Chromosome 15 Abnormalities and the Prader-Willi Syndrome: a Follow-Up Report of 40 Cases

Am J Hum Genet 34:278-285. 1982

Chromosome 15 Abnormalities and the Prader-Willi Syndrome: A Follow-up Report of 40 Cases

DAVID H. LEDBETTER," J. T. MASCARELLO,' VINCENT M. RICCARDI, VIRGINIA D. HARPER,. SUSAN D. AIRHART,' AND RICHARD J. STROBEL

SUMMARY High-resolution chromosome analysis and multiple banding techniques were performed on blood samples from 40 patients with Prader-Willi syndrome (PWS) as a follow-up to our recent report in which we found interstitial deletions of 15q in four of five patients with this syndrome. Of the 40 new patients, 19 had interstitial del(l5q), one had an apparently balanced 15;15 translocation, and one was mos46,XX/47,XX+idic(15) (pter-qI 1::ql 1-pter). These data confirm our previous report and dem- onstrate that half of all patients with the clinical diagnosis of PWS have chromosome abnormalities involving chromosome 15 detectable by high-resolution methods. Although the majority of these involve a specific deletion of bands lSq I I -q12, other alterations of chromosome 15 may be present.

INTRODUCTION Since 1976, 11 cases of Prader-Willi syndrome (PWS) have been reported with translocations involving chromosome 15 (table 1). Five of these were 15;15 Rob- ertsonian translocations [1-4], while five involved chromosome 15 and other autosomes [5-9]. The eleventh case was mosaic for three cell lines, each with a different balanced translocation between chromosome 15 and another autosome [10]. In addition to translocations of chromosome 15, six cases of PWS have been reported with an extra, small metacentric chromosome [11-15]. In two of these cases, the extra chromosome was derived from chromosome 15 [14, 15].

Received June 12, 1981; revised July 24, 1981. ' Kleberg Cytogenetics Laboratory, Baylor College of Medicine, Houston, Texas. 2 Department of Medicine, Baylor College of Medicine. I Genetics Laboratory, Children's Hospital and Health Center, San Diego, Calif. © 1982 by the American Society of Human Genetics. All rights reserved. 0002-9297/82/3402-0011$02.00 278 PRADER-WILLI SYNDROME 279 TABLE I CHROMOSOME ABNORMALITIES IN PWS

Reference Karyotype Comments

Ridler et al. [11] ...... 47,XX,+mar Hawkey and Smithies [1] ...... 45,XY,t(15q15q) No breakpoints assigned Emberger et al. [2] ...... 45,XX,t(15; 1 5)(p 1l;q 11) Deletion 15q11-15pter Fraccaro et al. [3]: Case I ...... 45,XY,t(15;15) Dicentric Case 2 ...... 45,XY,t(l;15) Monocentric Zuffardi et al. [5] ...... 45,XY,t(9; 15) Deletion 15q 11-1l5pter Aurias et al. [6] ...... 46,XY,t(3;15)(q29;qll) Balanced KucerovA et al. [7] ...... 45,XY,t(3;15)(p25;q15) Deletion 15ql5-l5pter Fleischnick et al. [4] ...... 45,XX,t(15;15) Dicentric Lejeune et al. [10] ...... mos46,XY,t(5;15)(pter;q12)/ Balanced 46,XY,t(8; I5Xqter;q 12)/ 46,XY,t(12;1 5)(qter;q 12) Michaelson et al. [12]: Case I .. 46,XY/47,XY,+mar ... Case 2 ...... 46,XY/47,XY,+mar Guanti [8] ...... 45,XY,t(9;15)(p24;q 11) Deletion 15q11-I5pter Kousseff [13] ...... 46,XY/47,XY,+mar ... Wisniewski et al. [14] ...... 47,XY,+inv dup(15) Fujita et al. [15] ...... 47,XX,+idic( l5p)jqI1) ...I Stnith and Noel [9] ...... 45,XXt(14;15)(pl 1;qIl ) Deletion 15ql 1-I5pter

Recently we reported that high-resolution chromosome analysis revealed subtle interstitial deletions of proximal 15q in four of five patients with PWS [16]. We suggested that deletion of the region 15q1l-q12 might be a cause of the PWS, and that prophase banding analysis was necessary for all PWS patients. We predicted that at least some patients previously reported to have normal karyotypes would, in fact, exhibit small interstitial deletions, and that PWS patients with apparently balanced translocations might actually have subtle deletions in proximal 15q. To further assess the frequency of del(15q) in PWS, we applied high-resolution methods to 40 additional patients.

