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

Abstract

Objective 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 in which gonadal generation the mutation occurred.MethodsWe 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.ResultsIn 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.ConclusionsOur 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.

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics

“…Reproductive genomic medicine and associated counseling, including pre-implantation genetic diagnosis (PGD), relies heavily on the ability to haplotype or phase alleles in embryos, patients, and parents. Long reads enable direct phasing of amplicons from targeted loci which can be used to determine parent-of-origin alleles in embryos or patients ( 66 , 67 ). In a family having one child with Treacher Collins syndrome, SMRT amplicons sequencing was used to confirm the paternal transmission of a TCOF1 variant that affects splicing of the gene and potentially causes the disease ( 67 ).…”

“…Long reads enable direct phasing of amplicons from targeted loci which can be used to determine parent-of-origin alleles in embryos or patients ( 66 , 67 ). In a family having one child with Treacher Collins syndrome, SMRT amplicons sequencing was used to confirm the paternal transmission of a TCOF1 variant that affects splicing of the gene and potentially causes the disease ( 67 ). For apparent de novo mutations that are a result of germ line mosaicism, determining the frequency of damaging alleles is informative in predicting recurrence in future offspring.…”

“…For apparent de novo mutations that are a result of germ line mosaicism, determining the frequency of damaging alleles is informative in predicting recurrence in future offspring. For a couple with multiple miscarriages and suspected Noonan syndrome in the fetuses, SMRT amplicon sequencing identified a disease causing PTPN11 variant in 37% of the father's sperm ( 67 ). Digital Droplet PCR showed no signs of the variant in the father's blood, but confirmed the 40% frequency in the fathers sperm ( 67 ).…”

“…For a couple with multiple miscarriages and suspected Noonan syndrome in the fetuses, SMRT amplicon sequencing identified a disease causing PTPN11 variant in 37% of the father's sperm ( 67 ). Digital Droplet PCR showed no signs of the variant in the father's blood, but confirmed the 40% frequency in the fathers sperm ( 67 ). This therefore enabled an estimate of recurrent risk for subsequent pregnancies.…”

Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

“…Long-read sequencing is becoming increasingly used in clinical research 58 . Advantages over short-read methods have been noted for applications such as identifying SVs 28,59 , resolving complex SVs 29 , phasing alleles 60 , sequencing repetitive or highly homologous regions 61 and inferring methylation state 30 . The fast turn-around time of Nanopore sequencing in particular has already led to the development of rapid diagnostic assays for specific cancers 30,31 .…”