Validation of Next-Generation Sequencer for 24-Chromosome Aneuploidy Screening in Human Embryos.

BACKGROUND: Next-Generation Sequencing (NGS) is the latest approach for preimplantation genetic diagnoses (PGD). AIM: The purpose of this study was to standardize and validate an NGS method for comprehensive chromosome screening and to investigate its applicability to PGD. METHODS: Embryo biopsy, whole-genome amplification, array comparative genomic hybridization (aCGH), and semiconductor sequencing were employed. RESULTS: A total of 204 whole-genome-amplified products were tested with an NGS-based protocol, from which 100 samples were used for standardization and to evaluate the quality of the results produced by this new technique. The remaining 104 samples tested by NGS were previously analyzed by using the aCGH protocol to determine the sensitivity and specificity of this new technique. In total, 4896 chromosomes were assessed, out of which 196 carried a copy number imbalance. NGS sensitivity and specificity for calling aneuploidy was 100%. CONCLUSION: This is the first study reporting preclinical validation and accuracy assessment of the Ion Torrent Personal Genome Machine (PGM) NGS-based comprehensive chromosome screening method using blastomeres and blastocysts. The NGS proved to be a robust methodology and is ready for clinical application in reproductive medicine, with the major advantage of low cost and enhanced precision when compared with other technologies used for comprehensive chromosome screening.

Mutation analysis of the CFTR gene in 225 children: identification of five novel severe and seven reported severe mutations.

BACKGROUND: Cystic fibrosis (CF) is the most common inherited disorder in Caucasian populations, with more than 1400 cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations. The type of mutations and their distributions varies widely between different countries and/or ethnic groups. METHODS: We characterized the mutations in the CFTR gene by single-strand conformation polymorphism followed by sequencing in CF patients. RESULTS: Twelve mutations were found in 79/225 (35.1%) patients. The most frequent mutations were F508 deletion (31.1%), p.R1162× (2.2%), p.M1T (0.8%), and S559N (0.8%). Five novel severe mutations (p.R80N11fs*11, p.R75G, p.Y577×, p.Y808Yfs*10, and p.I331×) and three reported mutations (p.C343×, p.Ile1000×, p.M469V) were detected. CONCLUSION: The protocol for identification of mutations in cases of CF in developing countries would have to include a different set of mutations than those reported from western countries.

Mutation studies in the CFTR gene in Asian Indian subjects with congenital bilateral absence of vas deferens: report of two novel mutations and four novel variants.

BACKGROUND: Congenital bilateral absence of vas deferens (CBAVD) is a form of male infertility in which mutations occur in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The molecular basis of CBAVD is not completely understood, especially in developing countries. METHODS: We characterized the mutations/variants in the CFTR gene by single strand conformation polymorphism followed by sequencing in 35 CBAVD patients. None of the patients had systemic manifestations of cystic fibrosis. Fifty normal subjects were studied as controls. RESULTS: Mutations/variants in the CFTR gene were found in all CBAVD patients. Five mutations and 10 variants were detected in 35 patients. The most frequent severe mutation was F508del (34.2%) and the most common variant was IVS8-5T (54.2%). Two novel severe mutations (p.E217Gfs*11 and p.A1285V) and four novel variants (pT438A, c.4095+30insCT, c.-737G>A, and c.2909-92A>G) were detected. CONCLUSION: The protocol for identification of mutations in cases of CBAVD in developing countries would have to include a different set of mutations than those reported from western countries.