A 5-year-old white girl with Prader-Willi syndrome and a submicroscopic deletion of chromosome 15q11q13

We report on a 5-year-old white girl with Prader-Willi syndrome (PWS) and a submicroscopic deletion of 15q11q13 of approximately 100–200 kb in size. High resolution chromosome analysis was normal but fluorescence in situ hybridization (FISH), Southern hybridization, and microsatellite data from the 15q11q13 region demonstrated that the deletion was paternal in origin and included the SNRPN, PAR-5, and PAR-7 genes from the proximal to distal boundaries of the deletion segment. SNRPN and PW71B methylation studies showed an abnormal pattern consistent with the diagnosis of PWS and supported the presence of a paternal deletion of 15q11q13 or an imprinting mutation. Biparental (normal) inheritance of PW71B (D15S63 locus) and a deletion of the SNRPN gene were observed by microsatellite, quantitative Southern hybridization, and/or FISH analyses. Our patient met the diagnostic criteria for PWS, but has no reported behavior problems, hyperphagia, or hypopigmentation. Our patient further supports SNRPN and possibly other genomic sequences which are deleted as the cause of the phenotype recognized in PWS patients. © 1996 Wiley-Liss, Inc.     

Paternally derived de novo interstitial duplication of proximal 15q in a patient with developmental delay

Interstitial duplications of proximal 15q containing the Prader-Willi syndrome/Angelman syndrome (PWS/AS) region have been found in patients with autism or atypical autism. In these cases with an abnormal phenotype, the duplications were maternally derived. Paternal origin of the duplication has been associated with a normal phenotype. We report on a patient who presented with nonspecific developmental delay and partial agenesis of the rostral corpus callosum. Fluorescence in situ hybridization (FISH) studies using probes specific for the PWS/AS region demonstrated a double signal on one chromosome 15, indicating the presence of an interstitial duplication of proximal 15q involving the PWS/ AS region in the patient. Parental chromosomes were normal with FISH studies. Methylation analysis at exon alpha of the SNRPN locus showed a maternal band at 4.2 kb and a paternal band of apparent double intensity at 0.9 kb, suggestive of one copy of the maternal allele and two copies of the paternal allele in the patient. Microsatellite analysis was informative at the GABRB3 locus in the family, which showed the inheritance of two different paternal alleles and a maternal allele in the patient consistent with the origin of this duplication from an unequal crossing over between the two chromosome 15 homologs in the father. This is the first report of an abnormal phenotype associated with a paternally derived duplication of proximal 15q shown to contain the PWS/AS region by molecular techniques.     

A rapid, PCR based test for differential molecular diagnosis of Prader-Willi and Angelman syndromes.

Approximately 98% of Prader-Willi syndrome (PWS) and 80% of Angelman syndrome (AS) cases have deletions at a common region in chromosome 15q11-13, uniparental disomy for chromosomes 15 (UPD15), or mutations affecting gene expression in this region. The resulting clinical phenotype (PWS or AS) in each class of mutation depends upon the parent of origin. Both disorders are characterised at the molecular level by abnormal methylation of imprinted genes at 15q11-q13 including the small nuclear ribonucleoprotein N gene (SNRPN). Current diagnostic strategies include high resolution cytogenetics, fluorescence in situ hybridisation (FISH), Southern blot hybridisation, or microsatellite typing. We have developed a novel and rapid diagnostic test for PWS and AS based on differential digestion of expressed (paternally imprinted) SNRPN sequences by the methylation sensitive endonuclease NotI or repressed (maternally imprinted) SNRPN sequences by the methylation requiring nuclease McrBC, followed by PCR amplification of the SNRPN promoter. We have evaluated this test by blinded analysis of 60 characterised DNA samples (20 PWS, 20 AS, and 20 unaffected controls). SNRPN sequences could not be amplified from PWS patient DNA which had been digested with McrBC, nor from AS patient DNA which had been digested with NotI. We were able to make a correct diagnosis of PWS, AS, or unaffected in all 60 samples tested. This novel test is rapid and has a high specificity and sensitivity for deletion and UPD15 cases. These features make this new test suitable as the initial step in a molecular diagnostic strategy for PWS/AS.     

