A genetic model for the Prader-Willi syndrome and its implication for angelman syndrome

Summary Sporadic cases of Prader-Willi syndrome (PWS) are associated with the physical absence of the paternal Prader-Willi chromosome region (PWCR) by deletion 15q11–13, by segmental maternal heterodisomy or by chromosome rearrangements resulting in homozygosity for maternal PWCR. In isolated/familial cases, it is proposed that the expression of PWS depends on the functional absence caused by mutated gene(s) within the paternal PWCR. The same mutation on a maternally derived chromosome 15 is not able to express PWS. An epigenetic mechanism associated with the paternal meiosis is essential. In the Angelman syndrome (AS), inverse mechanisms are postulated. There is convincing evidence for specific PWS and AS genes or alleles within PWCR. This is compatible with the observations of interstitial chromosome deletions of the critical region in normal individuals or in probands with phenotypes other than PWS or AS. The new ideas of the model stated here are: (1) the proposed epigenetic mechanism in PWCR is obviously common in humans, but is usually of no phenotypic relevance; (2) interactions with specific chromosomal or gene mutations are required for the clinical expression of PWS or AS; (3) each factor alone is not able to produce an abnormal phenotype.     

The impact of imprinting: Prader-Willi syndrome resulting from chromosome translocation, recombination, and nondisjunction.

Prader-Willi syndrome (PWS) is most often the result of a deletion of bands q11.2-q13 of the paternally derived chromosome 15, but it also occurs either because of maternal uniparental disomy (UPD) of this region or, rarely, from a methylation imprinting defect. A significant number of cases are due to structural rearrangements of the pericentromeric region of chromosome 15. We report two cases of PWS with UPD in which there was a meiosis I nondisjunction error involving an altered chromosome 15 produced by both a translocation event between the heteromorphic satellite regions of chromosomes 14 and 15 and recombination. In both cases, high-resolution banding of the long arm was normal, and FISH of probes D15S11, SNRPN, D15S10, and GABRB3 indicated no loss of this material. Chromosome heteromorphism analysis showed that each patient had maternal heterodisomy of the chromosome 15 short arm, whereas PCR of microsatellites demonstrated allele-specific maternal isodisomy and heterodisomy of the long arm. SNRPN gene methylation analysis revealed only a maternal imprint in both patients. We suggest that the chromosome structural rearrangements, combined with recombination in these patients, disrupted normal segregation of an imprinted region, resulting in uniparental disomy and PWS.     

A de novo unbalanced reciprocal translocation identified as paternal in origin in the Prader-Willi syndrome

Summary Interstitial cytogenetic deletions involving the paternally derived chromosome 15q11–13 have been described in patients with the Prader-Willi syndrome (PWS). We report a child with PWS and a de novo unbalanced karyotype –45,XY,–9,–15,+der(9)t(9;15)(q34;q13). Molecular studies with the DNA probe pML34 confirmed that only a single Prader Willi critical region (PWCR: 15q11.2-q12) copy was present. Hybridisation of patient and parental DNA with the multi-allelic probe CMW1, which maps to pter-15q13, showed that the chromosome involved in the translocation was paternal in origin. This is the first example of a paternally-derived PWCR allele loss caused by an unbalanced translocation that has arisen de novo.     

