Maternal origin of 15q11-13 deletions in Angelman syndrome suggests a role for genomic imprinting

Six persons with the classical Angelman syndrome (AS) phenotype and de novo deletions of chromosome 15q11-q13 were studied to determine the parental origin of the chromosome deletion. Four of the 6 patients had informative cytogenetic studies and all demonstrated maternal inheritance of the deletion. These findings, together with other reported cases of the origin of the chromosome 15 deletion in AS, suggest that deletion of the maternally contributed chromosome leads to the AS phenotype. This contrasts with the Prader-Willi syndrome (PWS) in which a similar deletion of the paternally contributed chromosome 15 is observed. In deletion cases, a parental gamete effect such as genomic imprinting may be the best model to explain why apparently identical 15q11-q13 deletions may develop the different phenotypes of AS or PWS.     

Maternal origin of 15q11–13 deletions in Angelman syndrome suggests a role for genomic imprinting

Six persons with the classical Angelman syndrome (AS) phenotype and de novo deletions of chromosome 15q11-q13 were studied to determine the parental origin of the chromosome deletion. Four of the 6 patients had informative cytogenetic studies and all demonstrated maternal inheritance of the deletion. These findings, together with other reported cases of the origin of the chromosome 15 deletion in AS, suggest that deletion of the maternally contributed chromosome leads to the AS phenotype. This contrasts with the Prader-Willi syndrome (PWS) in which a similar deletion of the paternally contributed chromosome 15 is observed. In deletion cases, a parental gamete effect such as genomic imprinting may be the best model to explain why apparently identical 15q11-q13 deletions may develop the different phenotypes of AS or PWS.     

Interstitial duplications of chromosome region 15q11q13: Clinical and molecular characterization

Duplications of chromosome region 15q11q13 often occur as a supernumerary chromosome 15. Less frequently they occur as interstitial duplications [dup(15)]. We describe the clinical and molecular characteristics of three patients with de novo dup(15). The patients, two males and one female (ages 3–21 years), had nonspecific findings that included autistic behavior, hypotonia, and variable degrees of mental retardation. The extent, orientation, and parental origin of the duplications were assessed by fluorescent in situ hybridization, microsatellite analyses, and methylation status at D15S63. Two patients had large direct duplications of 15q11q13 [dir dup(15)(q11q13)] that extended through the entire Angelman syndrome/Prader-Willi syndrome (AS/PWS) chromosomal region. Their proximal and distal breaks, at D15S541 or D15S9 and between D15S12 and D15S24, respectively, were comparable to those found in the common AS/PWS deletions. This suggests that duplications and deletions may be the reciprocal product of an unequal recombination event. These two duplications were maternally derived, but the origin of the chromatids involved in the unequal crossing over in meiosis differs. In one patient, the duplication originated from two different maternal chromosomes, while in the other patient it arose from the same maternal chromosome. The third patient had a much smaller duplication that involved only D15S11 and parental origin could not be determined. There was no obvious correlation between phenotype and extent of the duplication in these patients. Am. J. Med. Genet. 79:82–89, 1998. © 1998 Wiley-Liss, Inc.     

Cloning of the breakpoints of a submicroscopic deletion in an Angelman syndrome patient

The majority of cases of the two distinct disorders Prader–Willisyndrome (PWS) and Angelman syndrome (AS) result from cytogeneticdeletions of chromosome 15q11–q13. These deletions areexclusively of maternal origin in AS but of paternal originin PWS indicating that the 15q11–q13 region is subjectto genomic imprinting. Transmission of a submicroscopic deletionin one three generation family resulted in AS only upon maternaltransmission of the deletion with no clinical phenotype associatedwith paternal transmission (1, 2). The breakpoint of this submicroscopicdeletion has been cloned and sequenced. This is the first deletionjunction from the AS/PWS region which has been so characterized.The nucleotide sequence of the deletion junction revealed a19 bp insertion of unknown origin with no evidence of repetitiveelements. A probe from the proximal deletion breakpoint, PB11,lies within the currently defined minimum region of deletionoverlap in PWS, which contains the SNRPN and D15S63 locl. Ourresults suggest that the imprinted gene(s) responsible for thePWS phenotype are proximal of pB11 in this deletion overlapregion.     

Familial translocations involving 15q11-q13 can give rise to interstitial deletions causing Prader-Willi or Angelman syndrome.

