Prader-Willi-Syndrom und Angelman-Syndrom

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two distinct neurogenetic disorders caused by the loss of function of imprinted genes in the chromosomal region 15q11q13. An approximately 2 Mb region inside 15q11q13 is subject to genomic imprinting. As a consequence the maternal and paternal copies in this region are different in DNA methylation and gene expression. The most frequent genetic lesions in both disorders are an interstitial de novo deletion of the chromosomal region 15q11q13, uniparental disomy 15, an imprinting defect or, in the case of AS, a mutation of the UBE3A gene. Microdeletions in a small number of patients with PWS and AS with an imprinting defect have led to the identification of the chromosome 15 imprinting centre (IC) upstream of the SNURF-SNRPN gene, which acts in cis to regulate imprinting in the whole 15q imprinted domain. The IC consists of two critical elements: one in the more centromeric part which is deleted in patients with AS and which is thought to be responsible for the establishment of imprinting in the female germ line, and one in the more telomeric part which is deleted in patients with PWS and which is required to maintain the paternal imprint.     

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.     

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     

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 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.     

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.     

A modified MS-PCR approach to diagnose patients with Prader-Willi and Angelman syndrome

Prader-Willi (PWS) and Angelman (AS) syndromes are clinically distinct neurodevelopmental genetic diseases with multiple phenotypic manifestations. They are one of the most common genetic syndromes caused by non-Mendelian inheritance in the form of genomic imprinting, and can be attributable to the loss of gene expression due to imprinting within the chromosomal region 15q11-q13. Clinical diagnosis of PWS and AS is challenging, and the use of molecular and cytomolecular studies is recommended to help in determining the diagnosis of these conditions. The methylation analysis is a sensible approach; however there are several techniques for this purpose, such as the methylation-sensitive polymerase chain reaction (MS-PCR). This study aims to optimize the MS-PCR assay for the diagnosis of potential PWS and AS patients using DNA modified by sodium bisulfite. We used the MS-PCR technique of PCR described by Kosaki et al. (1997) adapted with betaine. All different concentrations of betaine used to amplify the methylated and unmethylated chromosomal region 15q11-q13 on the gene SNRPN showed amplification results, which increased proportionally to the concentration of betaine. The methylation analysis is a technically robust and reproducible screening method for PWS and AS. The MS-PCR assures a faster, cheaper and more efficient method for the primary diagnosis of the SNRPN gene in cases with PWS and AS, and may detect all of the three associated genetic abnormalities: deletion, uniparental disomy or imprinting errors.     

Imprinting defects on human chromosome 15

The Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two distinct neurogenetic diseases that are caused by the loss of function of imprinted genes on the proximal long arm of human chromosome 15. In a few percent of patients with PWS and AS, the disease is due to aberrant imprinting and gene silencing. In patients with PWS and an imprinting defect, the paternal chromosome carries a maternal imprint. In patients with AS and an imprinting defect, the maternal chromosome carries a paternal imprint. Imprinting defects offer a unique opportunity to identify some of the factors and mechanisms involved in imprint erasure, resetting and maintenance. In approximately 10% of cases the imprinting defects are caused by a microdeletion affecting the 5' end of the SNURF-SNRPN locus. These deletions define the 15q imprinting center (IC), which regulates imprinting in the whole domain. These findings have been confirmed and extended in knock-out and transgenic mice. In the majority of patients with an imprinting defect, the incorrect imprint has arisen without a DNA sequence change, possibly as the result of stochastic errors of the imprinting process or the effect of exogenous factors.     

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 targeted deletion upstream of <Emphasis Type="Italic">Snrpn</Emphasis> does not result in an imprinting defect

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) result from the disturbance of imprinted gene expression within human chromosome 15q11–q13. Some cases of PWS and AS are caused by microdeletions near the SNRPN gene that disrupt a regulatory element termed the imprinting center (IC). The IC has two functional components; an element at the promoter of SNRPN involved in PWS (PWS-IC) and an element 35 kilobases (kb) upstream of SNRPN involved in AS (AS-IC). To further understand the function of the IC, we sought to create a mouse model for AS-IC mutations. We have generated two deletions at a location analogous to that of the human AS-IC. Neither deletion produced an imprinting defect as indicated by DNA methylation and gene expression analyses. These results indicate that no elements critical for AS-IC function in mouse reside within the 12.8-kb deleted region and suggest that the specific location of the AS-IC is not conserved between human and mouse. Camilynn I. Brannan was Deceased     

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.     

