Assessment of parental mosaicism in SCN1A-related epilepsy by single-molecule molecular inversion probes and next-generation sequencing

Abstract

BackgroundDravet syndrome is a severe genetic encephalopathy, caused by pathogenic variants in SCN1A. Low-grade parental mosaicism occurs in a substantial proportion of families (7%–13%) and has important implications for recurrence risks. However, parental mosaicism can remain undetected by methods regularly used in diagnostics. In this study, we use single-molecule molecular inversion probes (smMIP), a technique with high sensitivity for detecting low-grade mosaic variants and high cost-effectiveness, to investigate the incidence of parental mosaicism of SCN1A variants in a cohort of 90 families and assess the feasibility of this technique.MethodsDeep sequencing of SCN1A was performed using smMIPs. False positive rates for each of the proband’s pathogenic variants were determined in 145 unrelated samples. If parents showed corresponding variant alleles at a significantly higher rate than the established noise ratio, mosaicism was confirmed by droplet digital PCR (ddPCR).ResultsSequence coverage of at least 100× at the location of the corresponding pathogenic variant was reached for 80 parent couples. The variant ratio was significantly higher than the established noise ratio in eight parent couples, of which four (5%) were regarded as true mosaics, based on ddPCR results. The false positive rate of smMIP analysis without ddPCR was therefore 50%. Three of these variants had previously been considered de novo in the proband by Sanger sequencing.ConclusionsmMIP technology combined withnext generation sequencing (NGS) performs better than Sanger sequencing in the detection of parental mosaicism. Because parental mosaicism has important implications for genetic counselling and recurrence risks, we stress the importance of implementing high-sensitivity NGS-based assays in standard diagnostics.

Alpers- and MNGIE-like disease with disturbed CSF folate transport and an unusual mode of genetic transmission of POLG mutations: a case report

“…As Sanger sequencing is known to have a detection limit of 10% to 20% [13], low-grade somatic mosaicisms might only be detectable by more sensitive methods. It could be more frequent than previously assumed as deep sequencing of parental samples from children with de novo variants in genes causative of epileptic encephalopathy revealed low levels of somatic mosaicism ranging from 1-10% in blood in 5-6% of cases [14,15]. However, as deep sequencing was negative in our patient s father, we rather suggest a germline mutation as the underlying cause.…”

Genetic Landscape of Rett Syndrome Spectrum: Improvements and Challenges

“…Recent studies have shown that germline and somatic mosaicism is present in genes related with severe encephalopathies such as Dravet syndrome [24,25], focal cortical dysplasia [26] and intellectual disability [27]. Mosaicism is being postulated as the cause to explain differential phenotype expression of the disease among patients (somatic mosaicism due to a postzygotic mutation) and to explain recurrent mutations in the same family assumed de novo (due to low-grade parental mosaicism).…”

Detection of germline mosaicism in fathers of children with intellectual disability syndromes caused by de novo variants

“…Previous cohort studies of de novo variants have reported varying prevalence of parental mosaicism (Table 3) (Breuss et al, 2020;Campbell, Yuan, et al, 2014;de Lange et al, 2019;Hu et al, 2019;Jónsson et al, 2018;Legrand et al, 2019;Møller et al, 2019;Myers et al, 2018;Nakayama et al, 2018;Xu et al, 2015;Yang et al, 2017Yang et al, , 2019. In contrast to parental mosaicism studies on peripheral blood, sperm has only been investigated in three cohorts previously (Breuss et al, 2020;Yang et al, 2017Yang et al, , 2019.…”

“…Parental investigations most often depend on genetic analysis of blood‐derived DNA. Currently, a recurrence risk of 1% is commonly used during counselling (Campbell, Stewart, et al, 2014; de Lange et al, 2019; Myers et al, 2018; Röthlisberger & Kotzot, 2007). However, the recurrence risk may be higher or lower depending on if one of the parents is a germline mosaic or not.…”