Single-Molecule Real-Time Sequencing for MUC1 VNTR Variation to Improve Autosomal Dominant Tubulointerstitial Kidney Disease Diagnosis

Background: ADTKD- MUC1 is caused by frameshift mutations in the MUC1 gene, producing a frameshifted neoprotein (MUC1fs) toxic to kidney cells. The gene’s variable number of tandem repeats (VNTR), with ∼80% guanine/cytosine content, has made it largely inaccessible to standard short-read sequencing, leaving the reference sequence and natural variation poorly defined and complicating mutation detection. Methods: Using Single Molecule, Real-Time (SMRT) sequencing, we characterized MUC1 VNTR in 300 individuals, including 279 from 143 families suspected of having ADTKD- MUC1 , assessing VNTR length, repeat structure, and frameshift mutations. Results were compared with the CLIA-approved probe-extension assay, detecting the prevalent 59dupC mutation, and with MUC1fs immunohistochemistry, which detects the pathogenic protein independent of the underlying genomic change. Results: We identified 215 unique VNTR alleles composed of 80 distinct repeat units, 46 (58%) of which were novel, and nine distinct frameshift mutations present on 52 mutated alleles. Overall, MUC1 frameshift mutations were identified in 71 of 143 families (50%) with suspected ADTKD- MUC1 , comprising 135 affected individuals (48%). The SMRT assay outperformed the probe-extension assay by identifying frameshift mutations in two families with previously inconclusive results and in eight additional families whose mutations were undetectable by the probe-extension design. When successful, SMRT assay showed 100% concordance with probe-extension assay at the family level and 98% at the individual level, with discordance attributable to allelic dropout inherent to long-read sequencing. Analysis of the mutational spectrum confirmed 59dupC as the most prevalent mutation, affecting ∼90% of families, while the other eight mutation types occurred at most twice. Conclusions: The SMRT assay outperformed the CLIA-approved probe-extension assay by detecting essentially all VNTR-associated frameshift mutations. The probe-extension assay identified ∼90% of affected families. MUC1fs immunohistochemistry added diagnostic value in genetically unresolved cases by detecting the pathogenic protein independent of the underlying mutation.