SNP array-based whole genome homozygosity mapping as the first step to a molecular diagnosis in patients with Charcot-Marie-Tooth disease

Considerable non-allelic heterogeneity for autosomal recessively inherited Charcot-Marie-Tooth (ARCMT) disease has challenged molecular testing and often requires a large amount of work in terms of DNA sequencing and data interpretation or remains unpractical. This study tested the value of SNP array-based whole-genome homozygosity mapping as a first step in the molecular genetic diagnosis of sporadic or ARCMT in patients from inbred families or outbred populations with the ancestors originating from the same geographic area. Using 10 K 2.0 and 250 K Nsp Affymetrix SNP arrays, 15 (63%) of 24 CMT patients received an accurate genetic diagnosis. We used our Java-based script eHoPASA CMT—easy Homozygosity Profiling of SNP arrays for CMT patients to display the location of homozygous regions and their extent of marker count and base-pairs throughout the whole genome. CMT4C was the most common genetic subtype with mutations detected in SH3TC2, one (p.E632Kfs13X) appearing to be a novel founder mutation. A sporadic patient with severe CMT was homozygous for the c.250G > C (p.G84R) HSPB1 mutation which has previously been reported to cause autosomal dominant dHMN. Two distantly related CMT1 patients with early disease onset were found to carry a novel homozygous mutation in MFN2 (p.N131S). We conclude that SNP array-based homozygosity mapping is a fast, powerful, and economic tool to guide molecular genetic testing in ARCMT and in selected sporadic CMT patients

Tissue specific characteristics of cells isolated from human and rat tendons and ligaments

<p>Abstract</p> <p>Background</p> <p>Tendon and ligament injuries are common and costly in terms of surgery and rehabilitation. This might be improved by using tissue engineered constructs to accelerate the repair process; a method used successfully for skin wound healing and cartilage repair. Progress in this field has however been limited; possibly due to an over-simplistic choice of donor cell. For tissue engineering purposes it is often assumed that all tendon and ligament cells are similar despite their differing roles and biomechanics. To clarify this, we have characterised cells from various tendons and ligaments of human and rat origin in terms of proliferation, response to dexamethasone and cell surface marker expression.</p> <p>Methods</p> <p>Cells isolated from tendons by collagenase digestion were plated out in DMEM containing 10% fetal calf serum, penicillin/streptomycin and ultraglutamine. Cell number and collagen accumulation were by determined methylene blue and Sirius red staining respectively. Expression of cell surface markers was established by flow cytometry.</p> <p>Results</p> <p>In the CFU-f assay, human PT-derived cells produced more and bigger colonies suggesting the presence of more progenitor cells with a higher proliferative capacity. Dexamethasone had no effect on colony number in ACL or PT cells but 10 nM dexamethasone increased colony size in ACL cultures whereas higher concentrations decreased colony size in both ACL and PT cultures. In secondary subcultures, dexamethasone had no significant effect on PT cultures whereas a stimulation was seen at low concentrations in the ACL cultures and an inhibition at higher concentrations. Collagen accumulation was inhibited with increasing doses in both ACL and PT cultures. This differential response was also seen in rat-derived cells with similar differences being seen between Achilles, Patellar and tail tendon cells. Cell surface marker expression was also source dependent; CD90 was expressed at higher levels by PT cells and in both humans and rats whereas D7fib was expressed at lower levels by PT cells in humans.</p> <p>Conclusion</p> <p>These data show that tendon & ligament cells from different sources possess intrinsic differences in terms of their growth, dexamethasone responsiveness and cell surface marker expression. This suggests that for tissue engineering purposes the cell source must be carefully considered to maximise their efficacy.</p

Modeling of Environmental Effects in Genome-Wide Association Studies Identifies SLC2A2 and HP as Novel Loci Influencing Serum Cholesterol Levels

Genome-wide association studies (GWAS) have identified 38 larger genetic regions affecting classical blood lipid levels without adjusting for important environmental influences. We modeled diet and physical activity in a GWAS in order to identify novel loci affecting total cholesterol, LDL cholesterol, HDL cholesterol, and triglyceride levels. The Swedish (SE) EUROSPAN cohort (NSE = 656) was screened for candidate genes and the non-Swedish (NS) EUROSPAN cohorts (NNS = 3,282) were used for replication. In total, 3 SNPs were associated in the Swedish sample and were replicated in the non-Swedish cohorts. While SNP rs1532624 was a replication of the previously published association between CETP and HDL cholesterol, the other two were novel findings. For the latter SNPs, the p-value for association was substantially improved by inclusion of environmental covariates: SNP rs5400 (pSE,unadjusted = 3.6×10−5, pSE,adjusted = 2.2×10−6, pNS,unadjusted = 0.047) in the SLC2A2 (Glucose transporter type 2) and rs2000999 (pSE,unadjusted = 1.1×10−3, pSE,adjusted = 3.8×10−4, pNS,unadjusted = 0.035) in the HP gene (Haptoglobin-related protein precursor). Both showed evidence of association with total cholesterol. These results demonstrate that inclusion of important environmental factors in the analysis model can reveal new genetic susceptibility loci