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.     

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 findings in a prospective series of patients with DiGeorge anomaly.

High-resolution cytogenetics analysis of peripheral blood lymphocytes was done prospectively on 27 of 28 patients with features of DiGeorge anomaly. Twenty-two patients (81%) had normal chromosome studies with no detectable deletion in chromosome 22. Five patients (18%) had demonstrable chromosome abnormalities. Three patients had monosomy 22q11, one due to a 4q;22q translocation, one due to a 20q;22q translocation, and one due to an interstitial deletion of 22q11. One patient had monosomy 10p13, and one patient had monosomy 18q21.33, although the latter had subsequent resolution of T-cell defects. These findings are consistent with the heterogeneity of DiGeorge anomaly but confirm the association with monosomy 22q11 in some cases. However, monosomy 10p13 may also lead to this phenotype. Because of these associated chromosome findings, cytogenetic analyses should be done on patients with suspected DiGeorge anomaly. This is particularly important since many of the abnormalities involving chromosome 22 are translocations that can be familial with a higher recurrence risk. Since only one subtle, interstitial deletion of chromosome 22 was observed, it is not clear whether high-resolution cytogenetic analysis is cost beneficial for all such patients.     

Unbalanced translocation t(15;22) in "severe" Prader-Willi syndrome

A 13-year-old girl with an unbalanced karyotype 45,XX,-15,der(22)t(15;22)(q13;q13.3) de novo had Prader-Willi syndrome (PWS), (score 13.5), but with features of mental and physical retardation more severe than usually seen in PWS. The clinical diagnosis of PWS was confirmed by methylation analysis that showed absence of the paternal band. With GTG banding, the cytogenetic breakpoint on chromosome 15q13, with 15q14 intact, encompassed the PWS region, while the breakpoint on 22q was terminal. Investigations with FISH utilised ten different probes/combinations, namely SNRPN/PML, TUPLE1/22q13.3, TUPLE/ARSA, GABRB3, three YAC clones and one cosmid for specific regions within chromosome 15q, painting probes for the long arm of chromosomes 15 and 22 and a pantelomere probe. Deletion of SNRPN,TYAC 9 (at 15q11-12), TYAC19 (at 15q13) and GABRB3 (within the PWS locus), was evident on the derivative (22) chromosome, while TYAC10 (at 15q22), cos15-5 (at 15q22) and PML (15q22) were not deleted. On the der(22), 22q13.3 and ARSA were not deleted, but the most distal non specific pantelomeric probe was deleted. Thus, the severe phenotype could be attributable to deletion on chromosome 15q extending beyond q13 to q14, (further than the usual chromosome 15q deletion (q11-13) in PWS), or be related to loss of the very terminal 22q region (from ARSA to the pantelomere) or be due to genetic factors elsewhere in the genome.     

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

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

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.     

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.     

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).     

A blind prometaphase study of Prader-Willi syndrome: frequency and consistency in interpretation of del 15q.

Controversy continues to exist concerning the proportion of individuals with Prader-Willi syndrome who have a chromosome 15 deletion and concerning the reliability with which a cytogenetic service laboratory can accurately perform the appropriate analysis. Blind prometaphase cytogenetic study of 13 individuals from a Prader-Willi syndrome clinic and seven controls has revealed that approximately 70% of accurately diagnosed clinically typical patients with this disorder have an evident deletion of at least 15q12. Blind analysis of panels of chromosome 15 pairs from all cases in this study by the directors of four independent cytogenetic service laboratories demonstrated substantial interobserver consistency in interpretation of results. The possibility of euploid mosaicism for del 15q was investigated, but remains unresolved.     

Clinical,cytogenetical and molecular analyses of Angelman syndrome

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

Parental Imbalances Involving Chromosomes 15q and 22q May Predispose to the Formation of De Novo Pathogenic Microdeletions and Microduplications in the Offspring

Microarray-based comparative genomic hybridization (array-CGH) led to the discovery of genetic abnormalities among patients with complex phenotype and normal karyotype. Also several apparently normal individuals have been found to be carriers of cryptic imbalances, hence the importance to perform parental investigations after the identification of a deletion/duplication in a proband. Here, we report the molecular cytogenetic characterization of two individuals in which the microdeletions/duplications present in their parents could have predisposed and facilitated the formation of de novo pathogenic different copy number variations (CNVs). In family 1, a 4-year-old girl had a de novo pathogenic 10.5 Mb duplication at 15q21.2q22.2, while her mother showed a 2.262 Mb deletion at 15q13.2q13.3; in family 2, a 9-year-old boy had a de novo 1.417 Mb deletion at 22q11.21 and a second paternal deletion of 247 Kb at 22q11.23 on the same chromosome 22. Chromosome 22 at band q11.2 and chromosome 15 at band q11q13 are considered unstable regions. We could hypothesize that 15q13.2q13.3 and 22q11.21 deletions in the two respective parents might have increased the risk of rearrangements in their children. This study highlights the difficulty to make genetic counseling and predict the phenotypic consequences in these situations.     

