Technology for comprehensive DNA analysis in fragile X syndrome

Abstract

We have developed the technology for complex DNA diagnostics of fragile X syndrome, contemporaneous to the current level of the in DNA sequencing methods, assessment of the DNA regional copy number, and detection of abnormal DNA methylation. The technology includes targeted high-throughput parallel DNA sequencing, multiplex amplification of ligated probes (MLPA), and multiplex methylation sensitive PCR. The search for point mutations and short insertions / deletions in the FMR1 and FMR1-AS1 genes was performed using an NGS on the Ion Torrent PGM instrument. In the case of fragile X syndrome, the sequencing methods allow detecting not only single nucleotide substitutions, small insertions and deletions, but also extended deletions observed in probands in the hemizygotus state. Thus, the MLPA of the exons of the FMR1 gene is used within the framework of the present DNA diagnostic technology rather as a confirmatory than the main method. Methylation sensitive PCR is used to detect abnormal methylation of the promoter of the FMR1 gene. Thus, the new technology of complex DNA diagnosis of the fragile X syndrome is aimed at identifying all known molecular genetic abnormalities leading to the development of the disease.