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Long-Read Sequencing of the MUC1 VNTR: Genomic Variation, Mutational Landscape, and Its Impact on ADTKD Diagnosis and
Alena Vrbacká1, Anna Přistoupilová1, Kendrah O Kidd1,2
1Research Unit of Rare Diseases, Department of Paediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague, Czech Republic.
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.
Insights
Single Molecule, Real-Time (SMRT) sequencing improves diagnosis of Autosomal Dominant Tubulointerstitial Kidney Disease due to MUC1 (ADTKD-MUC1) mutations. MUC1 VNTR structures correlate with disease progression, offering prognostic insights.
Area of Science:
- Genetics
- Nephrology
- Molecular Biology
Background:
- Autosomal Dominant Tubulointerstitial Kidney Disease due to MUC1 (ADTKD-MUC1) is caused by MUC1 gene frameshift mutations.
- The MUC1 gene's variable number of tandem repeats (VNTR) region is GC-rich and difficult to sequence, hindering mutation detection and understanding of genotype-phenotype correlations.
Purpose of the Study:
- To characterize the MUC1 VNTR genomic sequence in individuals with suspected ADTKD-MUC1.
- To compare the diagnostic performance of Single Molecule, Real-Time (SMRT) sequencing with existing methods.
- To investigate the correlation between MUC1 VNTR structural features and kidney function decline.
Main Methods:
- Single Molecule, Real-Time (SMRT) sequencing was used to analyze the MUC1 gene in 300 individuals.
- Results were compared to those from a CLIA-approved mass spectrometry-based probe extension (PE) assay.
- MUC1 VNTR structures were correlated with the rate of kidney function decline.
Main Results:
- 205 unique MUC1 VNTR alleles and 52 distinct mutated VNTR alleles with 9 frameshift mutation types were identified.
- MUC1 frameshift mutations were found in 50% of families with suspected ADTKD-MUC1, affecting 48% of individuals.
- SMRT sequencing showed complete concordance with the PE assay, which detected mutations in 85% of affected families. A genotype-progression model was proposed based on VNTR structures.
Conclusions:
- SMRT sequencing is superior to current diagnostic methods for ADTKD-MUC1.
- MUC1 VNTR structures possess prognostic value for disease progression in ADTKD-MUC1.
- While modest, the contribution of VNTR structures to disease progression is biologically and clinically significant.
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