FISH detection of chromosome 15 deletions in Prader-Willi and Angelman syndromes

We have evaluated fluorescence in situ hybridization (FISH) analysis for the clinical laboratory detection of the 15q11-q13 deletion seen in Prader-Willi syndrome (PWS) and Angelman syndrome (AS) using probes for loci D15S11, SNRPN, D15S10, and GABRB3. In a series of 118 samples from patients referred for PWS or AS, 29 had deletions by FISH analysis. These included two brothers with a paternally transmitted deletion detectable with the probe for SNRPN only. G-banding analysis was less sensitive for deletion detection but useful in demonstrating other cytogenetic alterations in four cases. Methylation and CA-repeat analyses of 15q11-q13 were used to validate the FISH results. Clinical findings of patients with deletions were variable, ranging from newborns with hypotonia as the only presenting feature to children who were classically affected. We conclude that FISH analysis is a rapid and reliable method for detection of deletions within 15q11-q13 and whenever a deletion is found, FISH analysis of parental chromosomes should also be considered. © 1996 Wiley-Liss, Inc.     

Two Prader-Willi/Angelman syndrome loci present in an isodicentric marker chromosome

We found an abnormal 47,XX,+mar karyotype in a patient with developmental delay, hypotonia, microcephaly, failure to thrive, and cognitive delay. When metaphases were hybridized with Prader-Willi and Angelman loci-specific probes by the FISH technique, two sites were noted at opposite positions on the marker chromosome. The alphoid satellite DNA probe documented the isodicentric nature while retention of the p arms on both sides of the marker chromosome was demonstrated by beta satellite probe. The patient does not exhibit manifestations of either syndrome despite the presence of these loci in tetrasomic dose. The present investigation suggests that other marker chromosomes be reevaluated, as their clinical manifestations are quite variable. © 1994 Wiley-Liss, Inc.     

Sister chromatid exchange in families with Angelman or Prader-Willi syndrome

Using estimation of numbers of sister chromatid exchanges arising in 15q 11 q 13 as a measure, comparisons of the stability of the Prader-Willi syndrome critical region have been made. The groups studied included probands with Prader-Willi or Angelman syndromes either with or without a cytogenetically visible deletion in 15q11q13, their parents, specifically those parents who had passed on the homologue which had become deleted, and a control group. No significant differences were found between any of the four groups, indicating that there was no increase in the instability of the PWSCR region as measured by sister chromatid exchange.     

Cytogenetic and molecular studies in the Prader-Willi and Angelman syndromes: An overview

The majority of patients with Angelman syndrome and Prader-Willi syndrome have a cytogenetic and molecular deletion of chromosome 15q11q13 with the primary difference being in the parental origin of deletion. Our current understanding of the cytogenetics and molecular genetics of these 2 clinically distinct syndromes will be discussed in this review. © 1993 Wiley-Liss, Inc.     

Prenatal diagnosis of chromosome 15 abnormalities in the Prader-Willi/Angelman syndrome region by traditional and molecular cytogenetics

With improvements in culturing and banding techniques, amniotic fluid studies now achieve a level of resolution at which the Prader-Willi syndrome (PWS) and Angelman syndrome (AS) region may be questioned. Chromosome 15 heteromorphisms, detected with Q- and R-banding and used in conjunction with PWS/AS region-specific probes, can confirm a chromosome deletion and establish origin to predict the clinical outcome. We report four de novo cases of an abnormal-appearing chromosome 15 in amniotic fluid samples referred for advanced maternal age or a history of a previous chromosomally abnormal child. The chromosomes were characterized using G-, Q-, and R-banding, as well as isotopic and fluorescent in situ hybridization of DNA probes specific for the proximal chromosome 15 long arm. In two cases, one chromosome 15 homolog showed a consistent deletion of the ONCORPWS/AS region A and B. In the other two cases, one of which involved an inversion with one breakpoint in the PWS/AS region, all of the proximal chromosome 15 long arm DNA probes used in the in situ hybridization were present on both homologs. Clinical follow-up was not available on these samples, as in all cases the parents chose to terminate the pregnancies. These cases demonstrate the ability to prenatally diagnose chromosome 15 abnormalities associated with PWS/AS. In addition, they highlight the need for a better understanding of this region for accurate prenatal diagnosis. © 1995 Wiley-Liss, Inc.     

