Long-read genome sequencing resolves a complex structural variant involving TBCD and exposes a gap in existing variant classification frameworks

Intragenic duplications can lead to loss-of-function by disrupting the native gene sequence, however, understanding the precise structural conformation is important for accurate predictions of functional outcomes. Here, we describe an Amish kindred with a positive history of Galloway-Mowat and pontocerebellar hypoplasia syndromes. The youngest child also harboured a homozygous intragenic exon 8–13 duplication involving TBCD , initially reported as likely-pathogenic. HiFi long-read genome sequencing and cis morphism-based phasing (spans of 24bp–9.8kb), demonstrated the variant to be a non-tandem duplication-inversion-duplication (DUP-INV-DUP) that leaves the native TBCD gene intact. Integration of long-read data with founder-population frequency strongly indicated that this DUP-INV-DUP was benign, however a formal classification using ACGS/ACMG guidelines for interpreting CNVs was not possible, as none of the specified evidence criteria are applicable. These results highlight the utility of long-read genome sequencing for fully characterising complex structural variants for improved pathogenicity assessment and expose a gap in existing SV/CNV interpretation frameworks.