Sporadic imprinting defects in Prader-Willi syndrome and Angelman syndrome: implications for imprint-switch models, genetic counseling, and prenatal diagnosis.

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are caused by the loss of function of imprinted genes in proximal 15q. In approximately 2%-4% of patients, this loss of function is due to an imprinting defect. In some cases, the imprinting defect is the result of a parental imprint-switch failure caused by a microdeletion of the imprinting center (IC). Here we describe the molecular analysis of 13 PWS patients and 17 AS patients who have an imprinting defect but no IC deletion. Heteroduplex and partial sequence analysis did not reveal any point mutations of the known IC elements, either. Interestingly, all of these patients represent sporadic cases, and some share the paternal (PWS) or the maternal (AS) 15q11-q13 haplotype with an unaffected sib. In each of five PWS patients informative for the grandparental origin of the incorrectly imprinted chromosome region and four cases described elsewhere, the maternally imprinted paternal chromosome region was inherited from the paternal grandmother. This suggests that the grandmaternal imprint was not erased in the father's germ line. In seven informative AS patients reported here and in three previously reported patients, the paternally imprinted maternal chromosome region was inherited from either the maternal grandfather or the maternal grandmother. The latter finding is not compatible with an imprint-switch failure, but it suggests that a paternal imprint developed either in the maternal germ line or postzygotically. We conclude (1) that the incorrect imprint in non-IC-deletion cases is the result of a spontaneous prezygotic or postzygotic error, (2) that these cases have a low recurrence risk, and (3) that the paternal imprint may be the default imprint.     

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.     

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.     

Parent-of-origin specific histone acetylation and reactivation of a key imprinted gene locus in Prader-Willi syndrome

To examine the chromatin basis of imprinting in chromosome 15q11-q13, we have investigated the status of histone acetylation of the SNURF-SNRPN locus, which is a key imprinted gene locus in Prader-Willi syndrome (PWS). Chromatin immunoprecipitation (ChIP) studies revealed that the unmethylated CpG island of the active, paternally derived allele of SNURF-SNRPN was associated with acetylated histones, whereas the methylated maternally derived, inactive allele was specifically hypoacetylated. The body of the SNURF-SNRPN gene was associated with acetylated histones on both alleles. Furthermore, treatment of PWS cells with the DNA methyltransferase inhibitor 5-azadeoxycytidine (5-aza-dC) induced demethylation of the SNURF-SNRPN CpG island and restoration of gene expression on the maternal allele. The reactivation was associated with increased H4 acetylation but not with H3 acetylation at the SNURF-SNRPN CpG island. These findings indicate that (1) a significant role for histone deacetylation in gene silencing is associated with imprinting in 15q11-q13 and (2) silenced genes in PWS can be reactivated by drug treatment.     

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.     

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.     

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.     

Molecular characterization of Prader-Willi syndrome by real-time PCR

Prader-Willi syndrome (PWS) is a contiguous gene syndrome caused by the loss of function of genes situated within the 15q11-q13 region. The loss of function arises as a result of paternally derived mutations complemented by maternal imprinting. The molecular events underlying the disorder include interstitial deletions (70%), uniparental disomy (UPD) (25%), imprinting center defects (<5%), and rarely chromosomal translocations (<1%). The standard diagnosis of PWS is based on clinical observations and genetic investigations involving DNA methylation studies and fluorescence in situ hybridization (FISH) analysis. The absence of a paternal methylation pattern within 15q11 is sufficient for a diagnosis of PWS, and FISH analyses are used for the additional categorization of patients as either deletion or nondeletion. The main limitation of these investigations is that they neither determine the size of the molecular deletions nor permit detection of individuals with microdeletions in the PWS imprinting center that regulates imprinting in this region. We have designed and implemented a real-time PCR assay using genomic DNA and SYBR green I intercalating dye to determine the size of the chromosomal deletions in patients with PWS. This has been successfully performed on genomic DNA isolated from both peripheral blood leukocytes and buccal epithelial cells. Through this assay, the two common deletion classes in PWS were observed, and all results were 100% concordant with previous FISH assays performed on the same patients.     

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.     