Clinical spectrum and molecular diagnosis of Angelman and Prader-Willi syndrome patients with an imprinting mutation

Recent studies have identified a new class of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) patients who have biparental inheritance, but neither the typical deletion nor uniparental disomy (UPD) or translocation. However, these patients have uniparental DNA methylation throughout 15q11-q13, and thus appear to have a mutation in the imprinting process for this region. Here we describe detailed clinical findings of five AS imprinting mutation patients (three families) and two PWS imprinting mutation patients (one new family). All these patients have essentially the classical clinical phenotype for the respective syndrome, except that the incidence of microcephaly is lower in imprinting mutation AS patients than in deletion AS patients. Furthermore, imprinting mutation AS and PWS patients do not typically have hypopigmentation, which is commonly found in patients with the usual large deletion. Molecular diagnosis of these cases is initially achieved by DNA methylation analyses of the DN34/ZNF127, PW71 (D15S63), and SNRPN loci. The latter two probes have clear advantages in the simple molecular diagnostic analysis of PWS and AS patients with an imprinting mutation, as has been found for typical deletion or UPD PWS and AS cases. With the recent finding of inherited microdeletions in PWS and AS imprinting mutation families, our studies define a new class of these two syndromes. The clinical and molecular identification of these PWS and AS patients has important genetic counseling consequences. Am. J. Med. Genet. 68:195–206, 1997 © 1997 Wiley-Liss, Inc.     

Comparison of high resolution chromosome banding and fluorescence in situ hybridization (FISH) for the laboratory evaluation of Prader-Willi syndrome and angelman syndrome

The development of probes containing segments of DNA from chromosome region 15q11-q13 provides the opportunity to confirm the diagnosis of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) by fluorescence in situ hybridization (FISH). We have evaluated FISH studies and high resolution chromosome banding studies in 14 patients referred to confirm or rule out PWS and five patients referred to confirm or rule out AS. In four patients (three from the PWS category and 1 from the AS group) chromosome analysis suggested that a deletion was present but FISH failed to confirm the finding. In one AS group patient, FISH identified a deletion not detectable by high resolution banding. Review of the clinical findings in the discrepant cases suggested that the FISH results were correct and high resolution findings were erroneous. Studies with a chromosome 15 alpha satellite probe (D15Z) on both normal and abnormal individuals suggested that incorrect interpretation of chromosome banding may occasionally be attributable to alpha satellite polymorphism but other variation of 15q11-q13 chromosome bands also contributes to misinterpretation. We conclude that patients who have been reported to have a cytogenetic deletion of 15q11-q13 and who have clinical findings inconsistent with PWS and AS should be reevaluated by molecular genetic techniques. © 1994 Wiley-Liss, Inc.     

Prader-Willi syndrome in Black females

Reports of Black females with Prader-Willi have been rare. This communication describes two Black females with Prader-Willi syndrome. Chromosome analysis revealed a small deletion of the proximal portion of a chromosome 15 in one case and apparently normal chromosomes in the other.     

Methylation PCR analysis of Prader-Willi syndrome,Angelman syndrome,and control subjects

We report on a relatively large survey of Prader-Willi syndrome, Angelman syndrome, and control subjects with the newly described methylation polymerase chain reaction (PCR) method to determine its usefulness for molecular diagnosis. Sixty-one Prader-Willi syndrome (PWS) individuals (26 men and 35 women), 9 Angelman syndrome (AS) patients (5 men and 4 women), and 58 other individuals were studied with methylation PCR following sodium bisulfite treatment of genomic DNA. In addition, multiple tissues, including fetal tissue, were studied from several individuals to determine the effects of various tissues on methylation PCR results. The expected methylation PCR result was observed in each case. This PCR-based assay evaluates the methylation status of the CpG island of the SNRPN gene and allows for rapid molecular diagnosis of PWS or AS with less labor than Southern hybridization for methylation analysis. The PCR results were identical to those achieved by Southern hybridization in those individuals studied. Am. J. Med. Genet. 80:263–265, 1998. © 1998 Wiley-Liss, Inc.     

