BACKGROUND: ADTKD-MUC1 is caused by frameshift mutations in MUC1 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 MUC1 in 300 individuals including 279 individuals from 143 families suspected of having ADTKD-MUC1. 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 MUC1 VNTR with the rate of kidney function decline in affected individuals. RESULTS: We identified MUC1 consensus sequences for 205 unique VNTR alleles, with 9 distinct types of frameshift mutations present on 52 distinct mutated VNTR alleles. MUC1 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-MUC1 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.

B Braun Avitum Bratislava Slovakia

Biomedical Center Faculty of Medicine Charles University Pilsen Czechia

Broad Clinical Labs The Broad Institute Cambridge Massachusetts USA

Department of Medical Genetics Hospital de Santa Maria ULSSM Lisbon Portugal

Department of Medicine Royal College of Surgeons Dublin Ireland

Department of Microbiology Faculty of Medicine and University Hospital Pilsen Charles University Pilsen Czechia

Department of Nephrology 1st Faculty of Medicine Charles University Prague and General Teaching Hospital Prague Prague Czech Republic

Department of Nephrology and Renal Transplant Hospital de Santa Maria CHULN EPE Lisbon Portugal

Department of Nephrology Beaumont Hospital Dublin Ireland

Department of Nephrology Transplant Centre Institute for Clinical and Experimental Medicine Videnska 1958 Prague 14021 Czech Republic

Evangelismos Private Hospital Pafos Cyprus

Medical School University of Cyprus Nicosia Cyprus

Molecular Medicine Research Center biobank cy Center of Excellence in Biobanking and Biomedical Research University of Cyprus Nicosia Cyprus

Research Unit of Rare Diseases Department of Paediatrics and Inherited Metabolic Disorders 1st Faculty of Medicine Charles University Prague Czech Republic

School of Pharmacy and Biomolecular Sciences Royal College of Surgeons Dublin Ireland

Wake Forest University School of Medicine Section on Nephrology Winston Salem North Carolina USA

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