MATERIALS AND METHODS Chromosome preparations were made from routine 72-hr lymphocyte harvests or from cultures synchronized following a modification [17] of the Yunis technique [18]. Trypsin G-banding was used in all analyses, and BrdU-Acridine Orange R-banding [19], C-banding [20], distamycin-A/DAPI staining, and Ag-NOR staining [22] were used in selected cases. Chromosome analyses were carried out in the cytogenetics laboratories at the Children's Hospital and Health Center of San Diego and at Baylor College of Medicine. Because of the small size of the deletion, most of the patient samples (34/40) were analyzed in parallel with normal controls in a blinded fashion (see Ledbetter et al. [16]). Included as controls were a total of 23 parents and normal siblings of PWS patients. Because of the variability in contraction and staining one sees in normal 15s, and because of the polymorphic nature of 15p, determination of a deletion of the qI1 or q12 bands was made on the basis of a consistent absence of these bands in many cells, and a consistent difference in the length of the cen-ql4 region. By so restricting our criteria, however, a mosaic case might be misin- terpreted as normal. In some cases, polymorphisms of the satellites, stalks, or pl 1 bands could be exploited to identify individual homologs and thus insure that the length discrep- ancy observed was not merely random contraction differences. 280 LEDBETTER ET AL. Patients included in the study had been referred to one of the two laboratories with the purported diagnosis of PWS. No attempt will be made in the present report to give detailed clinical descriptions of the 40 patients, although all had at least three of the following classic features of PWS: muscular hypotonia, obesity, short stature, hypogonadism, mental retardation, small hands and feet, neonatal feeding difficulties, and, for older children, compul- sive eating tendencies.

RESULTS Of the 40 patients, 19 had interstitial deletions in 15q. Most of these (17/19) had breakpoints in qI 1 and q13 (fig. la), as in our four previous cases [16]. Two cases (both studied in a blinded fashion) appeared to have smaller deletions confined to the qI 1 band (fig. lb), so that the q12 was still present but had merged with the centromere. One patient had a 15;15 Robertsonian translocation (fig. Ic-f). G-banding demonstrated that the q12 and qI 1 bands were present in both arms of the translocation, and R-banding showed no difference in the size of the qI 1 bands on the two arms; apparently, no q arm material was deleted. C-banding and distamycin- A/DAPI staining were performed in an attempt to determine whether the translo-

FIG. 1.-Idiogram of chromosome 15 (adapted from Francke and Oliver [26]). G-banded partial karyotypes showing qI 1-12 deletion (a) and qI 1 deletion (b). Normal homolog is on the left and deleted homolog on the right in both cases. Partial karyotypes of the 15;15 translocation (c-fl: The G-banded translocation chromosome (c) has been cut in half for comparison of the two arms. The G-band positive q12 band and G-band negative q12 band are present in both arms. The R-banded translocation chromosome (d), also cut in half, shows no difference in size of the R-band positive qI 1 band in the two arms. C-banding (e) demonstrates a single C-band in the translocation chromosome. Distamycin- A/DAPI staining (f) shows a brightly fluorescent region, indicating presence of p 1 material. PRADER-WILLI SYNDROME 281 cation chromosome was monocentric or dicentric. However, since the centromere and p 11 band of chromosome 15 are both C-band positive, and because the pI 1 band is polymorphic, we were unable to determine whether one or two centro- meres were present. Only the mother of this patient was available for study, and C-banding of her normal 15s was not informative. One patient, who had intact 15s by prophase analysis, had an extra, small bisatellited chromosome (fig. 2a-c) in 16 of 20 cells analyzed. This marker chromosome was silver stain positive on both arms (fig. 2b). The distamycin-A/DAPI technique, which specifically stains the constitutive heterochromatin in chromosomes 1, 9, 15, and 16, and the Y, showed that both arms of the marker are derived from chromosome 15 (fig. 2c). In cells stained by the trypsin-Giemsa technique or the distamycin-A/DAPI method, the marker appeared to consist of a very thin negatively stained region separating two intensely stained regions. These regions were each separated from the satellites by the nonstaining stalk regions. The satellites appeared to be of equal size. In view of the symmetry exhibited by this chromosome by all staining techniques, it seems reasonable to conclude that it is an isochromosome and that the patient's karyotype can be designated 46,XX/47,XX+idic( 1 5)(pter-q 11 ::q I1 -pter). All of the controls, including all parents and siblings of PWS patients, had normal karyotypes. a )I b