FISH characterization of small supernumerary marker chromosomes in two Prader-Willi patients

A small supernumerary chromosome was observed in two Prader-Willi syndrome (PWS) patients. The clinical diagnosis of PWS was confirmed by the ascertainment of the deletion of region 15q11-13 in one case and uniparental disomy (UPD) of the same region in the other. The markers were negative for dystamycinA/DAPI banding, did not contain NOR-positive satellites, and had an appearance consistent with a very small ring chromosome. Fluorescent in situ hybridization (FISH) analysis with the “all human centromere” probe indicated the presence of centromeric sequences in both markers. Chromosomal in situ suppression hybridization with chromosome specific libraries demonstrated that the small markers in the deleted and UPD patient originated from chromosome 15 and X, respectively. To the best of our knowledge these are the only PWS patients reported with a supernumerary marker chromosome other than inv dup(15) characterized by FISH. Am. J. Med. Genet. 68:99–104, 1997 © 1997 Wiley-Liss, Inc.     

Difference in methylation patterns within the D15S9 region of chromosome 15qll–13 in first cousins with Angelman syndrome and Prader–Willi syndrome

Abnormalities of chromosome region 15qll–13 are associated with Angelman syndrome (AS) and Prader–Willi syndrome (PWS). Differences between the methylation patterns of the region of chromosome 15qll–13 which hybridizes to the highly conserved DNA, DN34, in normal individuals and in patients with AS and PWS have been described. We report on a family in which first cousins are affected by AS and PWS as a result of a familial paracentric inversion of 15qll–ql3. The results of the studies on this family demonstrate the differences in the methylation patterns in the 2 conditions and the phenomenon of genomic imprinting, whereby genetic information is expressed differently dependent on the parent of origin. © 1993 Wiley-Liss, Inc.     

Unexpected familial recurrence in Angelman syndrome

We report on two instances of familial recurrence of Angelman syndrome which, from pedigree analysis, appear incompatible with currently known mechanisms of inheritance of this disorder. In these two families, deletion-positive Angelman syndrome has recurred in cousins. Several established mechanisms for deletion-positive familial recurrence have been ruled out. In each family, molecular cytogenetic studies show typical chromosome 15 deletions, and DNA methylation analysis verifies the maternal origin of the deleted chromosomes in all four individuals. Since the mothers of the affected individuals in each family are not known to be related, these recurrences appear to be secondary to coincidental, de novo events. This conclusion is consistent with direct and indirect estimates of the population frequency of Angelman syndrome. Am. J. Med. Genet. 70:253–260, 1997. © 1997 Wiley-Liss, Inc.     

Unique karyotypes in two patients with Prader-Willi syndrome

A physical disruption of the Prader-Willi syndrome (PWS) chromosome region is thought to cause PWS. We describe 2 girls with PWS phenotype, who had unique chromosome 15 abnormalities. The first patient showed mosaicism: 45,XX,t(15;15)(qter→p11.1::q11.200→ qter)/46,XX,t(15;15)(qter → p11.1::q11.200→ qter), + mar. The band 15q11.2 apparently remained intact in the t(15;15) chromosome, and the mar chromosome was considered as r(15) (p11.1q11.1). The second patient had a karyo-type of 47,XX,del(15)(q11.200→q11.207), + idic (15)(pter → q11.1::q11.1→pter). The complex breakage and reunion involving the 15q11.2 regions of the father's homologous chromosomes 15 at meiosis appeared to have resulted in the idic(15) and the del(15) chromosomes. These cytogenetic findings suggest that the PWS chromosome region may be localized on the very proximal portion of band 15q11.2.     

