Targeted re-sequencing of linkage region on 2q21 identifies a novel functional variant for hip and knee osteoarthritis.

OBJECTIVE: The aim of the study was to identify genetic variants predisposing to primary hip and knee osteoarthritis (OA) in a sample of Finnish families. METHODS: Genome wide analysis was performed using 15 independent families (279 individuals) originating from Central Finland identified as having multiple individuals with primary hip and/or knee OA. Targeted re-sequencing was performed for three samples from one 33-member, four-generation family contributing most significantly to the LOD score. In addition, exome sequencing was performed in three family members from the same family. RESULTS: Genome wide linkage analysis identified a susceptibility locus on chromosome 2q21 with a multipoint LOD score of 3.91. Targeted re-sequencing and subsequent linkage analysis revealed a susceptibility insertion variant rs11446594. It locates in a predicted strong enhancer element region with maximum LOD score 3.42 under dominant model of inheritance. Insertion creates a recognition sequence for ELF3 and HMGA1 transcription factors. Their DNA-binding affinity is highly increased in the presence of A-allele compared to wild type null allele. CONCLUSION: A potentially novel functional OA susceptibility variant was identified by targeted re-sequencing. This variant locates in a predicted regulatory site and creates a recognition sequence for ELF3 and HMGA1 transcription factors that are predicted to play a significant role in articular cartilage homeostasis.

Identification of 33 candidate oncogenes by screening for base-specific mutations.

BACKGROUND: Genes with recurrent codon-specific somatic mutations are likely drivers of tumorigenesis and potential therapeutic targets. Hypermutable cancers may represent a sensitive system for generation and selection of oncogenic mutations. METHODS: We utilised exome-sequencing data on 25 sporadic microsatellite-instable (MSI) colorectal cancers (CRCs) and searched for base-specific somatic mutation hotspots. RESULTS: We identified novel mutation hotspots in 33 genes. Fourteen genes displayed mutations in the validation set of 254 MSI CRCs: ANTXR1, MORC2, CEP135, CRYBB1, GALNT9, KRT82, PI15, SLC36A1, CNTF, GLDC, MBTPS1, OR9Q2, R3HDM1 and TTPAL. A database search found examples of the hotspot mutations in multiple cancer types. CONCLUSIONS: This work reveals a variety of new recurrent candidate oncogene mutations to be further scrutinised as potential therapeutic targets.

Ectopic expression of Cdk6 circumvents transforming growth factor-beta mediated growth inhibition.

Transforming growth factor-beta (TGF-beta) induced growth arrest of cells involves regulation of the activities of both D- and E-type cyclin kinase complexes thought to be mediated primarily by the regulation of p15(Ink4b) and p27(Kip1) cyclin kinase inhibitors. We show here that TGF-beta downregulates Cdk6 and that transient and stable expression of Cdk6 in Mv1Lu mink epithelial cells overrides TGF-beta mediated arrest. The main effect of the ectopic Cdk6 expression was to sequester TGF-beta induced p15(Ink4b) and to maintain more p27(Kip1) in cyclin D-complexes preventing the complete shift of p27(Kip1) to Cdk2 invoked by TGF-beta. This led to the presence of an active cyclinD-Cdk6-p27(Kip1) complex and partially active cyclin E-Cdk2 complex and resulted in the failure of TGF-beta to fully arrest Mv1Lu cell growth. Though dominant negative Cdk6, expressed similarly in the cells, sequestered both p15(Ink4b) and p27(Kip1), it lacks kinase activity and was unable to override the TGF-beta arrest. The results demonstrate that downregulation of Cdk6 kinase is required for the enforcement of the G(1)-phase arrest by TGF-beta and results in changes in association of the p15(Ink4b) and p27(Kip1) inhibitors with D- and E-type cyclin kinase complexes.

A dominant gene for developmental dyslexia on chromosome 3.

