Novel MUC1 variant identified by massively parallel sequencing explains interstitial kidney disease in a large Dutch family

Chronic kidney disease (CKD) can be caused by various systemic and kidney disorders with overlapping or nonspecific clinical presentations. Despite a thorough diagnostic workup, the primary cause of CKD remains uncertain in 20% to 35% of affected individuals.1Kramer A. Pippias M. Noordzij M. et al.The European Renal Association – European Dialysis and Transplant Association (ERA-EDTA) Registry Annual Report 2015: a summary.Clin Kidney J. 2018; 11: 108-122Crossref PubMed Scopus (164) Google Scholar,2Boenink R. Astley M.E. Huijben J.A. et al.The ERA Registry Annual Report 2019: summary and age comparisons.Clin Kidney J. 2022; 15: 452-472Crossref PubMed Scopus (30) Google Scholar Recent studies have demonstrated that massively parallel sequencing (MPS) can be a useful additional tool in the diagnostic workup of patients with unexplained CKD, providing a molecular diagnosis in 11% to 56% of cases.3Lata S. Marasa M. 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Halleck F. et al.The underestimated burden of monogenic kidney disease in adults waitlisted for kidney transplantation.Genet Med. 2021; 23: 1219-1224Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Establishing the correct diagnosis through MPS may not only have therapeutic consequences but may also improve detection of extrarenal manifestations (reverse phenotyping), improve genetic counseling of patients and their relatives, and influence donor selection for (living-related) transplantation.S1 Here, we describe a large Dutch family with interstitial kidney disease of unknown origin in whom we identified a novel frameshift mutation in the MUC1 gene, encoding mucin 1, using an MPS-based multigene panel. The proband (VI-96) was a 46-year-old man who presented to our clinic in 2020 for evaluation for a second kidney transplant. In 2007, he first presented with headache, fatigue, hypertension (144/90 mm Hg), serum creatinine of 400 μmol/L, renal anemia, and secondary hyperparathyroidism. Family history (Figure 1a) revealed that many family members were affected with CKD (Supplementary Methods). This family had been followed up in outpatient nephrology clinics in the northern part of the Netherlands for >60 years with unexplained kidney failure classified as "hereditary nephropathy" and was the subject of a dissertation on hereditary idiopathic kidney diseases in 1967.S2 Urinalysis revealed no proteinuria or sediment abnormalities. Kidney ultrasonography showed increased echogenicity, with the left kidney measuring 11.2 cm and the right kidney measuring 10.3 cm. Kidney biopsy revealed sclerosed glomeruli and extensive interstitial fibrosis. Electron microscopy was not performed. The patient was diagnosed with hereditary idiopathic kidney disease. Shortly after presentation, he started dialysis and underwent living-unrelated kidney transplantation at the age of 35 years. He then had progressive loss of kidney function over the next 11 years, requiring evaluation for a second kidney transplantation. Summaries of the clinical presentation of other affected family members are provided in Supplementary Table S1 and Supplementary Data S1. Generally, family members presented between the ages of 30 and 65 years with unexplained kidney failure and histopathologic evidence of interstitial fibrosis and tubular atrophy. In VI-96, previous genetic testing restricted to UMOD and HNF1-β was negative in 2007. Therefore, additional genetic testing was performed using an MPS-based multigene panel (Supplementary Methods and Supplementary Table S2). This analysis revealed a novel frameshift variant (c.326_350dup, p.(Ser119Profs∗119); NM_001204286.1) in MUC1. This variant is located before the variable number of tandem repeats (VNTR) domain of MUC1 and leads to an insertion of 25 nucleotides, resulting in a 1-base frameshift in the open reading frame of the MUC1 mRNA and the introduction of a premature stop codon. The predicted C-terminal amino acid sequence of the resulting mutant protein, mucin 1 frame shift (MUC1fs), is the same as the previously reported pathogenic cytosine duplication within the VNTR (Figure 1b).S3 An additional restriction-specific enrichment assay of the MUC1 VNTR (Supplementary Methods) did not identify classic pathogenic variants in the VNTR. The variant cosegregated with the kidney phenotype in this family. No other (potentially) pathogenic variants were found. To determine if the variant resulted in the intracellular deposition of MUC1fs in a similar manner to autosomal dominant tubulointerstitial kidney disease–MUC1 (ADTKD-MUC1) due to the classic cytosine duplication, specific MUC1fs immunohistochemistry was performed in kidney tissue from VI-96 (Supplementary Methods). Granular MUC1fs positivity was detected in distal tubules and collecting ducts (Figure 2a and b ). In kidney tissue from an age-matched healthy control, MUC1fs immunostaining was negative (Figure 2c and d). We describe a large family with a novel mutation in MUC1 causing ADTKD. ADTKD-MUC1 is characterized