renaturation properties of repetitive DNA. In this type of chromosome banding, the Giemsa is believed to stain all of the repetitive DNA sequences irrespective of their base composition whereas the quinicrine mustard is con- sidered to bind repetitive DNA with a base composition spe- cificity (Gagné gt_gl,, 1971). By experimenting with modifications of this proce- dure, several investigators were able not only to achieve centromeric banding, but also banding in other parts of the chromatids. The denaturation and renaturation steps varied, as did the clarity and numbers of bands. The procedures generally consisted of a NaOH denaturation followed by an incubation at 50-65°C for several hours (1-72) in a saline sodium citrate buffer or a potassium phosphate-sodium phos- phate buffer (Hawkins, 1971; Schnedl, 1971; Ridler, 1971; Lomholt and Mohr, 1971; Drets and Shaw, 1971; Crossen, 1972). One technique, called the Giemsa 9 (Patil gt_al., 1971), ob- tained a differential staining by increasing the pH of the stain from 6.8 to 9. Another procedure, termed the ASG technique for acetic/saline/Giemsa (Sumner et_al,, 1971), fixed the slides in methanol and acetic acid, followed by an incubation for one hour at 60°C in 2xSSC, then stained in the Giemsa for 1.5 hours. This technique showed that the fixa- tion itself denatures the DNA. One procedure, however, reported by Dutrillaux and Lejeune (1971), displayed bands in the reverse order of the others and for this reason has been called the R band tech- nique. Slides are placed in a pH 6.5 phosphate buffer at 87°C for 10 minutes, fast cooled to 70°C, rinsed in tap water and stained in a pH 6.7 Giemsa solution. Even thongh the method is quite similar to the others, the banding results are just the opposite. While it was now possible to readily identify indivi- dual chromosome pairs by the position of their bands, an ex- planation of why the bands appeared as they do, remained a matter of speculation. Drets and Shaw (1971) believed that the centric heterochromatin represented the rapidly anneal- ing, highly repetitive DNA, while the bands scattered through- out the genome represented families of repeated sequences with fewer copies. The unstained interband regions could then be the sites of unique nucleotide sequences. Their hy- pothesis was further supported by the fact that a longer in— cubation period was necessary to reveal the bands. Schnedl (1971), believes that at least some of the bands might repre- sent chromosome regions occupied by reiterated DNA, since it is known from the work of Britten and Kohne that the repeti- tive DNA renatures at a faster rate than other DNA. It A should be pointed out, however, that the quinicrine fluores- cence technique, does not involve any sort of renaturation process, yet produces bands very similar to those described by the Giemsa technique (Sumner et_§1,, 1971). Thus, some factor other than repetition of DNA must be involved. A base-specific interaction would be a possibility, but there is no evidence this occurs. A more recent and more reliable technique is one de- scribed by Marina Seabright (1971; 1972) of the Cytogenetics Unit in Salisbury, England. She explored the possibility that the banding was due to differential patterns of DNA- protein association along the length of the chromosome. To test this hypothesis, she employed the proteolytic enzyme, trypsin, and achieved distinct bands comparable to those displayed by the other Giemsa techniques. The major advan- tage to this procedure, is its speed and consistency, since no incubation period is required and a greater proportion of the spreads are banded. Others, also using trypsin (Wang and Federoff, 1972), have postulated that the trypsin hy- drolyzes the protein component of the nucleoproteins which have been denatured by the fixation procedures. This then allows the Giemsa to react with the exposed DNA, producing the bands. The properties of Giemsa stain are undoubtedly im- portant for band formation. Drets and Shaw (1971) have shown that acetic orcein did not produce banding, and Crossen (1972) obtained negative results with cresyl violet. Sumner gt_al., (1971), have reported that methylene blue gives only weak banding and it would appear that the combination of eosin and methylene