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.     

Familial cryptic translocation resulting in Angelman syndrome:implications for imprinting or location of the Angelman gene?

Angelman syndrome (AS) is associated with a loss of maternal genetic information, which typically occurs as a result of a deletion at 15q11-q13 or paternal uniparental disomy of chromosome 15. We report a patient with AS as a result of an unbalanced cryptic translocation whose breakpoint, at 15q11.2, falls within this region. The proband was diagnosed clinically as having Angelman syndrome, but without a detectable cytogenetic deletion, by using high-resolution G-banding. FISH detected a deletion of D15S11 (IR4-3R), with an intact GABRB3 locus. Subsequent studies of the proband's mother and sister detected a cryptic reciprocal translocation between chromosomes 14 and 15 with the breakpoint being between SNRPN and D15S10 (3- 21). The proband was found to have inherited an unbalanced form, being monosomic from 15pter through SNRPN and trisomic for 14pter to 14q11.2. DNA methylation studies showed that the proband had a paternal-only DNA methylation pattern at SNRPN, D15S63 (PW71), and ZNF127. The mother and unaffected sister, both having the balanced translocation, demonstrated normal DNA methylation patterns at all three loci. These data suggest that the gene for AS most likely lies proximal to D15S10, in contrast to the previously published position, although a less likely possibility is that the maternally inherited imprinting center acts in trans in the unaffected balanced translocation carrier sister.     

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.     

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.     

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.     

Clinical,cytogenetical and molecular analyses of Angelman syndrome

A total of 95 patients suspected with the clinical diagnosis of AS were evaluated and 37 cases (39%) were confirmed by cytogenetic or molecular studies as affected by Angelman syndrome. The clinical analysis was performed according to a specific clinical protocol for the diagnosis of AS. Cytogenetical analysis was used to detect chromosome rearrangements by determining the karyotype in lymphocytes by GTG banding and revealed an abnormal karyotype in two cases (5.4%), both of them presenting a new pericentromeric inversion in chromosome 15. Molecular analyses included determination of DNA methylation within the 15q11-13 region by Southern blotting and microsattelite analysis within the 15q11-13 region by PCR and the UBE3A gene was also studied by mutational screening. In 16 cases (43.2%) a de novo deletion was detected in the maternal chromosome 15:3 cases (8.1%) presented imprinting defect at the 15q11-13 region; one case is due to a paternal uniparental dissomy (2.7%) and another two cases showed a inherited mutation at the UBE3A gene (5.4%). Thirteen cases (35.1%) showed no deletion, no UPD, no imprinting defect, no UBE3A mutation and the diagnosis of AS could be ruled out in 58 patients. The objective of the present work was to describe the clinical and laboratory protocols employed at our laboratory in order to establish the AS study. We conclude that the protocols employed here were efficient for the diagnosis of AS, a frequently underdiagnosed pathology.     

An unexpected recurrence of Angelman syndrome suggestive of maternal germ-line mosaicism of del(15)(q11q13) in a Finnish family

Angelman syndrome is a neuro-developmental disorder caused by genetic abnormalities affecting the maternal gene expression in the chromosome region 15q11-q13. In a study group of 45 Finnish Angelman patients, a recurrence of a del(15)(q11q13) was detected in one family. The mother's chromosomes 15 were structurally normal, whereas the patients and their unaffected brother shared an identical maternally derived haplotype outside the deletion region. These findings are suggestive of maternal germ-line mosaicism of del(15)(q11q13).     

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     

Angelman syndrome associated with an inversion of chromosome 15q11.2q24.3.

Angelman syndrome (AS) most frequently results from large (> or = 5 Mb) de novo deletions of chromosome 15q11-q13. The deletions are exclusively of maternal origin, and a few cases of paternal uniparental disomy of chromosome 15 have been reported. The latter finding indicates that AS is caused by the absence of a maternal contribution to the imprinted 15q11-q13 region. Failure to inherit a paternal 15q11-q13 contribution results in the clinically distinct disorder of Prader-Willi syndrome. Cases of AS resulting from translocations or pericentric inversions have been observed to be associated with deletions, and there have been no confirmed reports of balanced rearrangements in AS. We report the first such case involving a paracentric inversion with a breakpoint located approximately 25 kb proximal to the reference marker D15S10. This inversion has been inherited from a phenotypically normal mother. No deletion is evident by molecular analysis in this case, by use of cloned fragments mapped to within approximately 1 kb of the inversion breakpoint. Several hypotheses are discussed to explain the relationship between the inversion and the AS phenotype.     

