Whole genome sequencing in a Knobloch syndrome family confirms the molecular diagnosis

To establish the molecular diagnosis in two brothers presenting with the ocular features of Knobloch Syndrome using whole genome sequencing (WGS).

Clinical examination and ophthalmological phenotyping were completed under general anaesthesia. DNA samples were tested on a targeted retinal dystrophy next-generation sequencing panel. Subsequently, WGS was performed to identify additional variants.

Clinical examination confirmed the diagnosis of Knobloch Syndrome. Targeted sequencing identified a novel heterozygous frameshift pathogenic variant in COL18A1, c.2864dupC; p.(Gly956ArgfsX20), inherited from their mother. A second paternally inherited heterozygous missense variant was identified in both brothers, c.5014 G > A; p.(Asp1672Asn), which was initially considered to have too high frequency to be pathogenic (MAF 8.8%). This led to an in-depth analysis of the COL18A1 locus using WGS data, which confirmed that Asp1672Asn is a likely pathogenic hypomorphic allele.

To date, all confirmed genetic diagnoses of Knobloch syndrome are attributable to variants in COL18A1. The family described here has a heterozygous novel loss of function variant. Detailed analysis of WGS data combined with family segregation studies concluded that although Asp1672Asn has a high population frequency, it is the most likely second pathogenic variant in our family. This supports the hypothesis that this is a hypomorphic allele, which, in combination with a loss of function pathogenic variant, leads to Knobloch syndrome.To our knowledge, this is the first time that WGS has been used to confirm a molecular diagnosis of Knobloch syndrome in this way and has provided further insight into the molecular mechanisms in this rare disorder.