A de novo interstitial deletion of 15q11-q13 is the major cause of Prader-Willi syndrome (PWS) and Angelman syndrome (AS). Here we describe two unrelated PWS patients with a typical deletion, whose fathers have a balanced translocation involving the PWS/AS region. Microsatellite data suggest that the deletion is the result of an unequal crossover between the derivative chromosome 15 and the normal chromosome 15. We conclude that familial translocations involving 15q11-q13 can give rise to interstitial deletions causing PWS or AS and that prenatal diagnosis in such families should include fluorescence in situ hybridisation or microsatellite studies or both.     

Genomic inversions of human chromosome 15q11-q13 in mothers of Angelman syndrome patients with class II (BP2/3) deletions

Parental submicroscopic genomic inversions have recently been demonstrated to be present in several genomic disorders. These inversions are genomic polymorphisms that facilitate misalignment and abnormal recombination between flanking segmental duplications. Angelman syndrome (AS; MIM 105830) is associated with specific abnormalities of chromosome 15q11-q13, with about 70% of cases being mother-of-origin 4 Mb deletions. We present here evidence that some mothers of AS patients with deletions of the 15q11-q13 region have a heterozygous inversion involving the region that is deleted in the affected offspring. The inversion was detected in the mothers of four of six AS cases with the breakpoint 2-3 (BP2/3) 15q11-q13 deletion, but not in seven mothers of AS due to paternal uniparental disomy (UPD) 15. We have identified variable inversion breakpoints within BP segmental duplications in the inverted AS mothers, as well as in AS deleted patients. Interestingly, the BP2-BP3 region is inverted in the mouse draft genome sequence with respect to the human draft sequence. The BP2-BP3 chromosome 15q11-q13 inversion was detected in four of 44 subjects (9%) of the general population (P<0.004). The BP2/3 inversion should be an intermediate estate that facilitates the occurrence of 15q11-q13 BP2/3 deletions in the offspring.     

Parental and chromosomal origins of microdeletion and duplication syndromes involving 7q11.23, 15q11-q13 and 22q11

Non-allelic homologous recombination between chromosome-specific LCRs is the most common mechanism leading to recurrent microdeletions and duplications. To look for locus-specific differences, we have used microsatellites to determine the parental and chromosomal origins of a large series of patients with de novo deletions of chromosome 7q11.23 (Williams syndrome), 15q11-q13 (Angelman syndrome, Prader-Willi syndrome) and 22q11 (Di George syndrome) and duplications of 15q11-q13. Overall the majority of rearrangements were interchromosomal, so arising from unequal meiotic exchange, and there were approximately equal numbers of maternal and paternal deletions. Duplications and deletions of 15q11-q13 appear to be reciprocal products that arise by the same mechanisms. The proportion arising from interchromosomal exchanges varied among deletions with 22q11 the highest and 15q11-q13 the lowest. However, parental and chromosomal origins were not always independent. For 15q11-q13, maternal deletions tended to be interchromosomal while paternal deletions tended to be intrachromosomal; for 22q11 there was a possible excess of maternal cases among intrachromosomal deletions. Several factors are likely to be involved in the formation of recurrent rearrangements and the relative importance of these appear to be locus-specific.     

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.     

Difficulties of genetic counseling and prenatal diagnosis in a consanguineous couple segregating for the same translocation (14;15) (q11;q13) and at risk for Prader-Willi and Angelman syndromes

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are associated with a loss of function of imprinted genes in the 15q11-q13 region mostly due to deletions or uniparental disomies (UPD). These anomalies usually occur de novo with a very low recurrence risk. However, in rare cases, familial translocations are observed, giving rise to a high recurrence risk. We report on the difficulties of genetic counseling and prenatal diagnosis in a family segregating for a translocation (14;15)(q11;q13) where two consanguineous parents carry the same familial translocation in this chromosome 15 imprinting region. Both children of the couple inherited a chromosomal anomaly leading to PWS. However, a paternal 15q11-q13 deletion was responsible for PWS in the first child, whereas prenatal diagnosis demonstrated that PWS was associated with a maternal 15q11-q13 UPD in the fetus. This report demonstrates that both conventional and molecular cytogenetic parental analyses have to be performed when a deletion is responsible for PWS or AS in order not to overlook a familial translocation and to insure reliable diagnosis and genetic counseling.     

