A unique long-read genome sequencing approach to identify structural variants predisposing to hereditary breast cancer

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Mutations in the DNA repair genes BRCA1 and BRCA2 as well as several interaction partners and downstream targets predispose to hereditary breast and ovarian cancer (HBOC). However, only approximately 35% of cases with HBOC fulfilling the clinical criteria for molecular testing can be associated with causative mutations in described HBOC-genes. Surprisingly, negative testing results are also obtained in a substantial number of large families including numerous affected individuals, and also in cases presenting with a very early onset of the disease. In addition, recent molecular studies suggest the presence of genomic structural mutations, e.g. in regulatory regions of known and unknown HBOC-genes, which cannot be detected by short-read sequencing approaches. Based on the observation that (i) identified HBOC-associated genes explain the familial risk in only ~35% of patients and (ii) current strategies for the identification of causative mutations in novel HBOC-predisposing genes, namely WES in large cohorts of patients, have not been very successful, our newly established Lower Saxony consortium developed an innovative, new strategy for the identification of novel genes and novel molecular mechanisms in HBOC. Under the hypothesis that de novo or recessive mutations play an important causative role in the molecular pathogenesis of very early onset HBOC, we will perform both, low-coverage long-read (Pacific Biosciences) WGS and high-coverage short-read (Illumina) WGS in four selected trios (mother, father, HBOC index [negative for all known HBOC-genes]) with an early onset of breast cancer (<28 years of age) and negative family history. Long-read WGS aims to detect structural variants, and short-read WGS will detect causative single nucleotide variants. In addition to this trio approach, we have selected four large, HBOC-gene negative families with several affected individuals in different generations, and we aim to identify novel causative mutations and genes by the combination of PacBio- and Illumina-WGS. Both will be done in two individuals from different generations in each of the four families. The bioinformatics analysis will be provided by the teams in Düsseldorf and Cologne, which also includes the secondary analysis using the SMRT Link pipeline on the basis of a scientific cooperation. To ensure successful variant interpretation of WGS data sets, our MutationMining (MM) team at the Institute of Human Genetics in Göttingen will be involved in the project. The MM team is a unique team consisting of 21 clinicians and scientists with different expertise in e.g. clinical genetics, molecular genetics, molecular biology, biochemistry and bioinformatics, and it has a long-standing experience in NGS-based gene identification studies. Novel HBOC-associated genes and mutations profiles will be subsequently tested in larger HBOC cohorts in order to determine the frequency and relative risk of mutations.