Targeted VNTR long read sequencing resolves a diagnostic bottleneck in ADTKD and detects de novo ADTKD-MUC1

Kidney International

Abstract

INTRODUCTION: The molecular diagnosis of autosomal dominant tubulointerstitial kidney disease due to MUC1 variants (ADTKD-MUC1) using high-throughput (short-read) sequencing methods remains challenging due to the presence of a coding long variable-number tandem repeat (VNTR) region wherein most known pathogenic variants are located. METHODS: Here, we used targeted amplicon long-read sequencing to study the MUC1 VNTR in a retrospective cohort study of 78 individuals. Using a bioinformatic pipeline including newly developed specialized software, VNTRtools, we reconstructed patient-specific complete VNTR haplotypes, generated a synthetic VNTR reference, performed long-read realignment to this reference, and finally performed variant calling. RESULTS: VNTRtools proved efficient, requiring seconds or minutes per sample to accurately identify all pathogenic MUC1 frameshift variants in positive controls, including atypical variants. Ten new diagnoses of ADTKD-MUC1 were made. Furthermore, we report a confirmed de novo case of ADTKD-MUC1 in a 32-year-old patient. We were able to structurally resolve and phase the interindividually highly variable VNTRs in most probands, enabling the high-confidence detection of pathogenic frameshift variants in 24 individuals. We also detected 18 previously unreported VNTR repeat unit types, demonstrating the highly polymorphic nature of MUC1's VNTR. CONCLUSIONS: We propose a combined approach in which short-read VNTR analysis using the published alignment-free bioinformatic tools is used as a first-line test, followed by targeted long-read sequencing with VNTRtools analysis for confirmatory testing and in-depth VNTR characterization. This combined approach will lead to a higher diagnostic confidence in ADTKD-MUC1-especially in sporadic cases. Complete VNTR haplotype information will likely enable a better genetic understanding of this currently underdiagnosed disorder and may become relevant for future therapeutic approaches like targeted silencing of the pathogenic MUC1 allele.

Authors 14

1. Andrea Wenzel

   University of Cologne · University Hospital Cologne

   Affiliation as printed

   Institute of Human Genetics and Center for Rare and Hereditary Kidney Disease, Faculty of Medicine and University Hospital Cologne, Cologne, Germany

2. Björn Reusch

   University of Cologne · University Hospital Cologne

   Affiliation as printed

   Institute of Human Genetics and Center for Rare and Hereditary Kidney Disease, Faculty of Medicine and University Hospital Cologne, Cologne, Germany

3. Karl Xaver Knaup

   Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen

   Affiliation as printed

   Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany

4. Nikola Zagorec

   University of Zagreb · University Hospital Dubrava

   Affiliation as printed

   Department of Nephrology and Dialysis, Dubrava University Hospital, Zagreb, Croatia; Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia

5. Margareta Fištrek Prlić

   University Hospital Centre Zagreb

   Affiliation as printed

   Department of Nephrology, Faculty of Medicine and University Hospital Center Zagreb, Zagreb, Croatia

6. Kerstin Becker

   University of Cologne

   Affiliation as printed

   Cologne Center for Genomics, Medical Faculty, University of Cologne, Cologne, Germany; West German Genome Center, University of Cologne, Cologne, Germany

7. Vera Riehmer

   University of Cologne · University Hospital Cologne

   Affiliation as printed

   Institute of Human Genetics and Center for Rare and Hereditary Kidney Disease, Faculty of Medicine and University Hospital Cologne, Cologne, Germany

8. Roman-U. Müller Aachen Center of Excellence in Nephrology

   Düsseldorf University Hospital · Universitätsklinikum Aachen · Heinrich Heine University Düsseldorf

   Affiliation as printed

   Department of Nephrology, Medical Faculty, University Hospital Düsseldorf, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany; Center of Excellence in Nephrology, University Hospitals Aachen, Cologne, Dusseldorf, North Rhine-Westphalia, Germany

9. Francesca Pasutto

   Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen

   Affiliation as printed

   Institute of Human Genetics, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany

10. Julia Hoefele

    TUM Klinikum · Institut für Humangenetik · Technical University of Munich · Ludwig-Maximilians-Universität München

    Affiliation as printed

    Institute of Human Genetics, LMU University Hospital, LMU Medizin, Ludwig-Maximilians-Universität München, Munich, Germany; Institute of Human Genetics, Klinikum rechts der Isar, Technical University of Munich, TUM School of Medicine and Health, Munich, Germany

11. Michael Sean Wiesener

    Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen

    Affiliation as printed

    Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany

12. Bruno Hüettel

    Max Planck Institute for Plant Breeding Research

    Affiliation as printed

    Max Planck Genome Center, Max Planck Institute for Plant Breeding Research, Cologne, Germany

13. Florian Erger corresponding

    Universitätsmedizin Greifswald · Universität Greifswald

    Affiliation as printed

    University Medicine Greifswald, Institute for Molecular Genomics, Core Unit Genomics, Greifswald, Germany. Electronic address: Florian.Erger@med.uni-greifswald.de

14. Bodo B. Beck

    University of Cologne · University Hospital Cologne

    Affiliation as printed

    Institute of Human Genetics and Center for Rare and Hereditary Kidney Disease, Faculty of Medicine and University Hospital Cologne, Cologne, Germany. Electronic address: Bodo.Beck@uk-koeln.de