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Abstract

Abstract Accurate detection of low-level mosaicism in parents of children with seemingly de novo variants (DNV) is essential for personalized recurrence risk assessment and informed reproductive decision-making. Our study included 64 families with children affected by neurodevelopmental disorders due to DNVs. We extracted DNA from parental blood, buccal swab, hair follicles, and paternal semen. Using long-read amplicon sequencing, we phased the variants to identify the parental origin. Using ultra-deep spike-in amplicon short-read sequencing, we investigated for mosaicism in parental tissues. We estimated the mosaic variant allele frequency (VAF) using both a naive allele counting as well as a Bayesian model. Parental mosaicism was detected in seven of 64 families (11%): two (2/7, 29%) cases of mixed (i.e. in all tissues) maternal mosaicism, four (4/7, 57%) cases of paternal sperm-only mosaicism, and one (1/7, 14%) case of mixed paternal mosaicism. Mosaic naive VAFs ranged from 0.35–11.04%, with comparable results obtained using the Bayesian model. Phasing was successful in 42 families (66%); 39/42 (93%) of the phased variants were paternal and 3/42 (7%) maternal. We integrated the diagnostic workflow in routine practice. Our findings demonstrate that ultra-deep sequencing can identify clinically relevant parental mosaicism frequently missed by routine diagnostics. Although long-read sequencing and phasing were not successful in all cases and the female germline remains inaccessible, incorporating multi-tissue mosaicism testing into clinical practice enables more precise recurrence risk assessment, improves genetic counselling, and supports informed reproductive and prenatal testing decisions.