Identification of paternal germline mosaicism by MicroSeq and targeted next-generation sequencing

Prezygotic de novo mutations may be inherited from parents with germline mosaicism and are often overlooked when the resulting phenotype affects only one child. We aimed to identify paternal germline mosaicism in an index family and provide a strategy to determine germline mosaicism.'

Whole-exome sequencing was performed on an Alport syndrome-affected child. Variants were validated using Sanger sequencing in the pedigree analysis. An apparent de novo mutation was tested by next-generation sequencing (NGS) following chromosome microdissection of the mutant region (MicroSeq) to clarify its homologous chromosome source. Mosaic mutation in sperm samples was detected using targeted next-generation sequencing (TNGS). Self-prepared mosaic DNA samples of the 3% and 0.1% mutant fractions were used to evaluate the TNGS detection sensitivity.

Two novel heterozygous variants, maternally inherited c.1322delT (p.Ile441Thrfs*17) and the de novo mutation c.2939T>A (p.Leu980Ter), in the COL4A3 gene were discovered in the propositus. MicroSeq identified c.2939T>A in the paternal chromosome, which was in trans with c.1322delT. The frequency of c.2937A was 2.65% in the father's sperm sample. We also showed that a 500X depth coverage may detect a mosaic mutation with an allele frequency as low as 2%-3% using TNGS.

MicroSeq is a valuable tool to identify the allele source of de novo mutations in a single patient. TNGS can be used to assess the mosaic ratios of known sites. We provided a systematic algorithm to detect germinal mosaicism in a single patient. This algorithm may have implications for genetic and reproductive counseling on germline mosaicism.