blue is a factor in producing specific banding patterns. Classification of the Bands During the IVth International Congress of Human Genetics held in Paris in September, 1971, it was decided that because of the recent developments in human chromosome banding, that a conference on nomenclature be conducted in order to reconcile the many differences. The conference, sponsored by the National Foundation, will be published at a later date. The types of bands proposed by the committee are as follows 1) The 2) The 3) The 4) The (Hsu, 1972): Q bands C bands G bands R bands fluorescent bands revealed by quinicrine mustard heavily stained regions revealed by a denaturation-renaturation process, usually centromeric a variety of techniques revealing cross bands using Giemsa stain the reverse bands of Dutrillaux and Lejeune, previously discussed. MATERIALS AND METHODS Chromosome cultures were prepared from peripheral leukocytes, using the macro-method. Blood was drawn by vena puncture into a syringe containing 0.1 cc. heparin, to pre- vent coagulation. This was then left in an upright position in order for the white cells to separate out from the red cells. After approximately 1 1/2 hours, 2 cc. of leukocyte enriched plasma was added to 8 cc. of Grand Island Biological Company Chromosome Media 1A. The cultures were incubated for 3 days at 37°C. 0.2 cc. of .0048 colchicine was added to each culture to arrest the cells at metaphase. Incubation was then continued at 37° for an additional 3 hours. The harvesting procedure was a modification of that of Moorhead §t_al, (1960). The cultures were removed from in- cubation and spun at 1600 RPM for 3 minutes in a centrifuge, leaving a button of cells. The supernatant was drawn down to 1 ml. followed by the addition of 5 ml. of warm (37°C) .075 M KCl to each culture for 8 minutes. Fixation of the cells con- sisted of 4 washes with Carnoy's solution (3:1 methanol: gla- cial acetic acid) for 10 minutes each. Eight to ten drops of the chromosome suspension were dropped from arm's length onto slides pre-chilled in 95% ethyl alcohol. The slides were flame dried in order to rupture the cells. 10 11 Many different Giemsa banding procedures were at- tempted and it was decided that a modification of the Sea- bright procedure (1971) gave the most consistent and most reliable results. Freshly harvested slides were placed in a 0.25% trypsin - GKN solution (see Appendix) for 15-25 sec- onds. GKN is a Ca++ and Mg++ free balanced salt solution. The slides were then rinsed thoroughly in two washes of 0.85% NaCl. At this point, it was possible to scan the slides under phase contrast microscopy to assess the action of the enzyme. If the spreads appeared slightly swollen, the slides were ready for the stain. If not, they were ex- posed to the trypsin for a longer period of time. The stain was a 1:10 Fisher stock Giemsa (0.8 gm. powdered Giemsa, 50 ml. methanol, 50 ml. glycerol) to a 0.6 M NaZHPO4/KH2PO4 Sorensen buffer at pH 6.8 for 3 minutes. This was followed by a short rinse in the same pH 6.8 buffer. If the slides were understained, they could be returned to the Giemsa for a longer period of time and if they were over- stained, they were returned to the buffer for a more thor- ough rinse. The slides were scanned under bright-field on a stan- dard Zeiss photoscope and photographed without a coverslip. Adox 35 mm. film was used and this was developed in D-19 (1:4) for 3 minutes at 68°C. Prints were made on Fotorite FPI #4 photographic paper. RESULTS Before any patient could be analyzed karyotypically by the Giemsa banding procedure, 10 controls were studied in order to establish a set of "normally" banded chromosomes for the survey. The banding patterns attained compared fa- vorably with those of Seabright (1972), with the exception of the X, and the chromosomes were numbered according to her idiogram (Figure 1). Seabright's numbering corresponds to , that used by Caspersson (1971) for the fluorescent studies, since these had already been well established at the Orly- Paris Conference on human chromosome nomenclature. The following is a list of the individual chromosome pairs summarizing the banding patterns of each. The charac- teristics described are those which are most favorable for visual identification from photographic prints. The bands are discussed beginning with the distal end of the short arm and continuing to the distal end of the long arm. Chromosome 1 This chromosome which is easily recognized by its morphology has distinctive banding patterns in each arm. The distal portion of the short arm appears unbanded followed by 12 13 $3. I Seabright idiogram. Figure 1. 