Detection of a novel familial deletion of four genes between BP1 and BP2 of the Prader-Willi/Angelman syndrome critical region by oligo-array CGH in a child with neurological disorder and speech impairment

Two common classes of deletions are described in the literature in individuals with Prader-Willi/Angelman syndrome (PWS/AS): one between breakpoint 1 (BP1) to BP3 and the other between BP2 to BP3 of the PWS/AS critical region on chromosome 15q11-->q13. We present here a novel observation of an approximately 253-kb deletion between BP1 and BP2 on 15q11.2, in a 3(1/2)-year-old boy, who was referred to us with a clinical suspicion of having Angelman syndrome and presenting with mental retardation, neurological disorder, developmental delay and speech impairment. Karyotype and FISH results were found to be normal. The microdeletion between BP1 and BP2 includes four genes - NIPA1, NIPA2, CYFIP1 and TUBGCP5 which was detected by a high-resolution oligonucleotide array-CGH that was further validated by a Multiplex Ligation-dependent Probe Amplification (MLPA) assay. The same deletion was observed in the father who presented with similar but relatively milder clinical features as compared to the affected son. Methylation studies by methylation-specific MLPA (MS-MLPA) of the SNRPN imprinting center (IC) showed a normal imprinting pattern, both in the patient and the father. To our knowledge a microdeletion limited only to the BP1-BP2 region has not yet been reported. The familial genetic alteration together with the striking clinical presentation in this study are interesting, but from our single case study it is difficult to suggest if the deletion is causative of some of the abnormal features or if it is a normal variant. The study however further strengthens the fact that genome-wide analysis by array CGH in individuals with developmental delay and mental retardation is very useful in detecting such hidden interstitial chromosomal rearrangements.     

A novel, single nucleotide polymorphism-based assay to detect 22q11 deletions

Velocardiofacial syndrome, DiGeorge syndrome, and conotruncal anomaly face syndrome, now collectively referred to as 22q11deletion syndrome (22q11DS) are caused by microdeletions on chromosome 22q11. The great majority ( approximately 90%) of these deletions are 3 Mb in size. The remaining deleted patients have nested break-points resulting in overlapping regions of hemizygosity. Diagnostic testing for the disorder is traditionally done by fluorescent in situ hybridization (FISH) using probes located in the proximal half of the region common to all deletions. We developed a novel, high-resolution single-nucleotide polymorphism (SNP) genotyping assay to detect 22q11 deletions. We validated this assay using DNA from 110 nondeleted controls and 77 patients with 22q11DS that had previously been tested by FISH. The assay was 100% sensitive (all deletions were correctly identified). Our assay was also able to detect a case of segmental uniparental disomy at 22q11 that was not detected by the FISH assay. We used Bayesian networks to identify a set of 17 SNPs that are sufficient to ascertain unambiguously the deletion status of 22q11DS patients. Our SNP based assay is a highly accurate, sensitive, and specific method for the diagnosis of 22q11 deletion syndrome.     

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.     

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.     

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.     

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.     

Genome-wide examination of chromosomal aberrations in neuroblastoma SH-SY5Y cells by array-based comparative genomic hybridization

Most neuroblastoma cells have chromosomal aberrations such as gains, losses, amplifications and deletions of DNA. Conventional approaches like fluorescence in situ hybridization (FISH) or metaphase comparative genomic hybridization (CGH) can detect chromosomal aberrations, but their resolution is low. In this study we used array-based comparative genomic hybridization to identify the chromosomal aberrations in human neuroblastoma SH-SY5Y cells. The DNA microarray consisting of 4000 bacterial artificial chromosome (BAC) clones was able to detect chromosomal regions with aberrations. The SH-SY5Y cells showed chromosomal gains in 1q12 approximately q44 (Chr1:142188905-246084832), 7 (over the whole chromosome), 2p25.3 approximately p16.3 (Chr2:18179-47899074), and 17q 21.32 approximately q25.3 (Chr17:42153031-78607159), while chromosomal losses detected were the distal deletion of 1p36.33 (Chr1:552910-563807), 14q21.1 approximately q21.3 (Chr14:37666271- 47282550), and 22q13.1 approximately q13.2 (Chr22:36885764-4190 7123). Except for the gain in 17q21 and the loss in 1p36, the other regions of gain or loss in SH-SY5Y cells were newly identified.