Molecular characterization of two proximal deletion breakpoint regions in both Prader-Willi and Angelman syndrome patients.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct mental retardation syndromes caused by paternal and maternal deficiencies, respectively, in chromosome 15q11-q13. Approximately 70% of these patients have a large deletion of approximately 4 Mb extending from D15S9 (ML34) through D15S12 (IR10). To further characterize the deletion breakpoints proximal to D15S9, three new polymorphic microsatellite markers were developed that showed observed heterozygosities of 60%-87%. D15S541 and D15S542 were isolated from YAC A124A3 containing the D15S18 (IR39) locus. D15S543 was isolated from a cosmid cloned from the proximal right end of YAC 254B5 containing the D15S9 (ML34) locus. Gene-centromere mapping of these markers, using a panel of ovarian teratomas of known meiotic origin, extended the genetic map of chromosome 15 by 2-3 cM toward the centromere. Analysis of the more proximal S541/S542 markers on 53 Prader-Willi and 33 Angelman deletion patients indicated two classes of patients: 44% (35/80) of the informative patients were deleted for these markers (class I), while 56% (45/80) were not deleted (class II), with no difference between PWS and AS. In contrast, D15S543 was deleted in all informative patients (13/48) or showed the presence of a single allele (in 35/48 patients), suggesting that this marker is deleted in the majority of PWS and AS cases. These results confirm the presence of two common proximal deletion breakpoint regions in both Prader-Willi and Angelman syndromes and are consistent with the same deletion mechanism being responsible for paternal and maternal deletions. One breakpoint region lies between D15S541/S542 and D15S543, with an additional breakpoint region being proximal to D15S541/S542.     

A molecular and cytogenetic study in Finnish Prader-Willi patients

The Prader-Willi syndrome (PWS) is a developmental disorder caused by a deficiency of paternal contributions, arising from differently sized deletions, uniparental disomy or rare imprinting mutations, in the chromosome region 15q11–q13. We studied 41 patients with suspected PWS and their parents using cytogenetic and molecular techniques. Of the 27 clinically typical PWS patients, 23 (85%) had a molecular deletion that could be classified into four size categories. Only 15 of them (71%) could be detected cytogenetically. Maternal uniparental heterodisomy was observed in four cases. The rest of the patients showed no molecular defects including rare imprinting mutations. In our experience, the use of the methylation test with the probe PW71 (D15S63), together with the probe hN4HS (SNRPN), which distinguishes between a deletion and uniparental disomy, is the method of choice for the diagnosis of PWS.     

Molecular diagnosis of the Prader-Willi and Angelman syndromes by detection of parent-of-origin specific DNA methylation in 15q11-13

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are distinct genetic disorders that are caused by a deletion of chromosome region 15q11-13 or by uniparental disomy for chromosome 15. Whereas PWS results from the absence of a paternal copy of 15q11-13, the absence of a maternal copy of 15q11-13 leads to AS. We have found that an MspI/HpaII restriction site at the D15S63 locus in 15q11-13 is methylated on the maternally derived chromosome, but unmethylated on the paternally derived chromosome. Based on this difference, we have devised a rapid diagnostic test for patients suspected of having PWS and AS.     

Prader-Willi syndrome with an unusually large 15q deletion due to an unbalanced translocation t(4;15)

Prader-Willi syndrome (PWS) is a neurobehavioral disorder caused by deletions in the 15q11-q13 region, by maternal uniparental disomy of chromosome 15 or by imprinting defects. Structural rearrangements of chromosome 15 have been described in about 5% of the patients with typical or atypical PWS phenotype. An 8-year-old boy with a clinical diagnosis of PWS, severe neurodevelopmental delay, absence of speech and mental retardation was studied by cytogenetic and molecular techniques, and an unbalanced de novo karyotype 45,XY,der(4)t(4;15)(q35;q14),-15 was detected after GTG-banding. The patient was diagnosed by SNURF-SNRPN exon 1 methylation assay, and the extent of the deletions on chromosomes 4 and 15 was investigated by microsatellite analysis of markers located in 4qter and 15q13-q14 regions. The deletion of chromosome 4q was distal to D4S1652, and that of chromosome 15 was located between D15S1043 and D15S1010. Our patient's severely affected phenotype could be due to the extent of the deletion, larger than usually seen in PWS patients, although the unbalance of the derivative chromosome 4 cannot be ruled out as another possible cause. The breakpoint was located in the subtelomeric region, very close to the telomere, a region that has been described as having the lowest gene concentrations in the human genome.     

