Sequencing method and algorithm for analyzing methylation profiles based on NGS data to identify uniparental disomy

Uniparental disomy is an anomaly where both homologous chromosomes are inherited from the same parent. The pathological effects of this anomaly are associated with imprinting disorders and loss of heterozygosity. Traditional diagnostic methods, such as simple sequence repeats analysis and chromosomal microarray analysis, have limitations in diagnosing the anomaly in single-parent households and mosaic cases. The aim of this study is to evaluate the potential effectiveness of a diagnostic method for uniparental disomy and imprinting disorders based on DNA methylation profiling using next-generation sequencing data. In particular, a genomic DNA bank sample preparation was modified using the methyl-dependent endonuclease GlaI in combination with the restriction endonuclease Bst2UI to improve its informativeness. In addition, bioinformatic analysis algorithm was developed. The method was tested on a clinical case of maternal reciprocal translocation (3;19)(q12;q13.3) with suspected segmental uniparental disomy in the proband. Modification of the bank sample preparation protocol provided the coverage of approximately 5 million methylation sites. Bioinformatic analysis included definition of methylation status in clinically significant imprinting control regions and the search for regions of homozygosity. The method enabled the coverage of 2/3 of potential imprinting control regions. No signs of uniparental disomy were found in the proband, which is consistent with the results of chromosomal microarray analysis. Although the approach represents a cost-effective alternative to genomewide bisulfite sequencing, problems with normalizing the relative methylation levels remain. Further validation of biologically developed method on confirmed uniparental disomy cases is planned to definitively assess its suitability for identifying anomalies.