14 one faint band and two heavily stained bands nearer the cen— tromere.‘ The long arm is characterized by a dark staining band in the centromeric region, followed closely by a small dark band. This latter band is not easily distinguished from the centromeric band in most preparations and usually the two bands appear as one. These are followed by four bands throughout the rest of the length of the long arm, the second of which is the most prominent. Chromosome 2 Both arms of this chromosome have fairly uniform banding. The short arm has 4 equally spaced bands, the sec? ond band which is in the middle, being the most intensely stained. The bands on the long arm are difficult to iden- tify because they lie close together. According to Seabright there are 8 bands present, the two in the middle portion being the most intense. In this group of controls, the lar- gest number of bands observed in the long arm was 6, the two nearer the centromere being more lightly stained than the remaining 4. Chromosome 3 This chromosome has very distinct bands. Near the distal end of the short arm there is a strongly banded re— gion. Also at the centromere and on either side of it, there 15 is a very distinct, wide, dark stained band. The distal end of the long arm has two darkly stained bands which may ap- pear as one because of the intensity of the stain. Chromosome 4 There are two bands on the short arm, one in the mid- dle portion and one just above the centromere. The long arm has 5 bands, the first 3 which are prominent and separated from the distal pair by an unbanded region. The distal pair are very close together and may appear as one. Chromosome 5 The short arm bands very similar to chromosome 4, displaying 2 bands, one very near the distal end and one at the centromere. The long arm has a distinct band just be- neath the centromere, followed by a long heavily banded re- gion in the middle portion. According to Seabright, this represents 4 dark bands very close together. There is also a prominent band at the distal end. Chromosome 6 This chromosome is the largest member of the C group. The short arm has 2 distinct bands, one near the distal end and one at the centromere. These are separated by a clear unbanded region. The long arm also has a conspicuous band extending from the centromere. This is followed by two prom- inent bands and two pale distal bands. 16 Chromosome 7 The short arm displays a terminal dark band and a band near the centromere with a clear region between the two. The long arm has two very distinct bands on either side of the middle portion and a pale distal band. Chromosome 8 This very submetacentric chromosome has two bands in the short arm, one distal and one above the centromere. The long arm has 4 bands, two very near the centromere and two more distally located. Chromosome 9 The short arm has a dark terminal band and a band close to the centromere. The proximal portion of the long arm is unbanded followed by two darkly stained bands. Chromosome 10 The short arm has 2 bands, one near the distal end and one at the centromere. There are 3 bands on the long arm, the first being the most heavily stained and found just below the centromere, the second in the middle of the long arm and the third at the distal end. 17 Chromosome 11 This chromosome displays two bands in the short arm, one near the distal end and one above the centromere. The long arm has a very broad, prominent band in the middle por- tion with unbanded regions on either side. Chromosome 12 The pattern is very similar to chromosome 11, but ‘the short arm has only one band. This is also the most sub- Inetacentric of the C group chromosomes. The long arm has a small band adjacent to the centromere and a median dark, broad band. The X Chromosome Visually this chromosome does not fit the idiogram of Seabright. There is a prominent band in the middle of the short arm. The