A combination of five short tandem repeats of chromosome 15 significantly improves the identification of Prader-Willi syndrome etiology in the Argentinean population

Prader-Willi syndrome (PWS) is a multisystemic disorder caused by the loss of expression of paternally transcribed genes in the PWS critical region of chromosome 15. Various molecular mechanisms are known to lead to PWS: deletion 15q11-q13 (75% of cases), maternal uniparental disomy (matUPD15) (23%) and imprinting defects (2%). FISH and microsatellite analysis are required to establish the molecular etiology, which is essential for appropriate genetic counseling and care management. We characterized an Argentinean population, using five microsatellite markers (D15S1035, D15S11, D15S113, GABRB3, D15S211) chosen to develop an appropriate cost-effective method to establish the parental origin of chromosome 15 in nondeleted PWS patients. The range of heterozygosity for these five microsatellites was 0.59 to 0.94. The average heterozygosity obtained for joint loci was 0.81. The parental origin of chromosome 15 was established by microsatellite analysis in 19 of 21 non-deleted PWS children. We also examined the origin of the matUPD15; as expected, most of disomies were due to a maternal meiosis I error. The molecular characterization of this set of five microsatellites with high heterozygosity and polymorphism information content improves the diagnostic algorithm of Argentinean PWS children, contributing significantly to adequate genetic counseling of such families.     

Routine screening for microdeletions by FISH in 77 patients suspected of having Prader-Willi or Angelman syndromes using YAC clone 273A2 (D15S10)

About 70% of patients with Prader-Willi syndrome (PWS) and Angelman syndrome (AS) have a common interstitial de novo microdeletion encompassing paternal (PWS) or maternal (AS) loci D15S9 to D15S12. Most of the non-deletion PWS patients and a small number of non-deletion AS patients have a maternal or paternal uniparental disomy (UPD)15, respectively. Other chromosome 15 rearrangements and a few smaller atypical deletions, some of the latter being associated with an abnormal methylation pattern, are rarely found. Molecular and fluorescence in situ hybridization (FISH) analysis have both been used to diagnose PWS and AS. Here, we have evaluated, in a typical routine cytogenetic laboratory setting, the efficiency of a diagnostic strategy that starts with a FISH deletion assay using Alu-PCR (polymerase chain reaction)-amplified D15S10-positive yeast artificial chromosome (YAC) 273A2. We performed FISH in 77 patients suspected of having PWS (n = 66) or AS (n = 11) and compared the results with those from classical cytogenetics and wherever possible with those from DNA analysis. A FISH deletion was found in 16/66 patients from the PWS group and in 3/11 patients from the AS group. One example of a centromere 15 co-hybridization performed in order to exclude cryptic translocations or inversions is given. Of the PWS patients, 14 fulfilled Holm’s criteria, but two did not. DNA analysis confirmed the commmon deletion in all patients screened by the D15S63 methylation test and in restriction fragment length polymorphism dosage blots. In 3/58 non-deletion patients, other chromosomal aberrations were found. Of the non-deleted group, 27 subjects (24 PWS, 3 AS) were tested molecularly, and three patients with an uniparental methylation pattern were found in the PWS group. The other 24/27 subjects had neither a FISH deletion nor uniparental methylation, but two had other cytogenetic aberrations. Given that cytogenetic analysis is indispensable in most patients, we find that the FISH deletion assay with YAC 273A2 is an efficient first step for stepwise diagnostic testing and mutation-type analysis of patients suspected of having PWS or AS. Received: 14 November 1995     

A DNA methylation imprint, determined by the sex of the parent, distinguishes the angelman and Prader-Willi syndromes

The Angelman (AS) and Prader-Willi (PWS) syndromes are two clinically distinct disorders that are caused by a differential parental origin of chromosome 15q11-q13 deletions. Both also can result from uniparental disomy (the inheritance of both copies of chromosome 15 from only one parent). Loss of the paternal copy of 15q11-q13, whether by deletion or maternal uniparental disomy, leads to PWS, whereas a maternal deletion or paternal uniparental disomy leads to AS. The differential modification in expression of certain mammalian genes dependent upon parental origin is known as genomic imprinting, and AS and PWS represent the best examples of this phenomenon in humans. Although the molecular mechanisms of genomic imprinting are unknown, DNA methylation has been postulated to play a role in the imprinting process. Using restriction digests with the methyl-sensitive enzymes HpaII and HhaI and probing Southern blots with several genomic and cDNA probes, we have systematically scanned segments of 15q11-q13 for DNA methylation differences between patients with PWS (20 deletion, 20 uniparental disomy) and those with AS (26 deletion, 1 uniparental disomy). The highly evolutionarily conserved cDNA, DN34, identifies distinct differences in DNA methylation of the parental alleles at the D15S9 locus. Thus, DNA methylation may be used as a reliable, postnatal diagnostic tool in these syndromes. Furthermore, our findings demonstrate the first known epigenetic event, dependent on the sex of the parent, for a locus within 15q11-q13. We propose that expression of the gene detected by DN34 is regulated by genomic imprinting and, therefore, that it is a candidate gene for PWS and/or AS.     