Clinical Application of an Innovative Multiplex-Fluorescent-Labeled STRs Assay for Prader-Willi Syndrome and Angelman Syndrome

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two clinically distinct neurodevelopmental disorders caused by absence of paternally or maternally expressed imprinted genes on chr15q11.2-q13.3. Three mechanisms are known to be involved in the pathogenesis: microdeletions, uniparental disomy (UPD) and imprinting defects. Both disorders are difficult to be definitely diagnosed at early age if no available molecular cytogenetic tests. In this study, we identified 5 AS patients with the maternal deletion and 26 PWS patients with paternal deletion on chr15q11-q13 by using an innovative multiplex-fluorescent-labeled short tandem repeats (STRs) assay based on linkage analysis, and validated by the methylation-specific PCR and array comparative genomic hybridization techniques. More interesting, one of these PWS patients was confirmed as maternal uniparental isodisomy by the STR linkage analysis. The phenotypic and genotypic characteristics of these individuals were also presented. Our results indicate that the new linkage analysis is much faster and easier for large-scale screening deletion and uniparental disomy, thus providing a valuable method for early diagnosis of PWS/AS patients, which is critical for genetic diagnosis, management and improvement of prognosis.     

Evaluation of methylation analysis for diagnostic testing in 258 referrals suspected of Prader-Willi or Angelman syndromes

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurodevelopmental disorders with interrelated genetic mechanisms because genomic imprinting within the chromosome 15q11–13 region affects both the PWS and the AS locus. Methylation analysis is one method of distinguishing between the maternally and paternally inherited chromosome 15. Here we present clinical and molecular data on a large series of 258 referred patients, evaluated with methylation analysis: 115 with suspected PWS and 143 with suspected AS. In these patients, the clinical phenotype was graded into three groups: classical (group 1); not classical but possible (group 2); not classical and unlikely (group 3). For PWS, a fourth group consisted of hypotonic babies. DNA methylation analysis confirmed the diagnosis of PWS in 30 patients (26%) and AS in 28 patients (20%). For 21 PWS patients the mechanism was established: 15 had deletions, 4 had uniparental disomy (UPD) and 2 a presumed imprinting defect. Clinically all those with an abnormal methylation pattern had the classical phenotype and none of those with a normal methylation pattern had classical PWS. For 23 AS patients in whom a mechanism was established, 17 had a deletion, 3 had UPD and 3 had a presumed imprinting defect. There was greater clinical overlap in AS, with 26 classical AS patients having a normal methylation pattern while an abnormal methylation pattern was seen in one patient from group 2. In addition, there were a further 40 patients with a normal methylation pattern in whom AS was still a possible diagnosis. Our conclusion is that methylation analysis provides an excellent screening test for both syndromes, providing ∼99% diagnosis for PWS and for AS, a 75% diagnostic rate, supplemented for the remaining 25% with an essential basic starting point to further investigations. Received: 10 February 1998 / Accepted: 7 July 1998     

The Mouse Pink-Eyed Dilution Gene: Association with Hypopigmentation in Prader-Willi and Angelman Syndromes and with Human OCA2

Mutations at the mouse pink-eyed dilution locus, p, cause hypopigmentation. We have cloned the mouse p gene cDNA and the cDNA of its human counterpart, P. The region of mouse chromosome 7 containing the p locus is syntenic with human chromosome 15q11-q13, a region associated with Prader-Willi syndrome (PWS) and Angelman syndrome (AS), both of which involve profound imprinting effects. PWS patients lack sequences of paternal origin from 15q, whereas AS patients lack a maternal copy of an essential region from 15q. However, the critical regions for these syndromes are much smaller than the chromosomal region commonly deleted that often includes the P gene. Hypopigmentation in PWS and AS patients is correlated with deletions of one copy of the human P gene that is highly homologous with its mouse counterpart. A subset of PWS and AS patients also have OCA2. These patients lack one copy of the P gene in the context of a PWS or AS deletion, with a mutation in the remaining chromosomal homologue of the P gene. Mutations in both homologues of the P gene of OCA2 patients who do not have PWS or AS have also been detected.     

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.     

Reciprocal translocation between the proximal regions of the long arms of chromosomes 13 and 15 resulting in unbalanced offspring: characterization by fluorescence in situ hybridization and DNA analysis

