Allele-specific methylation of a functional CTCF binding site upstream of MEG3 in the human imprinted domain of 14q32.

The gene MEG3 is located in the imprinted human chromosomal region on 14q32. Imprinting of a structurally homologous region IGF2/H19 on 11p15 is mediated through cytosine methylation-controlled binding of the protein CTCF to target sites upstream of H19. We identified five new CTCF binding sites around the promoter of MEG3. Using an electrophoretic mobility shift assay, we showed that these sites bind CTCF in vitro. Using one of these sites, chromatin immunoprecipitation (ChIP) analysis confirmed CTCF binding in-vivo, and differential allele-specific methylation was demonstrated in seven individuals with either maternal or paternal uniparental disomy 14 (UPD14). The site was unmethylated on the maternally inherited chromosomes 14 and methylated on the paternally inherited chromosomes 14, suggesting parent-specific methylation of sequences upstream of MEG3. We speculate that this CTCF-binding region may provide a mechanism for the transcriptional regulation of MEG3 and DLK1.

Isolated left ventricular noncompaction is rarely caused by mutations in G4.5, alpha-dystrobrevin and FK Binding Protein-12.

Isolated left ventricular noncompaction (LVNC) is a form of cardiomyopathy that most commonly presents in infancy with a hypertrophic and dilated left ventricle characterized by deep trabeculations and intertrabecular recesses. Our goal was to determine the frequency of mutations in G4.5, alpha-dystrobrevin, and FK Binding protein-12 in isolated LVNC patients. No mutations were identified in 47 of the 48 patients studied, while a splice site acceptor site mutation of intron 10 of G4.5 was identified in one patient, resulting in the deletion of exon 10 from the mRNA.

Mutations in the muscle LIM protein and alpha-actinin-2 genes in dilated cardiomyopathy and endocardial fibroelastosis.

Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality. Two genes have been identified for the X-linked forms (dystrophin and tafazzin), while mutations in multiple genes cause autosomal dominant DCM. Muscle LIM protein (MLP) is a member of the cysteine-rich protein (CRP) family and has been implicated in both myogenesis and sarcomere assembly. In the latter role, it binds zyxin and alpha-actinin, both of which are involved in actin organization. An MLP-deficient mouse has been described; these mice develop dilated cardiomyopathy and heart failure. Based upon these data, and the recent descriptions of mutations in MLP in patients with DCM or hypertrophic cardiomyopathy, we screened patients for mutations in the MLP and alpha-actinin-2 genes. We identified a patient with DCM and EFE, having a mutation in MLP with the residue lysine 69 substituted by arginine (K69R). This is within a highly conserved region adjacent to the first LIM domain involved in alpha-actinin binding. Analysis in cell culture systems demonstrated that the mutation abolishes the interaction between MLP and alpha-actinin-2 and the cellular localization of MLP was altered. In another individual with DCM, a W4R mutation was identified. However, this mutation did not segregate with disease in this family. In another patient with DCM, a Q9R mutation was identified in alpha-actinin-2. This mutation also disrupted the interaction with MLP and appeared to inhibit alpha-actinin function in cultured cells, in respect to the nuclear localization of actinin and the initiation of cellular differentiation.

Subtelomeric FISH uncovers trisomy 14q32: lessons for imprinted regions, cryptic rearrangements and variant acrocentric short arms.

The recent development of a set of chromosome-specific, subtelomeric probes has proved useful in diagnosis and recurrence risk counseling of patients and families with mental retardation and in further characterization of known chromosomal abnormalities. Cases of cryptic, subtelomeric rearrangements may account for up to 7.5% of cases of idiopathic moderate-severe mental retardation. We present the molecular cytogenetic studies of trisomy 14q detected by subtelomeric fluorescence in situ hybridization (FISH). Our patient is a 3-year-old girl with growth and developmental delay, myelomeningocele, partial agenesis of the corpus callosum, hypertelorism, tented mouth, simple ears, small mandible, and congenital heart disease (atrial and ventricular septal defects with subaortic conus). G-banded chromosome analysis was apparently normal. A set of FISH-based, subtelomeric, region-specific probes revealed trisomy for 14q in the child. Parental FISH studies established that the mother is a balanced carrier for a half-cryptic translocation between the distal long arm of chromosome 14 and the short arm of chromosome 22. FISH analysis using two BAC clones that contain the imprinted genes MEG3 and DLK1, which localize to 14q32, established that our patient has two maternal copies of these genes. Because the child does not have features of the maternal UPD 14 syndrome, this case suggests that it is absence of expression of a paternally expressed gene, rather than overexpression of a maternally expressed gene, that is responsible for the maternal UPD 14 phenotype.

A case of segmental paternal isodisomy of chromosome 14.

Uniparental disomy of chromosome 14 (UPD 14) results in one of two distinct abnormal phenotypes, depending upon the parent of origin. This discordance may result from the reciprocal over-expression and/or under-expression of one or more imprinted genes. We report a case of segmental paternal isodisomy for chromosome 14 with features similar to those reported in other paternal disomy 14 cases. Microsatellite marker analysis revealed an apparent somatic recombination event in 14q12 leading to proximal biparental inheritance, but segmental paternal uniparental isodisomy distal to this site. Analysis of monochromosomal somatic cell hybrids containing either the paternally inherited or the maternally inherited chromosome 14 revealed no deletion of the maternally inherited chromosome 14 and demonstrated the presence of paternal sequences from D14S121 to the telomere on both chromosomes 14. Thus, the patient has paternal isodisomy for 14q12-14qter. Because the patient shows most of the features associated with paternal disomy 14, this supports the presence of the imprinted domain(s) distal to 14q12 and suggests that the proximal region of chromosome 14 does not contain imprinted genes that contribute significantly to the paternal UPD 14 phenotype.