Molecular, cytogenetic, and clinical investigations of Prader-Willi syndrome patients.

Thirty-seven patients presenting features of the Prader-Willi syndrome (PWS) have been examined using cytogenetic and molecular techniques. Clinical evaluation showed that 29 of these patients fulfilled diagnostic criteria for PWS. A deletion of the 15q11.2-q12 region could be identified molecularly in 21 of these cases, including several cases where the cytogenetics results were inconclusive. One clinically typical patient is deleted at only two of five loci normally included in a PWS deletion. A patient carrying a de novo 13;X translocation was not deleted for the molecular markers tested but was clinically considered to be "atypical" PWS. In addition, five cases of maternal heterodisomy and two of isodisomy for 15q11-q13 were observed. All of the eight patients who did not fulfill clinical diagnosis of PWS showed normal maternal and paternal inheritance of chromosome 15 markers; however, one of these carried a ring-15 chromosome. A comparison of clinical features between deletion patients and disomy patients shows no significant differences between the two groups. The parental ages at birth of disomic patients were significantly higher than those for deletion patients. As all typical PWS cases showed either a deletion or disomy of 15q11.2-q12, molecular examination should provide a reliable diagnostic tool. As the disomy patients do not show either any additional or more severe features than typical deletion patients do, it is likely that there is only one imprinted region on chromosome 15 (within 15q11.2-q12).     

FISH Variants with D15Z1

We present our experience with cross-hybridization of D15Z1, used in combination with D15S10, D15S11 or SNRPN, in 109 clinical cases referred for Angelman syndrome (AS), Prader-Willi syndrome (PWS), for autism to rule out duplication of 15q11.2, or to identify structural chromosome abnormalities thought to involve chromosome 15. Nine cases with normal karyotypes studied with at least one of these probe mixtures showed an extra signal with D15Z1 on a chromosome 14. One case showed absence of the D15Z1 signal from 15p and one case showed an extra signal with D15Z1 on both chromosome 14s. Sixteen cases from this series had structural abnormalities, which included ten cases with supernumerary markers, three of which had a D15Z1 signal on a chromosome 14. The remaining cases did not have an extra signal on chromosome 14, but included rearrangements between Y and 15, 15 and 19, and a r(15), all with breakpoints in 15p11.1 or p11.2. Of the three cases with a supernumerary marker and an extra D15Z1 signal on a chromosome 14, one was a maternally derived marker, while the variant 14 was paternal in origin. The other two markers were de novo. The high frequency of variant 14 in cases with supernumerary markers (30%) was not significant by Chi-square analysis compared to the overall frequency in 109 cases of 11.9%. The overall frequency is consistent with a previous report by Stergianou et al. (1993). We can now add that a false-negative result may occur slightly less than 1% of the time. The chance that both chromosome 14 homologs will be positive for D15Z1 is theoretically about 1 in 300. If associated with an abnormal phenotype, the possibility of uniparental disomy should be ruled out.     