Array-based comparative genomic hybridization analysis of recurrent chromosome 15q rearrangements

Genomic rearrangements of chromosome 15q11-q13 cause diverse phenotypes including autism, Prader-Willi syndrome (PWS), and Angelman syndrome (AS). This region is subject to genomic imprinting and characterized by complex combinations of low copy repeat elements. Prader-Willi and Angelman syndrome are caused primarily by 15q11-13 deletions of paternal and maternal origin, respectively. Autism is seen with maternal, but not paternal, interstitial duplications. Isodicentric 15q, most often of maternal origin, is associated with a complex phenotype often including autistic features. Limitations of conventional cytogenetic tests preclude a detailed analysis in most patients with 15q rearrangements. We have developed a microarray for comparative genomic hybridization utilizing 106 genomic clones from chromosome 15q to characterize this region. The array accurately localized all breakpoints associated with gains or losses on 15q. The results confirmed the location of the common breakpoints associated with interstitial deletions and duplications. The majority of idic(15q) chromosomes are comprised of symmetrical arms with four copies of the breakpoint 1 to breakpoint 5 region. Patients with less common breakpoints that are not distinguished by routine cytogenetic methods were more accurately characterized by array analysis. This microarray provides a detailed characterization for chromosomal abnormalities involving 15q11-q14 and is useful for more precise genotype-phenotype correlations for autism, PWS, AS, and idic(15) syndrome.     

High level of unequal meiotic crossovers at the origin of the 22q11. 2 and 7q11.23 deletions

Interstitial chromosomal deletions at 22q11.2 and 7q11.23 are detected in the vast majority of patients affected by CATCH 22 syndromes and the Williams-Beuren syndrome, respectively. In a group of 15 Williams- Beuren patients, we have shown previously that a large number of 7q11.23 deletions occur in association with an interchromosomal rearrangement, indicative of an unequal crossing-over event between the two homologous chromosomes 7. In this study, we show that a similar mechanism also underlies the formation of the 22q11.2 deletions associated with CATCH 22. In eight out of 10 families with a proband affected by CATCH 22, we were able to show that a meiotic recombination had occurred at the critical deleted region based on segregation analysis of grandparental haplotypes. The incidences of crossovers observed between the closest informative markers, proximal and distal to the deletion, were compared with the expected recombination frequencies between the markers. A significant number of recombination events occur at the breakpoint of deletions in CATCH 22 patients (P = 2.99x10(-7)). The segregation analysis of haplotypes in three- generation families was also performed on an extended number of Williams-Beuren cases (22 cases in all). The statistically significant occurrence of meiotic crossovers (P = 4.45x10(-9)) further supports the previous findings. Thus, unequal meiotic crossover events appear to play a relevant role in the formation of the two interstitial deletions. The recurrence risk for healthy parents in cases where such meiotic recombinations can be demonstrated is probably negligible. Such a finding is in agreement with the predominantly sporadic occurrence of the 22q11.2 and 7q11. 23 deletions. No parent-of-origin bias was observed in the two groups of patients with regard to the origin of the deletion and to the occurrence of inter- versus intrachromosomal rearrangements.      

Multiplex PCR of three dinucleotide repeats in the Prader-Willi/Angelman critical region (15q11-q13): molecular diagnosis and mechanism of uniparental disomy

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinctmental retardation disorders caused by a deficiency of paternal(PWS) or maternal (AS) contributions for chromosome 15 by eitherdeletion or uniparental disomy (UPD). To further study the molecularmechanisms involved in these disorders and to improve moleculardiagnostic methods, we have isolated three dinucleotide repeatmarkers in the PWS/AS critical region. An Alu-CA PCR methodwas used to isolate CA-repeat markers directly from yeast artificialchromosome (YAC) clones identified by probes IR4–3R (D15S11),LS6–1 (D15S113), and GABA_A receptor B3 (GABRB3). Threemarkers with 6–11 alleles and 73–83% heterozygositieswere identified and analyzed by multiplex PCR. Gene-centromeremapping was performed on a panel of ovarian teratomas of knownmeiotic origin, and showed the most proximal marker, IR4–3R,to be 13 cM (95% confidence limits: 7–19 cM) from thecentromere of chromosome 15. Molecular diagnostic studies wereperformed on 20 PWS and 9 AS patients. In 17 patients with deletions,the parental origin of deletion was determined. Ten PWS patientswere shown to have maternal heterodisomy. Since these markersare only 13 cM from the centromere, heterodisomy indicates thatmaternal meiosis I nondisjunction is involved in the originof UPD. In contrast, two paternal disomy cases of AS showedisodisomy for all markers tested along the length of chromosome15. This suggests a paternal meiosis II nondisjunction event(without crossing over) or, more likely, monosomic conception(due to maternal nondisjunction) followed by chromosome duplication.This latter mechanism would indicate that UPD in PWS and ASmay initiate as reciprocal products of maternal nondisjunctionevents.     

