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

Abstract

Background 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.‘ Methods 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. Results 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. Conclusion 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.

Evaluation of somatic and/or germline mosaicism in congenital malformation of the eye

“…Although it is the most used sample in routine, blood might not be the best-suited sample to detect parental mosaicism in ocular development diseases. In a family with an Alport syndrome-affected child, Dai et al 29 showed paternal germline mosaicism not found in blood sample but detected at 2.65% cells in the father's sperm sample. Thus, it would be interesting to look for parental mosaicism in other tissues (saliva, urine, sperm samples) to see if another easily accessible tissue is more appropriate for mosaics' detection.…”

Frequency of de novo variants and parental mosaicism in families with inactivating PTH/PTHrP signaling disorder type 2

“…As mentioned NGS technology allows for very high-fold coverage of sequenced fragments and the detection of low level mosaic mutations often classified as background noise and missed in Sanger sequencing ( 24 ). We use deep targeted NGS (average number of reads >500X) ( 41 , 42 ) to enhance sensitivity and accuracy of the detection of known variants at the GNAS gene. In addition, we selected the potential carrier parent based on the identification of the allele presenting the alteration in the proband.…”

Case Report: Identification of Maternal Low-Level Mosaicism in the Dystrophin Gene by Droplet Digital Polymerase Chain Reaction

“…Approximately, one third of the identified mutations are inherited from asymptomatic parents, which are defined as DNMs (Haldane, 2004 ; Wilfert et al, 2017 ). However, among all the DNMs pedigrees, the incidences of germline mosaicism are reportedly varied from 3.3 to 20% (Grimm et al, 2012 ; Qin et al, 2016 ; Dai et al, 2020 ).…”

“…In general, the sequencing capability depends on the depth of sequencing. Briefly, 200X depth coverage can reveal a mutation frequency of 3% and 500X depth coverage is believed to be stable enough for low-level mutation mosaicism detection (Dai et al, 2020 ; Lin et al, 2021 ). Comparing with routine whole-exon sequencing or whole-genome sequencing, which is 30-100X depth coverage on average, the depth coverage for low-level mutation mosaicism detection with NGS is costly (Postel et al, 2018 ).…”