Deep intronic variant causes aberrant splicing of ATP7A in a family with a variable occipital horn syndrome phenotype.

Genetic variants in ATP7A are associated with a spectrum of X-linked disorders. In descending order of severity, these are Menkes disease, occipital horn syndrome, and X-linked distal spinal muscular atrophy. After 30 years of diagnostic investigation, we identified a deep intronic ATP7A variant in four males from a family affected to variable degrees by a predominantly skeletal phenotype, featuring bowing of long bones, elbow joints with restricted mobility which dislocate frequently, coarse curly hair, chronic diarrhoea, and motor coordination difficulties. Analysis of whole genome sequencing data from the Genomics England 100,000 Genomes Project following clinical re-evaluation identified a deep intronic ATP7A variant, which was predicted by SpliceAI to have a modest splicing effect. Using a mini-gene splicing assay, we determined that the intronic variant results in aberrant splicing. Sanger sequencing of patient cDNA revealed ATP7A transcripts with exon 5 skipping, or inclusion of a novel intron 4 pseudoexon. In both instances, frameshift leading to premature termination are predicted. Quantification of ATP7A mRNA transcripts using a qPCR assay indicated that the majority of transcripts (86.1 %) have non-canonical splicing, with 68.0 % featuring exon 5 skipping, and 18.1 % featuring the novel pseudoexon. We suggest that the variability of the phenotypes within the affected males results from the stochastic effects of splicing. This deep intronic variant, resulting in aberrant ATP7A splicing, expands the understanding of intronic variation on the ATP7A-related disease spectrum.

Personalised virtual gene panels reduce interpretation workload and maintain diagnostic rates of proband-only clinical exome sequencing for rare disorders.

PURPOSE: The increased adoption of genomic strategies in the clinic makes it imperative for diagnostic laboratories to improve the efficiency of variant interpretation. Clinical exome sequencing (CES) is becoming a valuable diagnostic tool, capable of meeting the diagnostic demand imposed by the vast array of different rare monogenic disorders. We have assessed a clinician-led and phenotype-based approach for virtual gene panel generation for analysis of targeted CES in patients with rare disease in a single institution. METHODS: Retrospective survey of 400 consecutive cases presumed by clinicians to have rare monogenic disorders, referred on singleton basis for targeted CES. We evaluated diagnostic yield and variant workload to characterise the usefulness of a clinician-led approach for generation of virtual gene panels that can incorporate up to three different phenotype-driven gene selection methods. RESULTS: Abnormalities of the nervous system (54.5%), including intellectual disability, head and neck (19%), skeletal system (16%), ear (15%) and eye (15%) were the most common clinical features reported in referrals. Combined phenotype-driven strategies for virtual gene panel generation were used in 57% of cases. On average, 7.3 variants (median=5) per case were retained for clinical interpretation. The overall diagnostic rate of proband-only CES using personalised phenotype-driven virtual gene panels was 24%. CONCLUSIONS: Our results show that personalised virtual gene panels are a cost-effective approach for variant analysis of CES, maintaining diagnostic yield and optimising the use of resources for clinical genomic sequencing in the clinic.