Molecular dissection of the Prader-Willi/Angelman syndrome region (15q11-13) by YAC cloning and FISH analysis.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct mental retardation disorders associated with deletions of proximal 15q (q11-q13) of different parental origin. Yeast artificial chromosome (YAC) clones were isolated for 9 previously mapped DNA probes from this region, and for one newly derived marker, LS6-1 (D15S113). A YAC contig of 1-1.5 Mb encompassing four markers (ML34, IR4-3R, PW71, and TD189-1) was constructed. Multi-color fluorescence in situ hybridization (FISH) analysis of interphase nuclei was combined with YAC contig information to provide the following order of markers: cen-IR39-ML34-IR4-3R-PW71-TD189-1-LS6++ +-1-TD3-21-GABRB3-IR10-1-CMW1-tel. FISH analysis was performed on 8 cases of PWS and 3 cases of AS, including 5 patients with normal karyotypes. All eleven patients were deleted for YACs in the interval from IR4-3R to GABRB3. On the proximal side of the deletion interval, 10/10 breakpoints fell within a single ML34 YAC of 370 kb. On the distal side, 8/9 breakpoints fell within a single IR10-1 YAC of 200 kb. These results indicate a striking consistency in the location of the proximal and distal breakpoints in PWS and AS patients. FISH analysis on a previously reported case of familial AS confirmed a submicroscopic deletion including YACs corresponding to LS6-1, TD3-21 and GABRB3 and supports the separation of the PWS and AS critical regions. Since these three YACs do not overlap each other, the minimum size of the AS critical region is > or = 650 kb.     

Unequal interchromosomal rearrangements may result in elastin gene deletions causing the Williams-Beuren syndrome

Williams-Beuren syndrome (WBS) is generally the consequence of an interstitial microdeletion at 7q11.23, which includes the elastin gene, thus causing hemizygosity at the elastin gene locus. The origin of the deletion has been reported by many authors to be maternal in approximately 60% and paternal in 40% of cases. Segregation analysis of grandparental markers flanking the microdeletion region in WBS patients and their parents indicated that in the majority of cases a recombination between grandmaternal and grandpaternal chromosomes 7 at the site of the deletion had occurred during meiosis in the parent from whom the deleted chromosome stemmed. Thus, the majority of deletions were considered a consequence of unequal crossing-over between homologous chromosomes 7 (interchromosomal rearrangement) while in the remaining cases an intrachromosomal recombination (between the chromatids of one chromosome 7) may have occurred. These results suggest that the majority of interstitial deletions of the elastin gene region occur during meiosis, due to unbalanced recombination while a minority could occur before or during meiosis probably due to intrachromosomal rearrangements. The recurrence risk of the interchromosomal rearrangements for sibs of a proband with non-affected parents must be negligible, which fits well with the observation of sporadic occurrence of almost all cases of WBS.      

Origin of uniparental disomy 15 in patients with Prader-Willi or Angelman syndrome

Maternal uniparental disomy (UPD) accounts for approximately 25% of Prader-Willi patients (PWS) and paternal UPD for about 2-5% of Angelman syndrome (AS) patients. These findings and the parental origin of deletions are evidence of genomic imprinting in the cause of PWS and AS. The natural occurrence of UPD individuals allows the study of meiotic mechanisms resulting in chromosomal nondisjunction (ND). We selected patients with UPD15 from our sample of 30 PWS and 40 AS patients to study the origin of ND and the recombination along chromosome 15. These patients were analyzed with 10 microsatellites throughout the entire chromosome 15 (D15S541, D15S542, D15S11, D15S113, GABRB3, CYP19, D15S117, D15S131, D15S984, D15S115). The analysis disclosed seven heterodisomic PWS cases originating by meiosis I (MI) ND (four showed recombination and three no recombination), and one isodisomic PWS UPD15 originating by postzygotic duplication. Among the five paternal UPD15, we detected four isodisomies, three of which showed homozigosity for all markers, corresponding to a mitotic error, and one case originating from a paternal MII ND. Our results indicate that besides maternal MI and MII ND, paternal ND occurs when a PWS UPD15 patient originates from mitotic duplication of the maternal chromosome 15. ND events in AS are mainly due to mitotic errors, but paternal MII ND can occur and give origin to an AS UPD15 individual by two different mechanisms: rescue of a trisomic fetus or fertilization of a nullisomic egg with the disomic sperm, and in this case paternal and maternal ND are necessary.     