Developmental dyslexia is a neurofunctional disorder characterised by an unexpected difficulty in learning to read and write despite adequate intelligence, motivation, and education. Previous studies have suggested mostly quantitative susceptibility loci for dyslexia on chromosomes 1, 2, 6, and 15, but no genes have been identified yet. We studied a large pedigree, ascertained from 140 families considered, segregating pronounced dyslexia in an autosomal dominant fashion. Affected status and the subtype of dyslexia were determined by neuropsychological tests. A genome scan with 320 markers showed a novel dominant locus linked to dyslexia in the pericentromeric region of chromosome 3 with a multipoint lod score of 3.84. Nineteen out of 21 affected pedigree members shared this region identical by descent (corrected p<0.001). Previously implicated genomic regions showed no evidence for linkage. Sequencing of two positional candidate genes, 5HT1F and DRD3, did not support their role in dyslexia. The new locus on chromosome 3 is associated with deficits in all three essential components involved in the reading process, namely phonological awareness, rapid naming, and verbal short term memory.

Two translocations of chromosome 15q associated with dyslexia.

Developmental dyslexia is characterised by difficulties in learning to read. As reading is a complex cognitive process, multiple genes are expected to contribute to the pathogenesis of dyslexia. The genetics of dyslexia has been a target of molecular studies during recent years, but so far no genes have been identified. However, a locus for dyslexia on chromosome 15q21 (DYX1) has been established in previous linkage studies. We have identified two families with balanced translocations involving the 15q21-q22 region. In one family, the translocation segregates with specific dyslexia in three family members. In the other family, the translocation is associated with dyslexia in one family member. We have performed fluorescence in situ hybridisation (FISH) studies to refine the position of the putative dyslexia locus further. Our results indicate that both translocation breakpoints on 15q map within an interval of approximately 6-8 Mb between markers D15S143 and D15S1029, further supporting the presence of a locus for specific dyslexia on 15q21.

Accumulation of a form of p27(Kip1) not associated with Cdk-cyclin complexes in transforming growth factor-beta-arrested Mv1Lu cells.

The p27(Kip1) cyclin-dependent kinase inhibitor translocates in response to transforming growth factor-beta to a Cdk2-cyclin E complex inhibiting its catalytic activity, but the p27(Kip1) protein levels are unaffected [1]. We show here that transforming growth factor-beta induces the accumulation of a form of p27(Kip1) representing a subpopulation of total p27(Kip1) in growth-arrested Mv1Lu epithelial cells. The inducible p27(Kip1) is detectable only by a specific p27(Kip1) monoclonal antibody recognizing a native form of p27(Kip1). The increase in this subset of p27(Kip1) correlates with G(1) arrest and withdrawal of the cells from the cycle induced by transforming growth factor-beta, serum starvation, or contact inhibition. In contrast to the majority of p27(Kip1) in the cells, the transforming growth factor-beta-inducible p27(Kip1) is devoid of cyclin-dependent kinase/cyclin interactions. The results indicate that growth arresting treatments induce the accumulation of non-cyclin-dependent kinase-bound p27(Kip1), which may function as a reservoir for inhibition of Cdk2-cyclin E activities.

Clustering of private mutations in the congenital chloride diarrhea/down-regulated in adenoma gene.

An inherited defect in intestinal anion exchange, congenital chloride diarrhea (CLD), was recently shown to be caused by mutations in the down-regulated in adenoma (DRA) gene. A three base pair deletion resulting in the loss of an amino acid valine (V317del) in the predicted CLD/DRA protein was shown to be responsible for all CLD cases in a Finnish founder population. Two additional mutations, H124L and 344delT, were found in Polish CLD patients. Here, we screened for additional mutations in a set of 14 CLD families of Polish, Swedish, North American, and Finnish origin using primers that allowed mutation searches directly from genomic DNA samples. We found eight novel mutations in the CLD/DRA gene. The mutations included two transversions, one transition, one insertion, and four small deletions. Of 11 sequence alterations detected so far, nine lie clustered in three short segments that are 49 bp, 39 bp, and 65 bp in size, respectively. These short segments span only 6.7% of the total cDNA length, suggesting functional importance or mutation-prone DNA regions of the corresponding CLD/DRA protein domains.