A translocation breakpoint cluster disrupts the newly defined 3' end of the SNURF-SNRPN transcription unit on chromosome 15

Balanced translocations affecting the paternal copy of 15q11--q13 are a rare cause of Prader-Willi syndrome (PWS) or PWS-like features. Here we report on the cytogenetic and molecular characterization of a de novo balanced reciprocal translocation t(X;15)(q28;q12) in a female patient with atypical PWS. The translocation breakpoints in this patient and two previously reported patients map 70-80 kb distal to the SNURF-SNRPN gene and define a breakpoint cluster region. The breakpoints disrupt one of several hitherto unknown 3' exons of this gene. Using RT--PCR we demonstrate that sequences distal to the breakpoint, including the recently identified C/D box small nucleolar RNA (snoRNA) gene cluster HBII-85 as well as IPW and PAR1, are not expressed in the patient. Our data suggest that lack of expression of these sequences contributes to the PWS phenotype.     

Maternal disomy and Prader-Willi syndrome consistent with gamete complementation in a case of familial translocation (3;15) (p25;q11.2)

Maternal uniparental disomy (UPD) for chromosome 15 is responsible for an estimated 30% of cases of Prader-Willi syndrome (PWS). We report on an unusual case of maternal disomy 15 in PWS that is most consistent with adjacent-1 segregation of a paternal t(3;15)(p25;q11.2) with simultaneous maternal meiotic nondisjunction for chromosome 15. The patient (J.B.), a 17-year-old white male with PWS, was found to have 47 chromosomes with a supernumerary, paternal der(15) consisting of the short arm and the proximal long arm of chromosome 15, and distal chromosome arm 3p. The t(3;15) was present in the balanced state in the patient's father and a sister. Fluorescent in situ hybridization analysis demonstrated that the PWS critical region resided on the derivative chromosome 3 and that there was no deletion of the PWS region on the normal pair of 15s present in J.B. Methylation analysis at exon alpha of the small nuclear ribonucleoprotein-associated polypeptide N (SNRPN) gene showed a pattern characteristic of only the maternal chromosome 15 in J.B. Maternal disomy was confirmed by polymerase chain reaction analysis of microsatellite repeats at the gamma-aminobutyric acid receptor beta3 subunit (GABRB3) locus. A niece (B.B.) with 45 chromosomes and the derivative 3 but without the der(15) demonstrated a phenotype consistent with that reported for haploinsufficiency of distal 3 p. Uniparental disomy associated with unbalanced segregation of non-Robertsonian translocations has been reported previously but has not, to our knowledge, been observed in a case of PWS. Furthermore, our findings are best interpreted as true gamete complementation resulting in maternal UPD 15 and PWS. Am. J. Med. Genet. 78:134–139, 1998. © 1998 Wiley-Liss, Inc.     

Is there a higher incidence of maternal uniparental disomy 14 [upd(14)mat]? Detection of 10 new patients by methylation-specific PCR

Maternal uniparental disomy for chromosome 14 [upd(14)mat] is associated with a characteristic phenotype including pre- and postnatal growth retardation, muscular hypotonia, feeding problems, motor delay, small hands and feet, precocious puberty and truncal obesity. Patients with upd(14)mat show features overlapping with Prader-Willi syndrome (PWS) and are probably underdiagnosed. Maternal upd(14) is frequently described in carriers of a Robertsonian translocation involving chromosome 14, but is also found in patients with a normal karyotype. Based on the above mentioned criteria we have identified six patients with upd(14)mat including two patients with a normal karyotype, one patient with a de novo Robertsonian translocation (14;21), one patient with a familial Robertsonian translocation (13;14) and two patients with a marker chromosome. In addition, we analyzed a cohort of 33 patients with low birth weight, feeding difficulties and consecutive obesity in whom PWS had been excluded by methylation analysis of SNRPN. In four of these patients (12%) we detected upd(14)mat. For rapid testing of upd(14)mat we analyzed the methylation status of the imprinted MEG3 locus. In conclusion, we recommend considering upd(14)mat in patients with low birth weight, growth retardation, neonatal feeding problems, muscular hypotonia, motor delay, precocious puberty and truncal obesity as well as in patients with a PWS like phenotype presenting with low birth weight, feeding difficulties and obesity.     