Bloom Syndrome and Maternal Uniparental Disomy for Chromosome 15

Bloom syndrome (BS) is an autosomal recessive disorder characterized by increases in the frequency of sister-chromatid exchange and in the incidence of malignancy. Chromosome-transfer studies have shown the BS locus to map to chromosome 15q. This report describes a subject with features of both BS and Prader-Willi syndrome (PWS). Molecular analysis showed maternal uniparental disomy for chromosome 15. Meiotic recombination between the two disomic chromosomes 15 has resulted in heterodisomy for proximal 15q and isodisomy for distal 15q. In this individual BS is probably due to homozygosity for a gene that is telomeric to D15S95 (15q25), rather than to genetic imprinting, the mechanism responsible for the development of PWS. This report represents the first application of disomy analysis to the regional localization of a disease gene. This strategy promises to be useful in the genetic mapping of other uncommon autosomal recessive conditions.     

A candidate model for angelman syndrome in the mouse

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are well-recognized examples of imprinting in humans. They occur most commonly with paternal and maternal 15q11-13 deletions, but also with maternal and paternal disomy. Both syndromes have also occurred more rarely in association with smaller deletions seemingly causing abnormal imprinting. A putative mouse model of PWS, occurring with maternal duplication (partial maternal disomy) for the homologous region, has been described in a previous paper but, although a second imprinting effect that could have provided a mouse model of AS was found, it appeared to be associated with a slightly different region of the chromosome. Here, we provide evidence that the same region is in fact involved and further demonstrate that animals with paternal duplication for the region exhibit characteristics of AS patients. A mouse model of AS is, therefore, strongly indicated. Received: 15 December 1996 / Accepted: 31 January 1997     

Chromosome 15 uniparental disomy is not frequent in Angelman syndrome.

Genetic imprinting has been implicated in the etiology of two clinically distinct but cytogenetically indistinguishable disorders--Angelman syndrome (AS) and Prader-Willi syndrome (PWS). This hypothesis is derived from two lines of evidence. First, while the molecular extents of de novo cytogenetic deletions of chromosome 15q11q13 in AS and PWS patients are the same, the deletions originate from different parental chromosomes. In AS, the deletion occurs in the maternally inherited chromosome 15, while in PWS the deletion is found in the paternally inherited chromosome 15. The second line of evidence comes from the deletion of an abnormal parental contribution of 15q11q13 in PWS patients without a cytogenetic and molecular deletion. These patients have two maternal copies and no paternal copy of 15q11q13 (maternal uniparental disomy) instead of one copy from each parent. By qualitative hybridization with chromosome 15q11q13 specific DNA markers, we have now examined DNA samples from 10 AS patients (at least seven of which are familial cases) with no cytogenetic or molecular deletion of chromosome 15q11q13. Inheritance of one maternal copy and one paternal copy of 15q11q13 was observed in each family, suggesting that paternal uniparental disomy of 15q11q13 is not responsible for expression of the AS phenotype in these patients.     

A case of segmental paternal isodisomy of chromosome 14

Uniparental disomy of chromosome 14 (UPD 14) results in one of two distinct abnormal phenotypes, depending upon the parent of origin. This discordance may result from the reciprocal over-expression and/or under-expression of one or more imprinted genes. We report a case of segmental paternal isodisomy for chromosome 14 with features similar to those reported in other paternal disomy 14 cases. Microsatellite marker analysis revealed an apparent somatic recombination event in 14q12 leading to proximal biparental inheritance, but segmental paternal uniparental isodisomy distal to this site. Analysis of monochromosomal somatic cell hybrids containing either the paternally inherited or the maternally inherited chromosome 14 revealed no deletion of the maternally inherited chromosome 14 and demonstrated the presence of paternal sequences from D14S121 to the telomere on both chromosomes 14. Thus, the patient has paternal isodisomy for 14q12-14qter. Because the patient shows most of the features associated with paternal disomy 14, this supports the presence of the imprinted domain(s) distal to 14q12 and suggests that the proximal region of chromosome 14 does not contain imprinted genes that contribute significantly to the paternal UPD 14 phenotype.     

Imprinting mutations suggested by abnormal DNA methylation patterns in familial Angelman and Prader-Willi syndromes.