centromere is usually unstained, but in some preparations staining has been observed. The long arm has 3 equally spaced bands, the first two being the most prominent. .Qhromosome 13 None of the acrocentric chromosomes display distinct 'bands on the short arms. The long arm is best characterized by 3 heavy bands, two of which are near the distal end and because of the stain and close proximity, may appear as one. 18 Chromosome 14 The two darkly stained bands just below the centro- mere are characteristic for this chromosome. There is also an intense band at the distal end with a clear unbanded area between. Chromosome 15 This chromosome has one broad band in the long arm somewhat near the centromere, with the distal portion ap- pearing unbanded. Chromosome 16 The short arm has one faint band near the distal end. The long arm has a characteristic intense band just beneath the centromere followed by two bands of lesser in- tensity. Chromosome 17 The short arm is banded only at the centromere. The long arm is also banded at the centromere and has a band near the distal end. Chromosome 18 The short arm appears unbanded. Like chromosome 17, the long arm has 2 bands, but the centromere is 19 non-staining. There is one prominent band below the centro- mere and one at the distal end. Chromosome 19 This chromosome bands only at the centromere with the remainder of the Chromosome appearing very pale. Chromosome 20 There is a faint band in the medial portion of the short arm, a banded centromere, and a faint band in the me- dial portion of the long arm. Chromosome 21 The long arm of this chromosome is almost entirely banded, with only the most distal portion unbanded. Chromosome 22 This chromosome is readily identified by its darkly stained centromeric region. Thus, there is no difficulty in distinguishing chromosomes 21 and 22. The Y Chromosome Besides being recognized morphologically, this chrom- osome has a fairly heavy band on the distal half of the long arm . 20 A representative karyotype of one of the controls, displaying the bands as they would appear in an apparently normal individual, is shown in Figure 2. Use of the Banding Technique in Cytogenetic Diagnosis Since the Giemsa banding procedure utilizing trypsin, has made it possible to distinguish individual chromosome pairs and to recognize their structural variations, this technique was applied to 14 patients displaying transloca- tions, deletions and trisomies. In each case it was possible to specifically identify the abnormal chromosomes involved.. A summary of the patients, together with the results from routine staining and Giemsa banding is given in Table 1. Specific information including clinical findings and inter- pretation of the Giemsa bands will be analyzed for each uni- que case separately. Case #1: K. H. born 11-12-57 (See Figure 3) A mildly retarded girl, with growth retardation and microcephalus was referred for a possible chromosomal abnor- mality. Buccal smear revealed 37% with two sex chromatin bodies and karyotype analysis showed a 47,XXX. Autoradio- graphy was considered but not performed. Giemsa banding confirmed the diagnosis demonstrating the extra C group chromosome to be an X. Saw .9, (313‘ 21 Na x in"? , '- ‘1". l. ’36: £6 6 lb H n :26 6'3 l3 AA u f. 3. R. t! 20 H Normal male karyotype, 46,XY. Figure 2. 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I. am .. J. b. I 1.. m. 2 u. = o. r u r a. 2 .2 3 z x s n u _ Oil-D ‘I ’9‘ 0", (ad in» ‘a‘ .m madman 26 Case #2: J. G. born 11-27-51 (See Figure 4) This patient was initially referred for chromosomal analysis as a possible XYY. His clinical findings included tall stature, thrombophlebitis, acne and an essential tremor. These are all consistent with the diagnosis of XYY. The pa- tient displayed no tendency towards aggressive behavior, but did experience occasional, uncontrollable outbursts of tem- per. He was also chronically depressed with suicidal tenden- cies. His intelligence was estimated to be low average al- though no psychometric testing was performed. Routine chromosomal analysis revealed a modal count of 47 with 6 chromosomes in the G group, the extra one con- sidered to be a Y. Giemsa banding confirmed this diagnosis, showing two Y chromosomes, both heavily banded on the distal half of the long arm. Case #3: C. K. born 7-5—22 (See Figure 5) Standard karyotype analysis on C. K. displayed a possible mosaic Klinefelter's, 47,XXY/48,XXYY. The patient exhibited no gynecomastia and had normal external genitalia. He does have an essential tremor and varicose veins. He is the ninth of 11 children, has an IQ of 41 and a history of repeated escapes from the state home in which he is institu- tionalized. Ill-tempered at times, he is destructive and difficult to control. 