Summary We describe two female siblings with similar clinical features consisting of hydrocephalus, scaphocephaly, hypotonia, mongoloid eye slant, blepharophimosis, micrognathia, supernumerary mouth frenula and mental retardation. Routine cytogenetic studies in the elder patient did not reveal any abnormality, and initially it was assumed that the syndrome had an autosomal recessive inheritance. However, a slightly larger chromosome 13 was seen in routine G-banded metaphases of the mother and the youngest of the two siblings. A shorter chromosome 15 was detected in the mother only. High resolution banding showed that the abnormal chromosome 13 contained an extra G-positive band at 13q12. The short chromosome 15 in the mother appeared to have a deletion of band q12. Fluorescence in situ hybridization using DNA markers specific to chromosomes 13 and 15 unequivocally showed that the mother was a carrier of a balanced reciprocal translocation t(13;15)(q12;q13), whereas the youngest sibling's karyotype was 46,XX,-13,+der(15)t(13;15)(q12;q13)mat, resulting in partial monosomy 13pterq12 and partial trisomy 15pterq13. The proband is thus trisomie for the critical region responsible for Prader-Willi syndrome and Angelman syndrome; this was confirmed by DNA analysis demonstrating one paternal and two maternal alleles from multiallelic marker loci mapping to 15q11-q13. This report illustrates the sensitivity and specificity offered by fluorescence in situ hybridization and its usefulness in the diagnosis and delineation of subtle chromosomal rearrangements.     

A 5-kb imprinting center deletion in a family with Angelman syndrome reduces the shortest region of deletion overlap to 880 bp

Imprinting on human chromosome 15q11-q13 is controlled by a bipartite imprinting center (IC) that maps to the SNRPN locus. Deletions of the IC result in an imprinting defect and Prader-Willi syndrome or Angelman syndrome (AS). We have now identified a 5-kb IC deletion in an English AS patient (AS-LO); this represents the smallest microdeletion found in AS and narrows down the shortest region of deletion overlap to 880 bp.     

Methylation-specific multiplex ligation-dependent probe amplification analysis of subjects with chromosome 15 abnormalities

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurodevelopmental disorders caused by loss of expression of imprinted genes from the 15q11-q13 region. They arise from similar defects in the region but differ in parent of origin. There are two recognized typical 15q11-q13 deletions depending on size and several diagnostic assays are available but each has limitations. We evaluated the usefulness of a methylation-specific multiplex ligation-dependent probe amplification (MLPA) kit consisting of 43 probes to detect copy number changes and methylation status in the region. We used the MLPA kit to genotype 82 subjects with chromosome 15 abnormalities (62 PWS, 10 AS and 10 individuals with other chromosome 15 abnormalities) and 13 with normal cytogenetic findings. We developed an algorithm for MLPA probe analysis which correctly identified methylation abnormalities associated with PWS and AS and accurately determined copy number in previously assigned genetic subtypes including microdeletions of the imprinting center. Furthermore, MLPA analysis identified copy number changes in those with distal 15q deletions and ring 15s. MLPA is a relatively simple, cost-effective technique found to be useful and accurate for methylation status, copy number and analysis of genetic subtype in PWS and AS, as well as other chromosome 15 abnormalities.     

Molecular mechanism of angelman syndrome in two large families involves an imprinting mutation.

Patients with Angelman syndrome (AS) and Prader-Willi syndrome with mutations in the imprinting process have biparental inheritance but uniparental DNA methylation and gene expression throughout band 15q11-q13. In several of these patients, microdeletions upstream of the SNRPN gene have been identified, defining an imprinting center (IC) that has been hypothesized to control the imprint switch process in the female and male germlines. We have now identified two large families (AS-O and AS-F) segregating an AS imprinting mutation, including one family originally described in the first genetic linkage of AS to 15q11-q13. This demonstrates that this original linkage is for the 15q11-q13 IC. Affected patients in the AS families have either a 5.5- or a 15-kb microdeletion, one of which narrowed the shortest region of deletion overlap to 1.15 kb in all eight cases. This small region defines a component of the IC involved in AS (ie., the paternal-to-maternal switch element). The presence of an inherited imprinting mutation in multiple unaffected members of these two families, who are at risk for transmitting the mutation to affected children or children of their daughters, raises important genetic counseling issues.     

Epimutations in Prader-Willi and Angelman syndromes: a molecular study of 136 patients with an imprinting defect

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurogenetic disorders that are caused by the loss of function of imprinted genes in 15q11-q13. In a small group of patients, the disease is due to aberrant imprinting and gene silencing. Here, we describe the molecular analysis of 51 patients with PWS and 85 patients with AS who have such a defect. Seven patients with PWS (14%) and eight patients with AS (9%) were found to have an imprinting center (IC) deletion. Sequence analysis of 32 patients with PWS and no IC deletion and 66 patients with AS and no IC deletion did not reveal any point mutation in the critical IC elements. The presence of a faint methylated band in 27% of patients with AS and no IC deletion suggests that these patients are mosaic for an imprinting defect that occurred after fertilization. In patients with AS, the imprinting defect occurred on the chromosome that was inherited from either the maternal grandfather or grandmother; however, in all informative patients with PWS and no IC deletion, the imprinting defect occurred on the chromosome inherited from the paternal grandmother. These data suggest that this imprinting defect results from a failure to erase the maternal imprint during spermatogenesis.     

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.