Parental origin and functional relevance of a de novo UBE3A variant.

Sequence analysis of the imprinted UBE3A gene in a 3-year-old girl suspected of having Angelman syndrome had revealed a de novo 3bp in frame deletion predicted to encode a protein lacking the amino acid G538 (based on sequence NM_130838). In order to assess the clinical relevance of this unknown variant, we determined the parental origin and the functional consequences of the deletion. We separated the two chromosomes 15 by microdissection of metaphase spreads and used cytogenetic and molecular markers to demonstrate that the deletion is on the maternal chromosome. For determining the functional consequences of the deletion, we modelled the structure of the deletion mutant based on the wildtype X-ray structure and simulated the molecular dynamics of the wildtype and mutant protein in complex with UcbH7. Our simulations showed that deletion of G538 destroys a network of salt bridges between highly conserved residues in the catalytic cleft of UBE3A. In conclusion, our results strongly suggest that the 3bp deletion is a loss-of-function mutation of the maternal UBE3A allele that has caused Angelman syndrome in our patient. Our study may serve as a paradigm to determine the parental origin of a de novo mutation.

Clinical features of maternal uniparental disomy 14 in patients with an epimutation and a deletion of the imprinted DLK1/GTL2 gene cluster.

Maternal uniparental disomy 14 [upd(14)mat] is associated with a recognizable phenotype that includes pre- and postnatal growth retardation, neonatal hypotonia, feeding problems and precocious puberty. Chromosome 14 contains an imprinted gene cluster, which is regulated by a differentially methylated region (IG-DMR) between DLK1 and GTL2. Here we report on four patients with clinical features of upd(14)mat who show a maternal-only methylation pattern, but biparental inheritance for chromosome 14. In three of the patients loss of paternal methylation appears to be a primary epimutation, whereas the other patient has a paternally derived deletion of -1 Mb that includes the imprinted DLK1-GTL2 gene cluster. These findings demonstrate that the upd(14)mat phenotype is caused by altered expression of genes within this cluster.

Somatic mosaicism for maternal uniparental disomy 15 in a girl with Prader-Willi syndrome: confirmation by cell cloning and identification of candidate downstream genes.

Although uniparental disomy often results from the postzygotic rescue of a meiotic non-disjunction event, mosaicism is usually confined to the placenta. We describe a girl with Prader-Willi syndrome (PWS) who is mosaic for normal cells and cells with maternal uniparental disomy 15 [upd(15)mat] in blood and skin. Somatic mosaicism was confirmed by cloning and genotyping of skin fibroblasts. X inactivation studies indicated that upd occurred prior to X inactivation. RNA samples from the cloned cells were used in DNA microarray experiments to study the effect of upd(15)mat on the gene expression pattern of fibroblasts. Proof of principle was obtained by detecting several chromosome 15 genes known to be imprinted. We did not obtain any evidence for novel 15q genes showing imprinted expression in fibroblasts. Differentially expressed genes on other chromosomes are candidates for downstream genes regulated by an imprinted gene and may play a role in the pathogenesis of PWS. The finding of strongly reduced mRNA levels in upd(15)mat cells of the gene encoding secretogranin II (SCG2), which is a precursor of the dopamine releasing factor secretoneurin, raises the question whether hyperphagia in patients with PWS might be due to a defect in dopamine-modulated food reward circuits.

Epimutations in Prader-Willi and Angelman syndromes: a molecular study of 136 patients with an imprinting defect.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurogenetic disorders that are caused by the loss of function of imprinted genes in 15q11-q13. In a small group of patients, the disease is due to aberrant imprinting and gene silencing. Here, we describe the molecular analysis of 51 patients with PWS and 85 patients with AS who have such a defect. Seven patients with PWS (14%) and eight patients with AS (9%) were found to have an imprinting center (IC) deletion. Sequence analysis of 32 patients with PWS and no IC deletion and 66 patients with AS and no IC deletion did not reveal any point mutation in the critical IC elements. The presence of a faint methylated band in 27% of patients with AS and no IC deletion suggests that these patients are mosaic for an imprinting defect that occurred after fertilization. In patients with AS, the imprinting defect occurred on the chromosome that was inherited from either the maternal grandfather or grandmother; however, in all informative patients with PWS and no IC deletion, the imprinting defect occurred on the chromosome inherited from the paternal grandmother. These data suggest that this imprinting defect results from a failure to erase the maternal imprint during spermatogenesis.

Methylation analysis of several tumour suppressor genes shows a low frequency of methylation of CDKN2A and RARB in uveal melanomas.

We have investigated the frequency of methylation of several tumour suppressor genes in uveal melanoma. As the loss of one copy of chromosome 3 (monosomy 3), which is found in about half of these tumours, is tightly associated with metastatic disease, a special emphasis was laid on genes located on this chromosome, including the fragile histidine triad (FHIT), von Hippel-Lindau (VHL), beta-catenin (CTNNB1), activated leukocyte cell adhesion molecule (ALCAM) and retinoic acid receptor-beta2 (RARB) genes. In addition, the methylation patterns of the CpG-rich regions 5' of the E-cadherin (CDH1), p16/cyclin-dependent kinase inhibitor 2 A (CDKN2A) and retinoblastoma (RB1) genes were analysed by bisulphite genomic sequencing or methylation-specific PCR (MSP). Furthermore, the SNRPN and D15S63 loci, which are located in the imprinted region of chromosome 15, were included in the study. Aberrant methylation was detected in nine of 40 tumours analysed: The imprinted SNRPN and D15S63 loci were hypermethylated in three tumours, all of which retained both copies of chromosome 3. Methylated RARB alleles were detected in three tumours, whereas in three other tumours CDKN2A was found to be methylated. As we did not find RARB and CDKN2A preferentially methylated in tumours with monosomy 3, which is a significant predictor of metastatic disease, we suggest that these genes may play a causative role in the formation of uveal melanoma but not in the development of metastases.