Karl X Knaup1, Thomas Hackenbeck1, Bernt Popp2, Johanna Stoeckert1, Andrea Wenzel3, Maike Büttner-Herold4, Frederick Pfister4, Markus Schueler1, Didem Seven2,5, Annette M May6, Jan Halbritter7, Hermann-Josef Gröne8, André Reis2, Bodo B Beck3, Kerstin Amann4, Arif B Ekici2, Michael S Wiesener1. 1. Department of Nephrology and Hypertension, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. 2. Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. 3. Institute of Human Genetics and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany. 4. Department of Nephropathology, Institute of Pathology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. 5. Cerrahpaşa Medical Faculty, Department of Medical Biology, Istanbul University, Istanbul, Turkey. 6. Institute for Surgical Pathology, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany. 7. Division of Nephrology, Department of Internal Medicine, University of Leipzig, Leipzig, Germany. 8. Department of Cellular and Molecular Pathology, Deutsches Krebsforschungszentrum Heidelberg, Heidelberg, Germany.
Abstract
BACKGROUND: Providing the correct diagnosis for patients with tubulointerstitial kidney disease and secondary degenerative disorders, such as hypertension, remains a challenge. The autosomal dominant tubulointerstitial kidney disease (ADTKD) subtype caused by MUC1 mutations (ADTKD-MUC1) is particularly difficult to diagnose, because the mutational hotspot is a complex repeat domain, inaccessible with routine sequencing techniques. Here, we further evaluated SNaPshot minisequencing as a technique for diagnosing ADTKD-MUC1 and assessed immunodetection of the disease-associated mucin 1 frameshift protein (MUC1-fs) as a nongenetic technique. METHODS: We re-evaluated detection of MUC1 mutations by targeted repeat enrichment and SNaPshot minisequencing by haplotype reconstruction via microsatellite analysis in three independent ADTKD-MUC1 families. Additionally, we generated rabbit polyclonal antibodies against MUC1-fs and evaluated immunodetection of wild-type and mutated allele products in human kidney biopsy specimens. RESULTS: The detection of MUC1 mutations by SNaPshot minisequencing was robust. Immunostaining with our MUC1-fs antibodies and an MUC1 antibody showed that both proteins are readily detectable in human ADTKD-MUC1 kidneys, with mucin 1 localized to the apical membrane and MUC1-fs abundantly distributed throughout the cytoplasm. Notably, immunohistochemical analysis of MUC1-fs expression in clinical kidney samples facilitated reliable prediction of the disease status of individual patients. CONCLUSIONS: Diagnosing ADTKD-MUC1 by molecular genetics is possible, but it is technically demanding and labor intensive. However, immunohistochemistry on kidney biopsy specimens is feasible for nongenetic diagnosis of ADTKD-MUC1 and therefore, a valid method to select families for further diagnostics. Our data are compatible with the hypothesis that specific molecular effects of MUC1-fs underlie the pathogenesis of this disease.
BACKGROUND: Providing the correct diagnosis for patients with tubulointerstitial kidney disease and secondary degenerative disorders, such as hypertension, remains a challenge. The autosomal dominant tubulointerstitial kidney disease (ADTKD) subtype caused by MUC1 mutations (ADTKD-MUC1) is particularly difficult to diagnose, because the mutational hotspot is a complex repeat domain, inaccessible with routine sequencing techniques. Here, we further evaluated SNaPshot minisequencing as a technique for diagnosing ADTKD-MUC1 and assessed immunodetection of the disease-associated mucin 1 frameshift protein (MUC1-fs) as a nongenetic technique. METHODS: We re-evaluated detection of MUC1 mutations by targeted repeat enrichment and SNaPshot minisequencing by haplotype reconstruction via microsatellite analysis in three independent ADTKD-MUC1 families. Additionally, we generated rabbit polyclonal antibodies against MUC1-fs and evaluated immunodetection of wild-type and mutated allele products in human kidney biopsy specimens. RESULTS: The detection of MUC1 mutations by SNaPshot minisequencing was robust. Immunostaining with our MUC1-fs antibodies and an MUC1 antibody showed that both proteins are readily detectable in humanADTKD-MUC1 kidneys, with mucin 1 localized to the apical membrane and MUC1-fs abundantly distributed throughout the cytoplasm. Notably, immunohistochemical analysis of MUC1-fs expression in clinical kidney samples facilitated reliable prediction of the disease status of individual patients. CONCLUSIONS: Diagnosing ADTKD-MUC1 by molecular genetics is possible, but it is technically demanding and labor intensive. However, immunohistochemistry on kidney biopsy specimens is feasible for nongenetic diagnosis of ADTKD-MUC1 and therefore, a valid method to select families for further diagnostics. Our data are compatible with the hypothesis that specific molecular effects of MUC1-fs underlie the pathogenesis of this disease.
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