Genomic imprinting: potential function and mechanisms revealed by the Prader-Willi and Angelman syndromes

The Prader-Willi (PWS) and Angelman (AS) syndromes are two clinically distinct syndromes which result from lack of expression of imprinted genes within chromosome 15q11-q13. These two syndromes result from 15q11-q13 deletions, chromosome 15 uniparental disomy (UPD), imprinting centre mutations and, for AS, probable mutations in a single gene. The differential phenotype results from a paternal genetic deficiency in PWS patients and a maternal genetic deficiency in AS patients. Within 15q11-q13, four genes (SNRPN, IPW, ZNF127, FNZ127) and two expressed sequence tags (PAR1 and PAR5) have been found to be expressed only from the paternally inherited chromosome, and therefore all must be considered candidate genes involved in the pathogenesis of PWS. A candidate AS gene (UBE3A) has very recently been identified. The mechanisms of imprinted gene expression are not yet understood, but it is clear that DNA methylation is involved in both somatic cell expression and inheritance of the imprint. The presence of DNA methylation imprints that distinguish the paternally and maternally inherited alleles is a common characteristic of all known imprinted genes which have been studied extensively, including SNRPN and ZNF127. Recently, several PWS and AS patients have been found that have microdeletions in a region upstream of the SNRPN gene referred to as the imprinting centre, or IC. Paternal IC deletions in PWS patients and maternal IC deletions in AS patients result in uniparental DNA methylation and uniparental gene expression at biparentally inherited loci. The IC is a novel genetic element which controls initial resetting of the parental imprint in the germline for all imprinted gene expression over a 1.5-2.5 Mb region within chromosome 15q11-q13.      

Prader-Willi syndrome resulting from an unbalanced translocation: characterization by array comparative genomic hybridization

Prader-Willi syndrome (PWS) is caused by lack of expression of paternally inherited genes on chromosome 15q11-->15q13. Most cases result from microdeletions in proximal chromosome 15q. The remainder results from maternal uniparental disomy of chromosome 15, imprinting center defects, and rarely from balanced or unbalanced chromosome rearrangements involving chromosome 15. We report a patient with multiple congenital anomalies, including craniofacial dysmorphology, microcephaly, bilateral cryptorchidism, and developmental delay. Cytogenetic analysis showed a de novo 45,XY,der(5)t(5;15)(p15.2;q13), -15 karyotype. In effect, the proband had monosomies of 5p15.2-->pter and 15pter-->15q13. Methylation polymerase chain reaction analysis of the promoter region of the SNRPN gene showed only the maternal allele, consistent with the PWS phenotype. The proband's expanded phenotype was similar to other patients who have PWS as a result of unbalanced translocations and likely reflects the contribution of the associated monosomy. Array comparative genomic hybridization (array CGH) confirmed deletions of both distal 5p and proximal 15q and provided more accurate information as to the size of the deletions and the molecular breakpoints. This case illustrates the utility of array CGH in characterizing complex constitutional structural chromosome abnormalities at the molecular level.     

Molecular breakpoint cloning and gene expression studies of a novel translocation t(4;15)(q27;q11.2) associated with Prader-Willi syndrome

Background  

Prader-Willi syndrome (MIM #176270; PWS) is caused by lack of the paternally-derived copies, or their expression, of multiple genes in a 4 Mb region on chromosome 15q11.2. Known mechanisms include large deletions, maternal uniparental disomy or mutations involving the imprinting center. De novo balanced reciprocal translocations in 5 reported individuals had breakpoints clustering in SNRPN intron 2 or exon 20/intron 20. To further dissect the PWS phenotype and define the minimal critical region for PWS features, we have studied a 22 year old male with a milder PWS phenotype and a de novo translocation t(4;15)(q27;q11.2).     

Molecular study of the Prader-Willi syndrome: deletion, RFLP, and phenotype analyses of 50 patients.

Deletion and RFLP studies with 5 cloned DNA markers localized at 15q11.2 were performed in 50 patients with the Prader-Willi syndrome (PWS). A one-copy density (deletion) for at least one of 4 loci, D15S9, D15S11, D15S10, D15S12, was detected in 32 (64%) of the 50 patients; deletions of each of the 4 loci were found in 29, 30, 29, and 28 patients, respectively. Three patients showed 4 or more copy density for D15S12 locus, in addition to deletions. The remaining 18 patients showed two-copy densities for each of the 4 loci. A common site of rearrangements among our 32 patients as well as the reported patients seemed to be confined to a segment between D15S9 and D15S11, suggesting the putative PWS gene locus in this segment. Of 6 patients who have cytologic deletions but did not show any molecular deletions, 3 have normal size of hands and feet, and 4 have normally pigmented skin and hair. The normal pigmentation was also observed in 3 patients who had small molecular deletions in the examined 5-locus segment. These observations may support the conception of contiguous gene syndrome. RFLP analysis demonstrated maternal uniparental isodisomy of chromosomes 15 in both a patient with 45,t(15q;15q) and a karyotypically normal patient. Based on the results of the present study, a new model is proposed to explain the occurrence of PWS with a variety of chromosome abnormalities, including partial monosomy, disomy, trisomy, and/or tetrasomy for 15q11.2. The normal development may require an even or more "number ratio" of paternally derived allele(s) to maternally derived allele(s) of the gene(s) localized at 15q11.2, and a disturbance of the ratio would lead to the PWS phenotype.     

