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     

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.     

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     

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.     

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.     

Cytogenetic and molecular characterization of inverted duplicated chromosomes 15 from 11 patients.

We have studied the inverted duplicated chromosomes 15 (inv dup(15)) from 11 individuals--7 with severe mental retardation and seizures, 3 with a normal phenotype, and 1 with Prader-Willi syndrome (PWS). Through a combination of FISH and quantitative DNA analyses, three different molecular sizes of inv dup(15) were identified. The smallest inv dup(15) was positive only for the centromeric locus D15Z1 (type 1); the next size was positive for D15Z1 and D15S18 (type 2); and the largest inv dup(15) was positive for two additional copies of loci extending from D15Z1 and D15S18 through D15S12 (type 3). Type 1 or type 2 was observed in the three normal individuals and the PWS patient. Type 3 was observed in all seven individuals with mental retardation and seizures but without PWS or Angelman Syndrome (AS). The PWS patient, in addition to being mosaic for a small inv dup(15), demonstrated at D15S63 a methylation pattern consistent with maternal uniparental inheritance of the normal chromosomes 15. The results from this study show (a) two additional copies of proximal 15q loci, D15S9 through D15S12, in mentally retarded patients with an inv dup(15) but without AS or PWS and (b) no additional copies of these loci in patients with a normal phenotype or with 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.     

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.     

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.     

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.     

DNA diagnosis of Prader-Willi and Angelman syndromes with the probe PW71 (D15S63)

Previously, 158 nuclear families with probands suspected of having either Prader Willi (PWS) or Angelman syndrome (AS) were analyzed with polymorphic DNA markers from the 15q11–13 region. These cases have been re-evaluated with the probe PW71 (D15S63), which detects parent-of-origin-specific alleles after digestion with a methylation-sensitive restriction enzyme (HpaII). Application of PW71 to DNA samples isolated from leucocytes, confirmed the deletions and uniparental disomies detected earlier by marker analysis, and resolved 50% of the previously uninformative (n=18) cases. PW71 and restriction fragment length polymorphism analysis indicated that, in all resolved cases, disomies of the 15q11–13 region were present. The use of PW71 increased the percentage of disomies detected in our PWS and AS patient groups. Almost 50% of our PWS patients and 17% of the AS patients showed a disomy of maternal or paternal origin, respectively. DNA of first trimester chorionic villi and of fibroblast cultures was not suitable for analysis with PW71 because of different methylation patterns. The application of PW71 is recommended for the diagnosis of the PWS and AS, with respect to DNA samples from blood.     

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.     

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.     

Distribution of three alpha-chain beta-hexosaminidase A mutations among Tay-Sachs carriers.

DNA from 176 carriers of the Tay-Sachs gene was tested for the presence of the three mutations most commonly found among Ashkenazi Jews: the so-called insertion, splice junction, and adult mutations. Among 148 Ashkenazi Jews tested, 108 had the insertion mutation, 26 had the splice junction mutation, five had the adult mutation, and nine had none of the three. Among 28 non-Jewish carriers tested, most of whom were obligate carriers, four had the insertion mutation, one had the adult mutation, and the remaining 23 had none of the three.     

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 novel NOD2/CARD15 haplotype conferring risk for Crohn disease in Ashkenazi Jews

Crohn disease (CD) exhibits a 2-4-fold increased frequency in Jews as compared with other ethnic/racial groups. Three coding variants of the NOD2/CARD15 have been reported as independent disease-predisposing mutations (DPMs), but these were found in only 30%-40% of patients with CD and could not account for all the linkage between CD and the IBD1 locus. The aim of the present study was to explore whether additional DPMs at the IBD1 locus exist in the high-risk Jewish group. Sixty-four Ashkenazi Jewish and 147 non-Jewish white families were studied. Six microsatellite markers spanning IBD1 were genotyped for linkage analysis in subgroups stratified on NOD2/CARD15 DPM status. SNPs in NOD2/CARD15 (R702W, G908R, 1007fs, and S268P) were then genotyped in family and independent case-control samples. On the basis of initial results, sequencing was done on NOD2/CARD15-translated regions in 12 Jewish individuals. Subsequently, a new NOD2/CARD15 variant was genotyped and analyzed. After excluding the influence of the three DPMs, significant linkage of IBD1 to CD in Jews remained with two peaks at D16S403 (mean allele sharing [MAS] = 0.70] and D16S411 (MAS = 0.59). Further, we observed an increased frequency of a haplotype carrying only the 268S variant in Jewish patients (OR = 3.13, P=.0023) but not in non-Jews, suggesting the existence of a Jewish-specific additional disease-predisposing factor on this haplotype. Sequencing of this haplotype revealed a new variant (IVS8+158; JW1). The 268S-JW1 combination exhibited a further increased risk (OR = 5.75, P=.0005) and the highest population-attributable risk (15.1%) for CD among reported DPMs in Jews. In Ashkenazi Jews, unrecognized population-specific predisposing factor(s) exist on the 268S-JW1 haplotype at the IBD1 locus. This factor may contribute to the higher risk for CD in Ashkenazi Jews as compared with non-Jews.     

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.     

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.     

Angelman syndrome in an inbred family

Angelman syndrome (AS) is characterized by severe mental retardation, absent speech, puppet-like movements, inappropriate laughter, epilepsy, and abnormal electroencephalogram. The majority of AS patients ( 65%) have a maternal deficiency within chromosomal region 15q11–q13, caused by maternal deletion or paternal uniparental disomy (UPD). Approximately 35% of AS patients exhibit neither detectable deletion nor UPD, but a subset of these patients have abnormal methylation at several loci in the 15q11–q13 interval. We describe here three patients with Angelman syndrome belonging to an extended inbred family. High resolution chromosome analysis combined with DNA analysis using 14 marker loci from the 15q11-q13 region failed to detect a deletion in any of the three patients. Paternal UPD of chromosome 15 was detected in one case, while the other two patients have abnormal methylation atD15S9, D15S63, andSNRPN. Although the three patients are distantly related, the chromosome 15q11-q13 haplotypes are different, suggesting that independent mutations gave rise to AS in this family.     

A 28-kb deletion spanning D15S63 (PW71) in five families: a rare neutral variant?

Methylation analysis with probe PW71 (D15S63) is an established procedure to test patients suspected of having Prader-Willi syndrome or Angelman syndrome. Using this test, we have identified a 28-kb deletion spanning D15S63 in five independent families. Sequence analysis revealed identical breakpoints in all the families. The haplotype data are compatible with a common ancestral origin of the deletion in at least two families. The deletion was not found in 1, 000 unrelated controls. Although the deletion maps within the imprinting-center region, neither maternal nor paternal inheritance of the deletion appears to affect imprinting in proximal 15q. We conclude that the deletion is a rare neutral variant that can lead to false-positive results in the PW71-methylation test.