15~~~~~~1c~~~~c

Flo, 2-Partial metaphascs from the mos46,XX/47,XX,+idic(15Xpter-qI ::qI I-pter) prepared by (a) G-banding, (b) Ag-NOR staining, and (c) distamycin-A/DAPI staining. 282 LEDBETTER ET AL. DISCUSSION Routine metaphase chromosome analysis of PWS patients has revealed a vari- ety of balanced and unbalanced translocations involving chromosome 15 and several cases with an additional marker chromosome (table 1). Presumably, most routine cytogenetic evaluations of PWS patients have revealed apparently normal karyotypes, and many of these would go unreported. The discovery of a subtle deletion in PWS patients detectable only with high-resolution techniques makes it necessary to survey a large group of PWS patients with high-resolution techniques to assess the frequency of the various types of chromosome abnormalities and normal karyotypes. Combining the present data with our initial observations on five patients, high- resolution chromosome analysis has now been performed on a total of 45 patients with a diagnosis of PWS (table 2). Over half (25/45) had chromosome abnormalities involving chromosome 15. Deletions of 15q11-12 Of the 45 patients studied with high-resolution techniques, 23 had interstitial deletions of proximal 15q-with slightly varying-breakpoints. In all 23 cases, part or all of band qI 1 was lost. In two cases, only band ql 1 was deleted. These results confirm our prediction [16] that many PWS cases are associated with a specific deletion of chromosome 15, and refine our assignment of the critical region to band 1 5q 11. Deletion of the band 1 5q 11 is also consistent with at least six of the 1 1 previously reported cases with chromosome 15 translocations (see table 1). Al- though Smith and Noel [9] referred to the 14;15 translocation in their case as balanced, the breakpoint in 15qi1 (if correct) would indicate a deletion. It is interesting that three normal family members had the same 14;15 translocation. The case reported by Kucerova' et al. [7], with a breakpoint in 15q15, remains the largest reported deletion in PWS. Of the 23 deletion cases, both parents were studied in four cases and a single parent studied in six cases, all of which had normal karyotypes. Although it should be expected that most interstitial deletions will be- de novo events, the possibility of a balanced insertional translocation cannot be ruled out and paren- tal studies are necessary.

TABLE 2 SUMMARY OF HIGH-RESOLUTION ANALYSIS OF 45 PWS PATIENTS

Reference Karyotype Male Female Total 1 Ledbetter et al. [16] ..... Normal 1 * del(15)(q Ilql3) * 4 4 Present study ...... Normal 9 10 19 del(l5XqlIql3) 13 4 17 del(l5Xq IqI 1) 1 1 2 015;15)(p11 Ip 1) I I nmos idic(15) (pter-qI l::ql 1-pter) 1 1 PRADER-WILLI SYNDROME 283 Balanced Rearrangements of Chromosome 15 Four of the 11 previously reported translocation PWS cases [3, 4, 6, 10] had no apparent deletion of the 15q arm. Two were 15;15 Robertsonian translocations determined to be dicentric by the presence of two C-bands [3, 4]. The case reported by Aurias et al. [6] was a balanced reciprocal translocation between chromosomes 3 and 15, with the breakpoint in 15q1 1. The case reported by Lejeune et al. [10] was mosaic for balanced translocations involving chromosome 15 and three different autosomes. The breakpoint in 15 was in the q12 band in all three cell lines. Although high-resolution techniques were not applied by any of these authors and subtle deletions cannot be ruled out, our extensive analysis of one 15;15 Robertsonian translocation by prophase methods and a combination of banding techniques also revealed no deletion of 15q arm material. Although a submicroscopic deletion may be present, alternative explanations should also be consid- ered. It is possible, although not very likely, that these balanced translocations of chromosome 15 are merely coincidental to PWS or that rearrangements involving the proximal 15q region may produce changes in gene expression via position effect, as was suggested by Lejeune et al. [10]. Little evidence for position effect exists in man, but many cases of de novo, apparently balanced translocations or inversions have been reported in association with abnormal phenotypes [23, 24]. Further analysis of these apparently balanced chromosome rearrangements among PWS patients may provide a model for the study of position effect and mechanisms of gene regulation in man. idic(15)(pter-qJ J::q II-pter) Our case mosaic for an extra chromosome that proved to be an idic(15) appears similar to two other reported cases [14, 15]. Interpretation of these cases with extra chromosomes and PWS is difficult. At least in the case reported here, prophase analysis of the two chromosome 15 homologs showed no apparent deletion in proximal 15q or aberrations of 15p. Thus, four copies of both 15p and a small segment of proximal 15q were present in 80% of the cells. The only common feature between this patient and the patients with deletions is the fact that some atteration of the region around 5qI 1 had taken place. Either the- chromosome abnormality is coincidental to the PWS phenotype, or some more complex mech- anism of genetic interaction is involved.