Robertsonian (15q;15q) translocation in a child with Angelman syndrome: Evidence of uniparental disomy

A balanced Robertsonian translocation 45,XY,t(15q15q) was detected in a patient with mental retardation, microcephaly, and hypertonia. Deletion of the 15q11q13 region was unlikely based on fluorescence in situ hybridization studies that revealed hybridization of appropriate DNA probes to both arms of the Robertsonian chromosome. Inheritance of alleles from 13 highly polymorphic DNA markers on chromosome 15 showed paternal uniparental isodisomy. The clinical, cytogenetic, and molecular results are consistent with a diagnosis of Angelman syndrome. © 1996 Wiley-Liss, Inc.     

Phenotypic differences in Angelman syndrome patients: Imprinting mutations show less frequently microcephaly and hypopigmentation than deletions

Angelman syndrome (AS) is a relatively frequent disorder of psychomotor development caused by loss of function of a gene from chromosome 15q11-q13, a region subject to genomic imprinting. The AS gene(s) is exclusively expressed from the maternal chromosome. Several kinds of mutations have been found to cause AS. More than half of the cases exhibit a deletion of the maternal 15q11-q13 region. Recently, we and others described a new mutation type, the imprinting mutation, characterised by normal, biparental inheritance but aberrant methylation patterns of the entire chromosomal region. In AS, a paternal imprint is found on the maternal chromosome probably leading to functional inactivation of the AS gene(s). We have now compared the phenotype of 9 AS patients with imprinting mutation to that of nine age-matched ones with a maternally derived deletion. Both groups were evaluated for 19 common AS symptoms. All patients, independently of their molecular findings, showed classical AS symptoms such as mental retardation, delayed motor development, and absent speech. In contrast, for two signs, hypopigmentation and microcephaly, a different distribution among both groups was observed. Only one of nine AS patients with an imprinting mutation, but seven of nine in the deletion control group showed either symptom. Our results suggest that imprinting mutations, in contrast to deletions, cause only incomplete loss of gene function or that maternally derived deletions affect also genes not subject to genomic imprinting. We conclude that AS is caused by loss of function of a major gene that is imprinted but that there are also other genes that contribute to the phenotype when in hemizygous condition. © 1996 Wiley-Liss, Inc.     

Prader-Willi and Angelman syndromes: Diagnosis with a bisulfite-treated methylation-specific PCR method

The putative promoter region of the SNRPN gene contains a CpG island which is heavily methylated in the maternally derived allele and unmethylated in the paternally derived allele. In patients with Prader-Willi syndrome (PWS) only the methylated allele is present, while in those with Angelman syndrome (AS) only the unmethylated allele is present. The purpose of this paper is to report a polymerase chain reaction (PCR)-based assay to evaluate methylation status of the CpG island of the SNRPN gene and to show that this assay allows rapid diagnosis of PWS and AS. Methylated cytosines in the CpG dinucleotide are resistant to chemical modification by sodium bisulfite. In contrast, bisulfite treatment converts all unmethylated cytosines to uracil. Based on this differential effect, the bisulfite-modified DNA sequence of a methylated allele was successfully distinguished from that of an unmethylated allele using 2 sets of allele-specific primer pairs: a methylated allele-specific primer pair (MET) and an unmethylated allele-specific primer pair (UNMET). Bisulfite-modified DNA from 10 patients with PWS amplified only with the MET pair while modified DNA from 5 patients with AS amplified only with the UNMET pair. Modified DNA from 50 normal unrelated individuals amplified with both primer pairs. In that methylation-specific PCR (MSPCR) can detect all presently testable causes of PWS and AS in a rapid and cost-effective fashion, serious consideration should be given to the use of this test in the initial evaluation of all patients in which PWS or AS is being considered. Am. J. Med. Genet. 73:308–313, 1997. © 1997 Wiley-Liss, Inc.     