The D15S9 and D15S63 loci in the Prader-Willi/Angelman syndrome region on chromosome 15 are subject to parent-of-origin-specific DNA methylation. We have found two Prader-Willi syndrome families in which the patients carry a maternal methylation imprint on the paternal chromosome. In one of these families, the patients have a small deletion encompassing the gene for the small nuclear ribonucleoprotein polypeptide N, which maps 130 kb telomeric to D15S63. Furthermore, we have identified a pair of nondeletion Angelman syndrome sibs and two isolated Angelman syndrome patients who carry a paternal methylation imprint on the maternal chromosome. These Angelman and Prader-Willi syndrome patients may have a defect in the imprinting process in 15q11-13. We propose a model in which a cis-acting mutation prevents the resetting of the imprinting signal in the germ line and thus disturbs the expression of imprinted genes in this region.     

Angelman syndrome: three molecular classes identified with chromosome 15q11q13-specific DNA markers

Angelman syndrome (AS) and Prader-Willi syndrome (PWS) share a cytogenetic deletion of chromosome 15q11q13. To determine the extent of deletion in AS we analyzed the DNA of 19 AS patients, including two sib pairs, with the following chromosome 15q11q13--specific DNA markers: D15S9-D15S13, D15S17, D15S18, and D15S24. Three molecular classes were identified. Class I showed a deletion of D15S9-D15S13 and D15S18; class II showed a deletion of D15S9-D15S13; and in class III, including both sib pairs, no deletion was detected. These molecular classes appear to be identical to those observed in PWS. High-resolution cytogenetic data were available on 16 of the patients, and complete concordance between the presence of a cytogenetic deletion and a molecular deletion was observed. No submicroscopic deletions were detected. DNA samples from the parents of 10 patients with either a class I or a class II deletion were available for study. In seven of the 10 families, RFLPs were informative as to the parental origin of the deletion. In all informative families, the deleted chromosome 15 was observed to be of maternal origin. This finding is in contrast to the paternal origin of the deletions in PWS and is currently the only molecular difference observed between the two syndromes.     

普拉德-威利综合征的分子遗传诊断:一种连锁分析方法的初步建立

普拉德-威利综合征(Prader-Willi Syndrome,PWS)是一种基因组印记相关的疾病,是引起肥胖最常见的遗传综合征。分子和细胞遗传学检查对于该病早期诊断非常重要。通过选择PWS典型缺失区域内、外的STR遗传标记,初步建立了一种适用于中国人群的PWS核心家庭连锁分析方法,并用该方法确定了一例缺失型和一例异源单亲二体型PWS患者,经甲基化特异性PCR和高分辨染色体核型分析验证上述结果正确。同时,该连锁分析方法可以具体区分PWS的分子发病类型,从而为PWS家庭的遗传咨询提供信息,并为进一步研究PWS基因型和表型的关系提供了可能。     

Combined cytogenetic and molecular analyses for the diagnosis of Prader-Willi/Angelman syndromes

Prader-Willi (PWS) and Angelman (AS) are syndromes of developmental impairment that result from the loss of expression of imprinted genes in the paternal (PWS) or maternal (AS) 15q11-q13 chromosome. Diagnosis on a clinical basis is difficult in newborns and young infants; thus, a suitable molecular test capable of revealing chromosomal abnormalities is required. We used a variety of cytogenetic and molecular approaches, such as, chromosome G banding, fluorescent in situ hybridization, a DNA methylation test, and a set of chromosome 15 DNA polymorphisms to characterize a cohort of 27 PWS patients and 24 suspected AS patients. Molecular analysis enabled the reliable diagnosis of 14 PWS and 7 AS patients, and their classification into four groups: (A) 6 of these 14 PWS subjects (44 %) had deletions of paternal 15q11-q13; (B) 4 of the 7 AS patients had deletions of maternal 15q11-q13; (C) one PWS patient (8 %) had a maternal uniparental disomy (UPD) of chromosome 15; (D) the remaining reliably diagnoses of 7 PWS and 3 AS cases showed abnormal methylation patterns of 15q11-q13 chromosome, but none of the alterations shown by the above groups, although they may have harbored deletions undetected by the markers used. This study highlights the importance of using a combination of cytogenetic and molecular tests for a reliable diagnosis of PWS or AS, and for the identification of genetic alterations.