PWS Patients with Normal Karyotypes The finding of 20 PWS patients with normal karyotypes suggests several possi- bilities: (1) Submicroscopic deletions, not detectable by our present techniques, might be present. Development of a gene marker or DNA probe will be necessary to explore further this possibility. (2) Even though a number of cells were analyzed for each patient, we cannot rule out mosaicism for an undetected population of cells with a 15q deletion. (3) PWS may be etiologically heterogeneous, and the chromosomally abnormal cases may constitute only one subtype. An autosomal 284 LEDBETTER ET AL. recessive mode of inheritance has been suggested for PWS [25], and it may ac- count for some karyotypically normal cases. (4) The patients in our study were referred as definite or probable cases of PWS. This diagnosis is often difficult to make, reflecting the age dependence of certain features and the lack of unequivo- cal criteria for confirming the diagnosis independent of cytogenetic findings. Thus, it may well turn out that some putative PWS patients without chromosome 15 aberrations have an alternative diagnosis, although it is still premature to exclude PWS and its complications on the basis of an ostensibly normal karyotype. Fur- ther consideration of this possibility must await a more detailed clinical comparison of the karyotypically abnormal and normal cases.

SUMMARY AND CONCLUSIONS Our data indicate that half of all patients with a clinical diagnosis of PWS have cytogenetic abnormalities involving chromosome 15. For the majority, a deletion involving bands qI 1-12 is present, either by interstitial deletion or unbalanced translocation. Other cases exhibit apparently balanced translocations of chromosome 15, or extra derivative chromosomes comprised of chromosome 15 material. While we favor the view that abnormalities involving chromosome 15 can produce the set of phenotypic features recognized as PWS, the variability in chromosome 15 abnormalities suggests the possibility that complex mechanisms of genetic regulation may be involved. It is unlikely that these chromosome abnormalities are coincidental to PWS. We recommend that high-resolution chromosome analysis with special attention to chromosome 15 be performed in all patients with the clinical diagnosis of PWS. The finding of a chromosome 15 abnormality would confirm this clinical diagnosis, although at present a normal karyotype does not rule it out. We anticipate that further clinical and cytogenetic studies will aid in elucidating the apparent etiologic heterogeneity of PWS. ACKNOWLEDGMENTS We thank Drs. George Bray, Marilyn Higginbottom, Jose M. Louro, Arthur L. Beaudet, and John D. Crawford for patient referrals. A special thanks to Dr. Gerald P. Holmquist for the distamycin-A/DAPI preparations, and to Marlene Brennand for expert manuscript preparation. REFERENCES 1. HAWKEY CJ, SMITHIES A: The Prader-Willi syndrome with a 15/15 translocation. JMed Genet 14:275-278, 1976 2. EMBERGER JM, RODIERE M, ASTRUC J, BRUNEL D: Syndrome de Prader-Willi et translocation 15-15. Ann Genet (Paris) 20:297-300, 1977 3. FRACCARO M, ZUFFARDI O, BUHLER EM, JURIK LP: 15/15 Translocation in Prader-Willi syndrome. J Med Genet 14:275-278, 1977 4. 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