Methods for verification of submicroscopic pathogenic copy number variations

Genomic variability is the basis of genetic diversity and evolution and includes sequence and structural variability. Structural variability refers to variations in the number of copies of DNA (copy number variations - CNVs), ranging from 1000 bp up to several megabases (Mb) in size. Among them, some submicroscopic CNVs up to 3 Mb, can lead to clinical signs such as developmental delay, intellectual disability, congenital malformations and/or dysmorphic features, as well as autism spectrum disorders. A wide range of methods with different resolution is used for CNVs analysis. To date, chromosomal microarray analysis (CMA) is a universal method for CNVs detection. However, with the advent methods of next-generation sequencing, their applicability for CNV analysis is increasingly being estimated. Therefore, with the development of genome-wide technologies and bioinformatic tools for CNV analysis, there is an increasing need to confirm the obtained data in order to establish the true values of their sensitivity and specificity. In addition, information only about localization and gene content of CNVs is not enough for genetic counseling for the family. It is necessary to define structure and origin of the detected CNV to assess accurate recurrence risk of chromosome imbalance. For this purpose, molecular genetics and molecular cytogenetic methods are used. There are some methods of molecular genetics based on PCR with sufficient resolution to confirm submicroscopic CNV longer than 1000 bp. Analysis of submicroscopic CNVs by various modifications of FISH-method is limited by the length and specificity of DNA fragments in probes used in conventional FISH-protocols. Therefore, application of DNA probes of the order of several kb in length becomes relevant. If both group of methods allow to confirm CNVs detected by wide-genome technologies, than the latter are used to estimate the structure of chromosomal imbalance. Possibilities, advantages and disadvantages of different methods for CNVs verification are discussed.

структурная вариабельность, CNV, FISH, PRINS, TSA, structural variability, CNV, FISH, PRINS, TSA