Abstract
Wilson disease (WD) is an autosomal recessive disorder of copper transport caused by bi-allelic pathogenic variants in the ATPase copper transporting beta gene (ATP7B). Results of standard genetic diagnostics remain inconclusive in 3%-20% of WD patients in part due to problematic assessment of variants of unknown or conflicting pathogenicity (synonymous variants included). Correct interpretation of potential effects of such variants can be substantially enhanced by RNA analyses. This strategy is, however, of limited utility in WD patients because of predominant liver expression of ATP7B. To avoid invasive bioptic liver collection and increase WD diagnostic yields, we searched for a surrogate tissue sample and identified profiles of ATP7B transcripts in nasopharyngeal swabs that were comparable to liver. Amplicons spanning ATP7B Exons 3-21 were prepared from the swab material and analysed by long-read nanopore sequencing to enable the detection of splicing changes and variant phasing. Diagnostic utility of this novel in vivo methodology was demonstrated by characterization of mRNA splicing abnormalities caused by synonymous ATP7B variants c.1488C>T (p.(Gly496=)), c.2241C>T (p.(Ile747=)), c.2292C>T (p.(Phe764=)), and a nonsense variant c.2336G>A (p.(Trp779Ter)) in four WD patients, who were not genetically resolved by standard techniques. Nasopharyngeal swab sampling is minimally invasive and allows effective analyses of mRNA to detect and/or validate effects of ATP7B variants in WD patients. Conclusive genetic diagnosis attained by this novel technique may facilitate family counselling and substantiate initiation of copper-chelation therapy in presymptomatic individuals.