by tubulointerstitial fibrosis, an autosomal dominant inheritance pattern, and no extrarenal manifestations.S4 Urinary sediment is usually bland, and disease severity and age of onset of kidney failure vary within and between families, with most patients presenting between the ages of 20 and 70 years.S5–S7 The phenotype of this family matches the ADTKD-MUC1 phenotype, and the variation in age at the start of renal replacement therapy in affected family members is consistent with this diagnosis. The most common cause of ADTKD-MUC1, a cytosine duplication within the VNTR, is responsible for >90% of ADTKD-MUC1 cases, whereas other frameshift mutations within the VNTR may also cause ADTKD-MUC1.S8 In contrast, affected members of our family carry a novel mutation located before the first VNTR repeat unit. The novel mutation in this family resulted in the same MUC1fs protein as reported in all previous ADTKD-MUC1 cases.S8 Although the pathophysiology of ADTKD-MUC1 is not fully understood, it is considered a toxic proteinopathy with accumulation of MUC1fs protein being central to the disease pathogenesis, as Muc1 knockout mice do not have kidney diseaseS9 and all known pathogenic variants in MUC1 lead to expression of the same MUC1fs.S3,S8,S10 Our findings support this hypothesis by showing that a new variant outside the VNTR results in the same MUC1fs protein. The high GC-rich content and variable length of the VNTR in MUC1 prevents identification of typical variants by MPSS11; therefore, ADTKD-MUC1 is not commonly diagnosed via MPS. The common cytosine duplication and other variants within the VNTR can be identified by multiple other techniques.S12–S15 In our family, common pathogenic variants in the VNTR of VI-96 were excluded. Another family with a frameshift MUC1 mutation immediately before the VNTR has previously been identified using MPS.S10 Genetic testing in patients with unexplained (interstitial) kidney disease or with suspected ADTKD should thus include both MPS-based genetic testing, which can identify pathogenic variants outside the VNTR, and analysis of mutations within the VNTR.S16 This latter analysis is not provided in routine panels or with whole exome or whole genome sequencing but can be obtained at several institutes. This study also demonstrates the broader significance of genetic testing in patients with kidney failure of unknown etiology. Identification of a monogenic kidney disease provided a diagnosis for a family who had experienced unexplained CKD for many generations. In addition, a genetic diagnosis allows counseling for family planning and can guide donor selection for living-related kidney donation. Predonation genetic testing in ADTKD-MUC1 families is pivotal, as urinary sediment and kidney ultrasound are often normal in this condition, and some affected individuals may have normal serum creatinine values up until the age of 30 years. In addition, promising leads to a treatment for ADKTD-MUC1 are emerging,S17 and affected individuals may be able to participate in clinical trials or receive treatments in the future. This study also underlines the importance of reevaluation and updated genetic testing several years after initial negative test results in familial kidney disease. Currently, there are 4 confirmed carriers of the MUC1 variant in this family, and clinical evaluation is ongoing to characterize and potentially identify more affected individuals in generations VI to VIII. If more carriers are confirmed, this large family could be useful for future genetic studies. For example, it would allow us to study the influence of genetic modifiers on the age of onset of kidney failure.S18 In conclusion, we identified a novel frameshift mutation in MUC1 using MPS, explaining severe interstitial kidney disease in a large family. This study highlights that MUC1 variants positioned before the VNTR region can lead to ADTKD and that genetic testing in patients with suspected ADTKD-MUC1 should also include sequencing of the region before the VNTR. In addition, it demonstrates the value of genetic retesting in patients with persistent suspicion of hereditary kidney disease. All the authors declared no competing interests. The data underlying this article cannot be shared publicly because of concerns regarding the privacy of individuals participating in this study. The data will be shared on reasonable request to the corresponding author. This work is cofunded by Sanofi Genzyme and the PPP Allowance made available by Health∼Holland, Top Sector Life Sciences and Health, to stimulate public-private partnerships (grants RVO/6320 and IMAGEN/LSHM20009). AMvE was supported by the Dutch Kidney Foundation (18OKG19). SK, MŽ, KK, and AJB were supported by the Ministry of Health of the Czech Republic (grant NU21-07-00033), the Ministry of Education of the Czech Republic (grant LTAUSA19068), and institutional programs of Charles University in Prague (UNCE/MED/007). Several authors of this article are members of the European Reference Network for Rare Kidney Diseases project identifier 739532. Download .docx (.05 MB) Help with docx files Supplementary File (PDF)