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BACKGROUND: ADTKD- is caused by frameshift mutations in gene that produce a frameshifted protein (MUC1fs) toxic to kidney cells. The gene's variable number of tandem repeats (VNTR), with high GC content, makes it largely inaccessible to standard sequencing. As a result, both the reference sequence and natural variation in this region remain poorly defined, complicating mutation detection and data interpretation. Standard methods also fail to pinpoint the exact VNTR unit affected, limiting insight into mutation mechanisms and genotype-phenotype correlations.METHODS: We employed Single Molecule, Real-Time (SMRT) sequencing and characterized the genomic sequence of in 300 individuals including 279 individuals from 143 families suspected of having ADTKD-. We compared these results to those obtained using the CLIA-approved mass spectrometry-based probe extension (PE) assay, which specifically detect the most prevalent 59dupC mutation. We correlated the structural features of the VNTR with the rate of kidney function decline in affected individuals.RESULTS: We identified consensus sequences for 205 unique VNTR alleles, with 9 distinct types of frameshift mutations present on 52 distinct mutated VNTR alleles. frameshift mutations were identified in 71 of 143 families (50%) with suspected ADTKD, comprising 135 genetically affected individuals (48%). The SMRT assay exhibited complete concordance and revealed that the PE assay is capable of detecting frameshift mutations in approximately 85% of affected families. The constellation of VNTR structures supports a genotype-progression model, in which fast progressors exhibit a significantly lower number of repeat units on the wild-type allele and a higher number of repeats on the mutation-bearing allele, including an increased number of frameshifted repeat units.CONCLUSIONS: SMRT sequencing outperforms current diagnostic methods for ADTKD- and reveals the prognostic value of VNTR structures. Although their contribution to disease progression is modest (~6% variance explained), it remains biologically and clinically meaningful.