Targeted VNTR long read sequencing resolves a diagnostic bottleneck in ADTKD and detects de novo ADTKD-MUC1

INTRODUCTION: The molecular diagnosis of autosomal dominant tubulointerstitial kidney disease due to MUC1 variants (ADTKD-MUC1) using high-throughput (short-read) sequencing methods remains challenging due to the presence of a coding long variable-number tandem repeat (VNTR) region wherein most known pathogenic variants are located. METHODS: Here, we used targeted amplicon long-read sequencing to study the MUC1 VNTR in a retrospective cohort study of 78 individuals. Using a bioinformatic pipeline including newly developed specialized software, VNTRtools, we reconstructed patient-specific complete VNTR haplotypes, generated a synthetic VNTR reference, performed long-read realignment to this reference, and finally performed variant calling. RESULTS: VNTRtools proved efficient, requiring seconds or minutes per sample to accurately identify all pathogenic MUC1 frameshift variants in positive controls, including atypical variants. Ten new diagnoses of ADTKD-MUC1 were made. Furthermore, we report a confirmed de novo case of ADTKD-MUC1 in a 32-year-old patient. We were able to structurally resolve and phase the interindividually highly variable VNTRs in most probands, enabling the high-confidence detection of pathogenic frameshift variants in 24 individuals. We also detected 18 previously unreported VNTR repeat unit types, demonstrating the highly polymorphic nature of MUC1's VNTR. CONCLUSIONS: We propose a combined approach in which short-read VNTR analysis using the published alignment-free bioinformatic tools is used as a first-line test, followed by targeted long-read sequencing with VNTRtools analysis for confirmatory testing and in-depth VNTR characterization. This combined approach will lead to a higher diagnostic confidence in ADTKD-MUC1-especially in sporadic cases. Complete VNTR haplotype information will likely enable a better genetic understanding of this currently underdiagnosed disorder and may become relevant for future therapeutic approaches like targeted silencing of the pathogenic MUC1 allele.