Single-Molecule Real-Time Sequencing for MUC1 Variable Number of Tandem Repeat Variation to Improve Autosomal Dominant Tubulointerstitial Kidney Disease Diagnosis

KEY POINTS: Single-molecule real-time sequencing with the PacMUC1 script resolved exact MUC1 variable tandem repeat structure and full allelic variation. In 300 individuals, the protocol identified 215 distinct MUC1 tandem repeat alleles with 80 repeat units and nine frameshift mutation types. Probe extension assay identified 90% of families with frameshift mutations, detection of frameshifted mucin-1 aided genetically unresolved cases. 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 approximately 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 Clinical Laboratory Improvement Amendments-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 both long-range PCR amplification and long-read sequencing. Analysis of the mutational spectrum confirmed 59dupC as the most prevalent mutation, affecting approximately 90% of families, while the other eight mutation types occurred at most twice. CONCLUSIONS: The SMRT assay outperformed the Clinical Laboratory Improvement Amendments-approved probe-extension assay by detecting essentially all VNTR-associated frameshift mutations. The probe-extension assay identified approximately 90% of affected families. MUC1fs immunohistochemistry added diagnostic value in genetically unresolved cases by detecting the pathogenic protein independent of the underlying mutation.