Generation of two iPSC lines from patient with Mucopolysaccharidosis IV B type and autosomal recessive non-syndromic hearing loss 12.

We generated two human induced pluripotency stem cell (hiPSC) lines, RCMGi011-A and 11-B, from skin fibroblast from patient with Mucopolysaccharidosis IV B type and autosomal recessive non-syndromic hearing loss 12 using non-integrating, viral CytoTune™-iPS 2.0 Sendai Reprogramming Kit. We verified variant c.808 T > G and insertion in GLB1 gene, as well as two mutations, c.6992 T > C and c.805C > T, in CDH23 gene which lead to autosomal recessive hearing loss type 12. We have demonstrated normal karyotype of hiPSCs and capacity for cell differentiation into three germ layers.

[Scanning for KRAS, NRAS, BRAF, and PIK3CA mutations by DNA melting analysis with TaqMan probes].

Scanning for mutations by DNA melting analysis (DMA) is based on asymmetric PCR followed by the melting of duplexes formed by single-stranded amplicons with TaqMan probes. The method is optimally suited for clinical genetic testing; it is easy to perform, high-throughput, and sensitive. The detection limit of mutant alleles by the DMA method is about 3%, which is much higher than the sensitivity of Sanger sequencing. In addition, the DMA method is realized in a closed-tube format, while 2-h assay is carried out in a single tube without any intermediate or additional procedures thereby minimizing the risk of cross contamination of the samples. The validation of the DMA method was performed by scanning for mutations of clinically significant genes KRAS, NRAS, BRAF, and   PIK3CA in 324 DNA samples from tumors of patients with melanoma, colorectal and lung cancer. DNA was isolated either directly from tumor tissues, or from formalin-fixed paraffin-embedded tumor tissues. The detected mutations were verified by Sanger sequencing. The spectra of mutations identified in each tumor type correspond to the literature data and, thus, validate the use of DMA.