Single molecule real time sequencing in ADTKD-MUC1 allows complete assembly of the VNTR and exact positioning of causative mutations

Andrea Wenzel,Holger Thiele,Michael Wiesener,Peter Nuernberg,Eduardo Salido,Bodo B Beck,Bernt Popp,André Reis,Alois Pannes,Matthias T F Wolf,Janine Altmueller,Richard Reinhardt,Arif B Ekici,Bruno Huettel,Simon Staubach,Patrick Rump,Kurt Stueber,Franz-Georg Hanisch

doi:10.1038/s41598-018-22428-0

Abstract

Recently, the Mucin-1 (MUC1) gene has been identified as a causal gene of autosomal dominant tubulointerstitial kidney disease (ADTKD). Most causative mutations are buried within a GC-rich 60 basepair variable number of tandem repeat (VNTR), which escapes identification by massive parallel sequencing methods due to the complexity of the VNTR. We established long read single molecule real time sequencing (SMRT) targeted to the MUC1-VNTR as an alternative strategy to the snapshot assay. Our approach allows complete VNTR assembly, thereby enabling the detection of all variants residing within the VNTR and simultaneous determination of VNTR length. We present high resolution data on the VNTR architecture for a cohort of snapshot positive (n = 9) and negative (n = 7) ADTKD families. By SMRT sequencing we could confirm the diagnosis in all previously tested cases, reconstruct both VNTR alleles and determine the exact position of the causative variant in eight of nine families. This study demonstrates that precise positioning of the causative mutation(s) and identification of other coding and noncoding sequence variants in ADTKD-MUC1 is feasible. SMRT sequencing could provide a powerful tool to uncover potential factors encoded within the VNTR that associate with intra- and interfamilial phenotype variability of MUC1 related kidney disease.

Highlights

The Mucin-1 (MUC1) gene has been identified as a causal gene of autosomal dominant tubulointerstitial kidney disease (ADTKD)
ADTKD-MUC1 constitutes the first kidney disorder where mutations reside within a coding variable number of tandem repeat (VNTR contained in exon 2)
Using a modified PCR protocol published by Fowler et al allowed stable generation of PCR amplicons across a wide range of MUC1 VNTR allele sizes that could be visualized by agarose gel electrophoresis (Fig. 2)[22]

Summary

Introduction

The Mucin-1 (MUC1) gene has been identified as a causal gene of autosomal dominant tubulointerstitial kidney disease (ADTKD). This study demonstrates that precise positioning of the causative mutation(s) and identification of other coding and noncoding sequence variants in ADTKD-MUC1 is feasible. ADTKD-MUC1 constitutes the first kidney disorder where mutations reside within a coding variable number of tandem repeat (VNTR contained in exon 2). The prototypic causative variant in MUC1 associated ADTKD, the insertion of a eighth cytosine base (insC) in a seven cytosine stretch within one unit of the VNTR composed of almost identical 60-mer units (according to HGVS nomenclature recommendations a duplication, but for the sake of clarity we stay with term insertion used in most publications), has been repeatedly found worldwide ( arbitrarily referred to as c.428dupC)[4,5]. Due to the nature of the VNTR, insC within different located 60-mer unit numbers as well as other small insertions or deletions located at different positions, would result in a frameshift leading to a similar, presumably toxic MUC1-neoprotein that is retained within the cell (Fig. 1)[5]

Methods

Results

Conclusion