A case of an atypically large proximal 15q deletion as cause for Prader–Willi syndrome arising from a de novo unbalanced translocation

We describe an 11 month old female with Prader–Willi syndrome (PWS) resulting from an atypically large deletion of proximal 15q due to a de novo 3;15 unbalanced translocation. The 10.6 Mb deletion extends from the chromosome 15 short arm and is not situated in a region previously reported as a common distal breakpoint for unbalanced translocations. There was no deletion of the reciprocal chromosome 3q subtelomeric region detected by either chromosomal microarray or FISH. The patient has hypotonia, failure to thrive, and typical dysmorphic facial features for PWS. The patient also has profound global developmental delay consistent with an expanded, more severe, phenotype.     

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.     

Deletions of proximal 15q and non-classical Prader-Willi syndrome phenotypes

A deletion of the long arm of chromosome 15 (usually involving bands 15q11-q12) has been seen in approximately 50% of Prader-Willi syndrome (PWS) patients [Ledbetter et al, 1982]. However, 14 patients with non-PWS (or atypical PWS) phenotype with 15q deletion indicate great clinical variability. A deletion was found in a propositus with a de novo translocation [45,XY, -15, -22, +rec(15;22) (22pter----22q13.2::15q14----15qter)], who had anomalies not normally observed in PWS patients. Activities of several enzymes mapped to the involved chromosomes were studied in the patient and control individuals. A 50% decrease in the level of arylsulfatase-A confirmed a small deletion in 22q(22q13.2----qter), and additional studies localized more precisely the loci for alpha-mannosidase (cytoplasmic) and beta-galactosidase.     

Molecular study of the Prader-Willi syndrome: Deletion,RFLP, and phenotype analyses of 50 patients

Deletion and RFLP studies with 5 cloned DNA markers localized at 15q11.2 were performed in 50 patients with the Prader–Willi syndrome (PWS). A one-copy density (deletion) for at least one of 4 loci, D15S9, D15S11, D15S10, D15S12, was detected in 32 (64%) of the 50 patients; deletions of each of the 4 loci were found in 29, 30, 29, and 28 patients, respectively. Three patients showed 4 or more copy density for D15S12 locus, in addition to deletions. The remaining 18 patients showed two-copy densities for each of the 4 loci. A common site of rearrangements among our 32 patients as well as the reported patients seemed to be confined to a segment between D15S9 and D15S11, suggesting the putative PWS gene locus in this segment. Of 6 patients who have cytologic deletions but did not show any molecular deletions, 3 have normal size of hands and feet, and 4 have normally pigmented skin and hair. The normal pigmentation was also observed in 3 patients who had small molecular deletions in the examined 5-locus segment. These observations may support the conception of contiguous gene syndrome. RFLP analysis demonstrated maternal uniparental isodisomy of chromosomes 15 in both a patient with 45,t(15q;15q) and a karyotypically normal patient. Based on the results of the present study, a new model is proposed to explain the occurrence of PWS with a variety of chromosome abnormalities, including partial monosomy, disomy, trisomy, and/or tetrasomy for 15q11.2. The normal development may require an even or more “number ratio” of paternally derived allele(s) to maternally derived allele(s) of the gene(s) localized at 15q11.2, and a disturbance of the ratio would lead to the PWS phenotype.     

Minimal phenotype in a girl with trisomy 15q due to t(X;15)(q22.3;q11.2) translocation

Few cases of de novo unbalanced X;autosome translocations associated with a normal or mild dysmorphic phenotype have been described. We report a 3-year-old dizygotic female twin with prenatally ascertained increased nuchal translucency. Prenatal chromosome studies revealed nearly complete trisomy 15 due to a de novo unbalanced translocation t(X;15)(q22;q11.2) confirmed postnatally. A mild phenotype was observed with normal birth measurements, minor facial dysmorphic features (hypertelorism, short broad nose, and a relatively long philtrum), and moderate developmental delay at the age of 3 years in comparison to her male fraternal twin. Replication timing utilizing BrdU and acridine-orange staining showed that the der(X) chromosome was late-replicating with variable spreading of inactivation into the translocated 15q segment. The der(X) was determined to be of paternal origin by analyses of polymorphic markers and CGG-repeat at FMR1. Methylation analysis at the SNRPN locus and analysis of microsatellites on 15q revealed paternal isodisomy with double dosage for all markers and the unmethylated SNRPN gene. The Xq breakpoint was mapped within two overlapping BAC clones RP11-575K24 and RP13-483F6 at Xq22.3 and the 15q breakpoint to 15q11.2, within overlapping clones RP11-509A17 and RP11-382A4 that are all significantly enriched for LINE-1 elements (36.6%, 43.0%, 26.6%, 22.0%, respectively). We speculate that the attenuated phenotype may be due to inactivation spreading into 15q, potentially facilitated by the enrichment of LINE-1 elements at the breakpoints. In silico analysis of breakpoint regions revealed the presence of highly identical low-copy repeats (LCRs) at both breakpoints, potentially involved in generating the translocation.     