An unexpected function of the Prader-Willi syndrome imprinting center in maternal imprinting in mice

Genomic imprinting is a phenomenon that some genes are expressed differentially according to the parent of origin. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurobehavioral disorders caused by deficiency of imprinted gene expression from paternal and maternal chromosome 15q11-q13, respectively. Imprinted genes at the PWS/AS domain are regulated through a bipartite imprinting center, the PWS-IC and AS-IC. The PWS-IC activates paternal-specific gene expression and is responsible for the paternal imprint, whereas the AS-IC functions in the maternal imprint by allele-specific repression of the PWS-IC to prevent the paternal imprinting program. Although mouse chromosome 7C has a conserved PWS/AS imprinted domain, the mouse equivalent of the human AS-IC element has not yet been identified. Here, we suggest another dimension that the PWS-IC also functions in maternal imprinting by negatively regulating the paternally expressed imprinted genes in mice, in contrast to its known function as a positive regulator for paternal-specific gene expression. Using a mouse model carrying a 4.8-kb deletion at the PWS-IC, we demonstrated that maternal transmission of the PWS-IC deletion resulted in a maternal imprinting defect with activation of the paternally expressed imprinted genes and decreased expression of the maternally expressed imprinted gene on the maternal chromosome, accompanied by alteration of the maternal epigenotype toward a paternal state spread over the PWS/AS domain. The functional significance of this acquired paternal pattern of gene expression was demonstrated by the ability to complement PWS phenotypes by maternal inheritance of the PWS-IC deletion, which is in stark contrast to paternal inheritance of the PWS-IC deletion that resulted in the PWS phenotypes. Importantly, low levels of expression of the paternally expressed imprinted genes are sufficient to rescue postnatal lethality and growth retardation in two PWS mouse models. These findings open the opportunity for a novel approach to the treatment of 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.     

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.     

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.     

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).     

Localization of the gene encoding the GABAA receptor β3 subunit to the Angelman/Prader-Willi region of human chromosome 15

Deletions of the proximal long arm of chromosome 15 (bands 15q11q13) are found in the majority of patients with two distinct genetic disorders, Angelman syndrome (AS) and Prader-Willi syndrome (PWS). The deleted regions in the two syndromes, defined cytogenetically and by using cloned DNA probes, are similar. However, deletions in AS occur on the maternally inherited chromosome 15, and deletions in PWS occur on the paternally derived chromosome 15. This observation has led to the suggestion that one or more genes in this region show differential expression dependent on parental origin (genetic imprinting). No genes of known function have previously been mapped to this region. We show here that the gene encoding the GABAA (gamma-aminobutyric acid) receptor beta 3 subunit maps to the AS/PWS region. Deletion of this gene (GABRB3) was found in AS and PWS patients with interstitial cytogenetic deletions. Evidence of beta 3 gene deletion was also found in an AS patient with an unbalanced 13;15 translocation but not in a PWS patient with an unbalanced 9;15 translocation. The localization of this receptor gene to the AS/PWS region suggests a possible role of the inhibitory neurotransmitter GABA in the pathogenesis of one or both of these syndromes.     

Mouse imprinting defect mutations that model Angelman syndrome

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurobehavioral disorders resulting from deficiency of imprinted gene expression from paternal or maternal chromosome 15q11-15q13, respectively. In humans, expression of the imprinted genes is under control of a bipartite cis-acting imprinting center (IC). Families with deletions causing PWS imprinting defects localize the PWS-IC to 4.3 kb overlapping with SNRPN exon 1. Families with deletions causing AS imprinting defects localize the AS-IC to 880 bp 35 kb upstream of the PWS-IC. We report two mouse mutations resulting in defects similar to that seen in AS patients with deletion of the AS-IC. An insertion/duplication mutation 13 kb upstream of Snrpn exon 1 resulted in lack of methylation at the maternal Snrpn promoter, activation of maternally repressed genes, and decreased expression of paternally repressed genes. The acquisition of a paternal epigenotype on the maternal chromosome in the mutant mice was demonstrated by the ability to rescue the lethality and growth retardation in a mouse model of a PWS imprinting defect. A second mutation, an 80-kb deletion extending upstream of the first mutation, caused a similar imprinting defect with variable penetrance. These results suggest that there is a mouse functional equivalent to the human AS-IC.