Molecular analysis of a reciprocal translocation t(5;11)(q11;p13) in a WAGR patient

Summary Most patients with the complex association aniridia — predisposition to Wilms' tumor (WAGR syndrome) present with a de novo constitutional deletion of band 11p13. We report a patient with WAGR syndrome and a reciprocal translocation between chromosomes 5 and 11 t(5;11)(q11;p13). High resolution banding cytogenetic analysis and molecular characterization using 11p13 DNA markers showed a tiny deletion encompassing the gene for CAT but sparing the gene for FSHB. This suggests that syndromes associated with apparently balanced translocations may be due to undetectable loss of material at the breakpoint(s) rather than to breakage in the gene itself.     

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 analysis of aniridia patients for deletions involving the Wilms' tumor gene

A human aniridia candidate (AN) gene on chromosome 11p13 has been cloned and characterized. The AN gene is the second cloned gene of the contiguous genes syndrome WAGR (Wilms' tumor, aniridia, genitourinary malformations, mental retardation) on chromosome 11p13, WT1 being the first gene cloned. Knowledge about the position of the AN and WT1 genes on the map of 11p13 makes the risk assessment for Wilms' tumor development in AN patients possible. In this study, we analyzed familial and sporadic aniridia patients for deletions in 11p13 by cytogenetic analyses, in situ hybridization, and pulsed field gel electrophoresis (PFGE). Cytogenetically visible deletions were found in 3/11 sporadic AN cases and in one AN/WT patient, and submicroscopic deletions were identified in two sporadic AN/WT patients and in 1/9 AN families. The exact extent of the deletions was determined with PFGE and, as a result, we could delineate the risk for Wilms' tumor development. Future analyses of specific deletion endpoints in individual AN cases with the 11p13 deletion should result in a more precise risk assessment for these patients.     

Molecular genetic study of the frequency of monosomy 22q11 in DiGeorge syndrome

It is well established that DiGeorge syndrome (DGS) may be associated with monosomy of 22q11-pter. More recently, DNA probes have been used to detect hemizygosity for this region in patients with no visible karyotypic abnormality. However, DGS has also been described in cases where the cytogenetic abnormality does not involve 22q11; for instance, four cases of 10p- have been reported. In this study we have prospectively analyzed patients, by using DNA markers from 22q11, to assess the frequency of 22q11 rearrangements in DGS. Twenty-one of 22 cases had demonstrable hemizygosity for 22q11. Cytogenetic analysis had identified interstitial deletion in 6 of 16 cases tested; in 6 other cases no karyotype was available. When these results are combined with those from our previous studies, 33 of 35 DGS patients had chromosome 22q11 deletions detectable by DNA probes.     

15q11.2 microdeletion and FMR1 premutation in a family with intellectual disabilities and autism

Genomic rearrangements of chromosome 15q11–q13 are responsible for diverse phenotypes including intellectual disabilities and autism. 15q11.2 deletion, implicating common PWS/AS breakpoints BP1–BP2, has been described in patients with delayed motor and speech development and behavioural problems. Here we report the clinical and molecular characterisation of a maternally inherited BP1–BP2 deletion in two siblings with intellectual, motor and speech delay, autistic syndrome disorder and several dysmorphic features. One of the patients was also a carrier of an FMR1 allele in the low premutation range. The four genes within the deletion were under-expressed in all deletion carriers but FMR1 mRNA levels remained normal. Our results suggest that BP1-BP2 deletion could be considered as a risk factor for neuropsychological phenotypes and that it presents with variable clinical expressivity.     

Prader-Willi syndrome: clinical genetics, cytogenetics and molecular biology

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder that arises from lack of expression of paternally inherited genes known to be imprinted and located in the chromosome 15q11-q13 region. PWS is considered the most common syndromal cause of life-threatening obesity and is estimated at 1 in 10,000 to 20,000 individuals. A de novo paternally derived chromosome 15q11-q13 deletion is the cause of PWS in about 70% of cases, and maternal disomy 15 accounts for about 25% of cases. The remaining cases of PWS result either from genomic imprinting defects (microdeletions or epimutations) of the imprinting centre in the 15q11-q13 region or from chromosome 15 translocations. Here, we describe the clinical presentation of PWS, review the current understanding of causative cytogenetic and molecular genetic mechanisms, and discuss future directions for research.     

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.     

普拉德-威利综合征的分子遗传诊断:一种连锁分析方法的初步建立

普拉德-威利综合征(Prader-Willi Syndrome,PWS)是一种基因组印记相关的疾病,是引起肥胖最常见的遗传综合征。分子和细胞遗传学检查对于该病早期诊断非常重要。通过选择PWS典型缺失区域内、外的STR遗传标记,初步建立了一种适用于中国人群的PWS核心家庭连锁分析方法,并用该方法确定了一例缺失型和一例异源单亲二体型PWS患者,经甲基化特异性PCR和高分辨染色体核型分析验证上述结果正确。同时,该连锁分析方法可以具体区分PWS的分子发病类型,从而为PWS家庭的遗传咨询提供信息,并为进一步研究PWS基因型和表型的关系提供了可能。