A novel approach using long‐read sequencing and ddPCR to investigate gonadal mosaicism and estimate recurrence risk in two families with developmental disorders

Maria Wilbe, Sanna Gudmundsson, Josefin Johansson, Adam Ameur, Eva‐Lena Stattin, Göran Annerén, Helena Malmgren, Carina Frykholm, Marie‐Louise Bondeson

De novo mutations contribute significantly to severe early-onset genetic disorders. Even if the mutation is apparently de novo there is a recurrence risk due to parental germ line mosaicism, depending on the gonadal generation the mutation occurred. We demonstrate the power of using SMRT sequencing and ddPCR to determine parental origin and allele frequencies of de novo mutations in germ cells in two families whom had undergone assisted reproduction. In the first family, a TCOF1 variant c.3156C>T was identified in the proband with Treacher Collins syndrome. The variant affects splicing and was determined to be of paternal origin. It was present in <1% of the paternal germ cells, suggesting a very low recurrence risk. In the second family, the couple had undergone several unsuccessful pregnancies where a de novo mutation PTPN11 c.923A>C causing Noonan syndrome was identified. The variant was present in 40% of the paternal germ cells suggesting a high recurrence risk. Our findings highlight a successful strategy to identify the parental origin of mutations and to investigate the recurrence risk in couples that have undergone assisted reproduction with an unknown donor or in couples with gonadal mosaicism that will undergo preimplantation genetic diagnosis.