27 t, {II lllllf .ohkuoxumx ouonEou .wwx.ne .N .o: omwu .v oHSMMm .J O «:3 c: O ' and O 28 .omxuokuwx oponEou .wwxx.mv .m .od omwu .m oHSwfim . i i 3']: R .'.:‘.' ._ AI. 0‘ ' ha?- 9” 4“ out ~’ 29 Giemsa banding revealed a modal count of 48 with no evidence for mosaicism, 48,XXYY. The extra C group chromo- some was identified as an X displaying one prominent band in the short arm, and 2 bands in the long arm. In this partic- ular case, the centromere was also banded. The extra G group chromosome by morphology and distinctive banding on the dis- tal region of the long arm was identified as a Y. Case #4: K. P. born 5-11-60 (See Figures 6a and 6b) This 12 year old female was referred for chromosome studies because of extreme short stature. She also displayed severe scoliosis, a cafe-au-lait spot, perceptual problems. and a wide carrying angle. Buccal smear results were not con- sistent with those of a normal female, therefore chromosomal analysis was done to entirely rule out the possibility of Turner's syndrome. Routine staining revealed a modal count of 46 with one of the C group chromosomes being completely metacentric, resembling an A3 by size. It was suggested that this was an isochromosome for the long arm of one of her X chromosomes. Thus, K. P. was trisomy for the long arm of the X and mono- somy for the short arm, 46,X iso x. Giemsa banding exhibited one normally banded X in the C group, and one metacentric chromosome banded identically in both arms, resembling the pattern in the long arm of the nor— mal X chromosome. It was easily identified from the #3 since 30 .omxuoxnmx ouonEoo .M omH x.ov .e .od omwu .3 a on r. . u c c R m . u 2 2 i u. 2. .. a t. 2 2 unex a r .wo oudmflm i 31 .omxuoxhwx Hwfluhmm .v .oe ommu ”x.amv~ I‘l- - 4r" a. «.31, r: 40:3 Out I m so: go: .no oeumflm 32 .the 3 has a heavily banded centromere and the iso X has an unbanded centromere. The banding patterns reinforced the routine analysis diagnosis. Case #5: N. C. born 12-24-71 (See Figure 7) This very odd looking child was noticed in the new born nursery to have a high-pitched, whining and weak cry. Physical examination revealed an inactive white male, small in size with microcephaly, micrognathia, epicanthal folds, hypotonia, bilateral simian creases, pale mottled complexion and possible mental retardation. Pulmonary problems charac- terized by wheezing, congestion and "bubbly" breath sounds. were also noticed. Karyotypic analysis was requested because of the odd cry and physical findings. It revealed an apparent deletion of the short arm of one member of the B group chromosomes, 46,XY,Bp-. Because of the phenotypic characteristics of Cri- du-Chat, the deletion was assumed to be a no. 5. Giemsa banding procedures revealed 3 normal B's, two of which show the pattern characteristic of a no. 4 and one which shows that of a no. 5. An extra deleted chromosome has a Giemsa pattern consistent with the long arm of a no. 5 show- ing a band just beneath the centromere followed by a long heavily banded region in the middle portion. Almost all of the entire short arm is missing. 33 v. v.5fl- .omxpokumx wauuwo ..mm.»x.oe .m .od omwu .n madmwm m...