Molecular and clinical study of 61 Angelman syndrome patients

We analyzed 61 Angelman syndrome (AS) patients by cytogenetic and molecular techniques. On the basis of molecular findings, the patients were classified into the following 4 groups: familial cases without deletion, familial cases with submicroscopic deletion, sporadic cases with deletion, and sporadic cases without deletion. Among 53 sporadic cases, 37 (70%) had molecular deletion which commonly extended from D15S9 to D15S12, although not all deletions were identical. Of 8 familial cases, 3 sibs from one family had a molecular deletion involving only 2 loci, D15S10 and GABRB3, which define the critical region for AS phenotypes. The parental origin of deletion, both in sporadic and familial cases, was exclusively maternal and consistent with a genomic imprinting hypothesis. Among sporadic and familial cases without deletion, no uniparental disomy was found and most of them were shown to inherit chromosomes 15 from both parents (biparental inheritance). A discrepancy between cytogenetic and molecular deletion was observed in 14 (26%) of 53 patients in whom cytogenetic analysis could be performed. Ten (43%) of 23 patients with a normal karyotype showed a molecular deletion, and 4 (13%) of 30 patients with cytogenetic deletion, del(15) (q11q13), showed no molecular deletion. Most clinical manifestations, including neurological signs and facial characteristics, were not distinct in each group except for hypopigmentation of skin or hair. Familial cases with submicroscopic deletion were not associated with hypopigmentation. These findings suggested that a gene for hypopigmentation is located is located outside the critical region of AS and is not imprinted. © 1994 Wiley-Liss, Inc.     

Deletion involving D15S113 in a mother and son without Angelman syndrome: Refinement of the Angelman syndrome critical deletion region

Deletions of 15q11-q13 typically result in Angelman syndrome when inherited from the mother and Prader-Willi syndrome when inherited from the father. The critical deletion region for Angelman syndrome has recently been restricted by a report of an Angelman syndrome patient with a deletion spanning less than 200 kb around the D15S113 locus. We report here on a mother and son with a deletion of chromosome 15 that includes the D15S113 locus. The son has mild to moderate mental retardation and minor anomalies, while the mother has a borderline intellectual deficit and slightly downslanting palpebral fissures. Neither patient has the seizures, excessive laughter and hand clapping, ataxia or the facial anomalies which are characteristic of Angelman syndrome. The proximal boundary of the deletion in our patients lies between the D15S10 and the D15S113 loci. Our patients do not have Angelman syndrome, despite the deletion of the D15S113 marker. This suggests that the Angelman syndrome critical deletion region is now defined as the overlap between the deletion found in the previously reported Angelman syndrome patient and the region that is intact in our patients. © 1995 Wiley-Liss, Inc.     

Clinical profile of Angelman syndrome at different ages

We describe 47 patients with Angelman syndrome (AS) from Belgium and the Netherlands, including the anamnestic data, the clinical and the behavioral attributes at different ages. The clinical picture of AS is most distinct between the ages of 2–16 years. Most patients of this age group show at least 8 of the major characteristics (bursts of laughter, happy disposition, hyperactive behaviour, microcephaly, brachycephaly, macrostomia, tongue protrusion, mandibular prognathism, widely spaced teeth, stiff and puppetlike movements, typical stature, wide based gait) beside the mental retardation and (almost) absence of speech, which is a universal trait. The diagnosis in infants is based on only a limited number of clinical characteristics or on anamnestic data. However, if these occur in combination, they are indicative of AS. In older patients, the diagnosis may be hampered in part because of the changing behavioral characteristics and the decreasing frequency of fits. Other manifestations, such as scoliosis, may become more pronounced with age. © 1995 Wiley-Liss, Inc.     

Angelman syndrome and prenatally diagnosed Prader-Willi syndrome in first cousins

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are caused by loss of function of imprinted genes in the 15q11-13 critical region. Reports of PWS and AS in close relatives within the same family are rare. We report on the diagnosis of a familial unbalanced 10;15 translocation causing AS in a child that led to the prenatal diagnosis of an unbalanced 10;15 translocation with resultant deletion of the Prader-Willi critical region in her maternal uncle's offspring.