Familial Prader-Willi syndrome: case report and a literature review

Prader-Willi syndrome (PWS) is a neurobehavioural disorder arising through a number of different genetic mechanisms. All involve loss of paternal gene expression from chromosome 15q11q13. Although the majority of cases of PWS are sporadic, precise elucidation of the causative genetic mechanism is essential for accurate genetic counselling as the recurrence risk varies according to the mechanism involved. A pair of siblings affected by PWS is described. Neither demonstrates a microscopically visible deletion in 15q11q13 or maternal disomy. Methylation studies at D15S63 and at the SNRPN locus confirm the diagnosis of PWS. Molecular studies reveal biparental inheritance in both siblings with the exception of D15S128 and D15S63 where no paternal contribution is present indicating a deletion of the imprinting centre. Family studies indicate that the father of the siblings carries the deletion which, he has inherited from his mother. The recurrence risk for PWS in his offspring is 50%.     

Paternal deletion from Snrpn to Ube3a in the mouse causes hypotonia, growth retardation and partial lethality and provides evidence for a gene contributing to Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is caused by paternal deficiency of human chromosome 15q11-q13. There is conflicting evidence from human translocations regarding the direct involvement of SNRPN in the pathogenesis of PWS and it is not known if the phenotypic features result from the loss of expression of a single imprinted gene or multiple genes. In an attempt to dissect genotype/phenotype correlations for the homologous region of mouse chromosome 7C, we prepared three mutant genotypes: (i) mice with a deletion of Snrpn exon 2, which removes a portion of a small, upstream open reading frame (ORF); (ii) mice with double targeting for Snrpn exon 2 and Ube3a; (iii) mice deleted from Snrpn to Ube3a, removing coding exons for both loci and intervening genes. Mice deleted for Snrpn exon 2 have no obvious phenotypic abnormalities and switching of the genomic imprint for the region is conserved. Mice carrying the Snrpn - Ube3a deletion on the paternal chromosome showed severe growth retardation, hypotonia and approximately 80% lethality before weaning. The surviving mice were fertile and were not obese up to 14 months of age. The deletion was transmitted for multiple generations and continued to cause partial lethality when inherited paternally, but not when inherited maternally. The normal imprinted expression and methylation patterns of necdin, a gene outside the deletion region, indicate that the deletion is not an imprinting mutation. The data suggest the presence of a paternally expressed structural gene between Snrpn and Ipw whose deficiency causes lethality, although other possibilities exist, including position effects on expression of imprinted genes or that simultaneous deficiency of both ORFs of Snrpn causes lethality.     

Cryptic insertion producing two NUP98/NSD1 chimeric transcripts in adult refractory anemia with an excess of blasts

We performed cytogenetic and molecular studies on an adult patient with refractory anemia with an excess of blasts with an add(11)(p15). Multicolor fluorescence in situ hybridization (FISH) identified the extra material on 11p as belonging to chromosome 15. Metaphase FISH with probes for chromosomes 5, 11, and 15 revealed a complex four-break rearrangement. Clone RP5-1173K1, containing exons 10-20 of the NUP98 gene, gave three fluorescence signals on the normal 11, the der(5), and the der(15). 3'-RACE-PCR identified an in-frame fusion between NUP98 and NSD1, which was confirmed by RT-PCR. Two different spliced forms, that is, NUP98 exon 11/NSD1 exon 6 and NUP98 exon 12/NSD1 exon 6, were detected. The reciprocal NSD1/NUP98 was not found. A dual-color experiment with RP5-1173K1 and CTC-549A4, spanning the entire NSD1 gene, indicated an insertion of NUP98 into the NSD1 locus. This is the first report of an adult with myelodysplastic syndrome (MDS) harboring an NUP98/NSD1 fusion resulting from insertion of 5'-NUP98 into the NSD1/5q35 locus.