“ . L i. 34 Case #6: N. F. born 4-11-42 (See Figures 8a and 8b) This individual, a resident at the Lapeer State Home and Training School, was initially referred for chromosomal analysis because she was thought to exhibit some of the Mon- goloid characteristics. There were no outstanding physical findings aside from moderate retardation. Chromosome studies displayed a 46 count with a dele- tion of the short arm of an B group chromosome, 46,XX,Ep-. Giemsa banding revealed a deletion of the entire short arm of a no. 18, the long arm consistently showing 2 distinct bands, one beneath the centromere and one at the distal end, 46,XX,18p-. Case #7: M. P. born 10-23-56 (See Figures 9a and 9b) Case #8: C. C. born 6-10-49 (See Figures 10a and 10b) Case #9: W. C. born 6-19-52 (See Figures lla and 11b) Cases 7 through 9 were all diagnosed as Down's syn- drome at birth. All three clinically present the classical features of Down's syndrome including epicanthal folds, slightly protruding tongue, simple ears, spade hands, simian crease, short stubby incurving little finger and hyperexten- sibility of all joints. Routine chromosomal analysis in each case produced a Inodal count of 46. Karyotypes showed only 3 chromosomes in. group G (excluding the Y) and an extra metacentric 35 X x .ooxuothm ouonEou .smma.xx.ov .0 .od omwu .ww ohsmam . L an .u a z I d 0‘ L .. I t a. a. z e W V .. . .1... ....... S 1 i .2 = 2 c u r 3 ‘ It «a. .l d A ‘ r .......... y“ M .— m I. M H. u 1 n a 1 l I 3 P 4. 1 ....~ .1. a, .... 1.. s O 4 CW 1 fl 2““ . 36 .omxuoxuwx wapuwm .o..od.ommU .nw oudwwm : L: 2 e O c t. L. . ‘. ... 0‘ G. a ' 0‘ \ q ‘ 60‘ .5 0.! .0" 37 .omhuoxpwx ouoamfiou .+m¢HN6HNuu.-HN.xNov .5 .od omwu .wm oudmam I||l1|4|1 .3 Jr a a r. . .m u c 3 K .. ,1 t 4. m. :. ........ d I .5... 12 a. = o. r a r J. o. '0 ‘50. . n.- H «u ah >> ”m . am Wm . .. . n N , 38 3a“ .omxuoxpdx waume .h .od ommu .nm enamam 10 XI IQ 39 ll l...“.1|l1| .omxuoxhwx \ a. no ,‘RX 2 an a. opoHQEOU .+fieHNeHNuu..HN.»x.oe .w .o: omau .aOH enemam J— v‘li all! "a! .3 a . a _ «a r. u;u . a u. v. a. 0 9 3th 1.54.. 4O .3 n. O .omxuothx Hmfiuuwm .m .~ I .o: ommu :- w L . U o. I a On a. U ‘ 0.. .noH musMHm 1| ’uh l1 41 .omxuo%uwx ouonEou .+mUHNoHNVu.-HN.>x.ov .m .0: omwu .mHa QHSMHm > as .43... a s i . ... a .. a .u. v. _ 2 r. 4. u. v. n. H .3 Z .2 1 _. fl. ,9. i a. 3 o. f u r J _w .. _. ; z: i H .. A”. AW _. , r. _. 1 ‘.#l. _ u n a . . .-. . . o. ’ «J. . . ~.. 0 — _’I-J _ o... :5“- .r....J... lo! _’J~€'J}F L min"; i J. VOW. .Jhr'oow a... be! 42 O, a! i. $3 ‘| II .3 H a»! In )L, It.II 58 my» ‘1 .omxuoxumx Hmwuumm .m .od omwu ll. .80 H .BHH wham.m 43 chromosome resembling members of the F group chromosomes. MOrphologically this abnormal G group chromosome can be the result of a 21/21 translocation, a 21/22 translocation or an isochromosome for the long arm of a no. 21. M. P. was the youngest of four children. Her mother and father were both shown to be karyotypically normal, in- dicating a sporadic translocation in the patient, 46,XX,G-, t(Gqu)+. C. C. and W. C. are siblings and the only children in the family. There is no report of miscarriages or still- births. Their mother is karyotypically normal, but their father has a 45 count and is a G/G translocation carrier.’ Because both children are affected, it was assumed that the father of C. C. and W. C. carried a 21/21 translocation or an isochromosome for the long arm of a no. 21. In either case, he could produce only Down's children. Both boys were given a cytogenetic diagnosis of 46,XY,G-,t(Gqu)+. Giemsa banding in all 3 cases revealed a 21/21 trans- location. The small metacentric resembling by size an F, was completely heavily banded. Thus, it could be easily dif- ferentiated from the F group chromosomes which display very little banding. Case #10: C. L. born 5-19-33 (See Figure 12) This patient was referred for chromosomal analysis because of his characteristic mongoloid features. He also 44 .omxuoxuwx oponEou .+mvHNdeHVu..va.>x.ov .oH .0: ommu .NH madman > .3 .u on r. _ c .. no a. H O O _ i a. c 2 2 5... z a 1 3 c 1N C v. ‘ ‘ . . i .2 = o. v a r 3 . 40 on“ can 1” S... i” F“ m u r n u . _ ‘J «an g- " Cl} ‘5 “if ‘33:: (Elli ‘gdfll. 45 had a speech problem and a behavioral disorder. This lat- ter is quite unusual for Down's children who are considered to be good natured individuals. Routine staining revealed a count of 46 with Snor- mal D group chromosomes and an extra submetacentric chromo- some resembling in size a C group chromosome. This addi- tional chromosome was thought to have originated through a translocation between a supernumerary no. 21 and a member of the D 13-15 group, 46,XY,D-,t(Dqu)+. Giemsa banding confirmed a D/G centric fusion and in addition specifically identified the chromosomes in ques- tion as the long arms of a no. 21 and a no. 14, 46,XY,l4-,t (l4q21q)+. The short arm of this submetacentric was actually the long arm of a no. 21, being nearly completely banded. The long arm of this chromosome was the long arm of a no. 14 displaying two dark bands just below the centromere and one dark band at the distal end. Case #11: M. H. born 1944 (See Figures 13a and 13b) Case #12: A. H. born 12-22-68 (See Figure 13b) Case #13: P. H. born 7-30-66 (See Figures 14a and 14b) Originally P. H. was clinically suspected to have Cornelia de Lange syndrome. At birth he had cleft lip and palate and bilateral polydactly on his hands and feet. When he was seen for chromosomal analysis his clinical manifestations 46 .omxpoxnwx oponEou .