Human chromosome 15q11-q14 regions of rearrangements contain clusters of LCR15 duplicons

Six breakpoint regions for rearrangements of human chromosome 15q11-q14 have been described. These rearrangements involve deletions found in approximately 70% of Prader-Willi or Angelman's syndrome patients (PWS, AS), duplications detected in some cases of autism, triplications and inverted duplications. HERC2-containing (HEct domain and RCc1 domain protein 2) segmental duplications or duplicons are present at two of these breakpoints (BP2 and BP3) mainly associated with deletions. We show here that clusters containing several copies of the human chromosome 15 low-copy repeat (LCR15) duplicon are located at each of the six described 15q11-q14 BPs. In addition, our results suggest the existence of breakpoints for large 15q11-q13 deletions in a proximal duplicon-containing clone. The study reveals that HERC2-containing duplicons (estimated on 50-400 kb) and LCR15 duplicons ( approximately 15 kb on 15q11-q14) share the golgin-like protein (GLP) genomic sequence. Through the analysis of a human BAC library and public databases we have identified 36 LCR15 related sequences in the human genome, most (27) mapping to chromosome 15q and being transcribed. LCR15 analysis in non-human primates and age-sequence divergences support a recent origin of this family of segmental duplications through human speciation.     

Molecular characterization of a unique de novo 15q deletion associated with Prader-Willi syndrome and central visual impairment

We report a 2-year-old boy with Prader-Willi Syndrome (PWS) caused by a deletion of the PWS critical region as a result of an unbalanced translocation t(3;15). Additional features, including central visual impairment, relative macrocephaly, retrognathia, preauricular tags, and bilateral club-feet, were noticed. The extension of the deletion was determined by fluorescence in situ hybridization (FISH) analysis using 11 region-specific YAC clones. Nine YACs were found to be deleted, allowing us to determine that the deletion is larger than in patients with typical PWS deletions. The karyotype of this patient can thus be designated: 45,XY,-15,der(3)t(3;15)(qter;q14).ish der(3)t(3;15)(qter;q14) (wcp3+,wcp15+,D15S10-,PML+,D15Z1-,D3S4560+,801_f_9x1, 815_e_6x2) de novo. Molecular analyses using seven polymorphic markers helped to narrow down the breakpoint between marker ACTC.PC3 and the distal end of the YAC 815_e_6. These results provide evidence that haploinsufficiency for genes in 15q13-q14, not affected in common PWS deletions, is associated with the additional features found in the patient, including a central visual impairment.     

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.     

Molecular and clinical overlap of Angelman and Prader-Willi syndrome phenotypes.

The Prader-Willi (PWS) and Angelman syndromes (AS) share the same apparent cytogenetic and molecular lesions of 15q11-13 and yet exhibit distinct clinical phenotypes. The etiology of PWS or AS appears to depend on the parental origin of the aberrant chromosome 15. Substantial clinical overlap has not been reported between deletion-positive PWS and AS patients. In the present study, we report the clinical, cytogenetic, and molecular findings in three AS patients. The first patient is a mentally retarded woman with a visible deletion of 15q11-13 with typical craniofacial, behavioral, and neurologic changes of AS. This patient is hyperphagic, and she is moderately obese for her height. Her hands and feet are small. These manifestations are more characteristic of PWS and not of AS. The molecular studies showed deletions of maternal origin for five distal PWCR loci. The most proximal locus, D15S18, was not deleted. These findings are identical to those found in our third AS patient who does not have any PWS features. To the best of our knowledge, this is the first report of concurrence of hyperphagia with consequent obesity and the AS phenotype in a patient with a del 15(q11-13) of maternal origin. These clinical findings suggest that overlap in the symptoms of PWS and AS can occur. Our second AS patient presents with atypical molecular findings in that he cannot be classed into any of the three proposed sub-groups of AS patients and may be representative of a fourth sub-group of AS patients.