+fldHNvavu..HN.pde.xx.we .HH .od omwu .me oudwflm an a oar}: 3 c. .. o a ... u i x c 2 r. 4. a. t. of... n. _fl .. ——_‘ w d a“ my .-_- a. s o. r II‘III .“klll “g - ”at I“ I!!! 93‘ O'I! " rt :33 C1- O~II 84’ a" I"ID hidl’ alIIB 01". ‘KINKJ. cii"lb 'n 47 .omxuoxmwx Hmfipnwa .NH use HH .0: momwu 7. «P. b {I .9». kt-v ~1: .EMH musmum 48 .omxpoxamx ouonEoo .+mcHNcmHVu.>x.me .mH .od ommu .wva madman r 3 . 5 Anita I a c 2. 4 a 0 i u a. r. 2 .. a. a. a. m a... n... a... a... a.“ = 2 2 z, _ L u. 2 9 .2 3 .M... s. 49 an .d .omxuothx Hmfluuwm .MH .0: omwu .nva oHSMMm A... 5 U 5 r. 9 1.. H7... .dll 50 included small stature, possible microcephaly, seizures and severe mental retardation. Routine chromosome studies revealed a 47 count with an extra acrocentric chromosome smaller than a D group, but larger than a G group. His father and younger brother were karyotypically normal. His mother and younger sister, M. H. and A. H. respectively, carried balanced translocations. They each had 5 normal D group chromosomes, 3 normal G group chromosomes, one deleted D and the acrocentric intermediate in size between a D and G. Both M. H. and A. H. are asymp- tomatic with a cytogenetic diagnosis of 46,XX,Dq‘,G-,t(Dqu)+. After determining the rearrangement in his mother and sister, P. H. was thought to be 47,XY,t(Dqu)+. Giemsa banding on A. H. and M. H. revealed one nor- mal no. 13, one normal no. 21 and one deleted distal portion of the long arm of a no. 13. The break in the no. 13 appears to have taken place between the two distal dark bands on the long arm. The acrocentric intermediate between a D and G was shown to be a translocated chromosome displaying almost all or perhaps the entire no. 21 in the centromeric position with the distal band of the long arm of a no. 13 attached to the no. 21,46,XX,l3q-,21-,t(13q21q)+. Thus, the translocated chromosome appears darkly banded at the centromere followed by a clear unbanded region and a heavy distal band. The banding patterns on P. H. revealed 46 normal chromosomes plus the acrocentric which was shown to be the 51 same translocated chromosome present in his mother and sis- ter, 47,XY,t(l3q21q)+. Thus, he is partially trisomic for the distal portion of the long arm of no. 13 and partially or completely trisomic for a no. 21. Case #14: J. K. born 8-?-69 (See Figures 15a and 15b) This child, the youngest of 3 pregnancies (the sec- ond ending in a full term stillborn), displayed a saddle nose, syndactly of the fourth and fifth fingers of the right hand, low set ears, high arched palate and gross motor and mental retardation. Routine chromosome analysis showed a complex de novo chromosomal rearrangement involving chromosomes of the Al, B and C groups. It was postulated that a double transloca- tion had taken place with the short arm fragment of the C going to either the no. 1 or the B and the long arm fragment going to the opposite chromosome, 46,XY,t(lq+,Bp+,Cp-q-). Giemsa banding studies confirmed a double transloca- tion involving chromosomes 1, 5 and 8. It appeared from the position of the bands that there were two breaks in chromo- some no. 8, involving approximately half of each arm, the long arm fragment translocating to the proximal arm of no. 5 and the short arm fragment to the distal arm of no. 1, 46,XY,t(lq+,5p+,8p-q-). Giemsa karyotypes on both parents were normal. 52 .omxuoxhwx ouonEoo .n+mm.+vH..d.mwvu.»x.ov .va .0: ommu .mmH madmam > AN 5 on r. A .. . ( .$ 0 . r a t ._ U 2 t 3. u. 1. n. M an a... an a... §a 1‘. x N. 2 o. r PLwa F J a a... H... . m... a z 3 T... .. n u .5; z E 2 2 é ? c . 8 O, % i 53 .omzuoxuwx Hmwuumm .VH .0: omwu .nma oHSMHm Li: a . m Lm . L; .. .m 3 a s cm . C1IL- gaulll :3 a DC. 0. 0.1-I3 le