Robust Detection of Somatic Mosaicism and Repeat Interruptions by Long-Read Targeted Sequencing in Myotonic Dystrophy Type 1.

Antoine Mangin,Laetitia Monteil,Mathilde Nizon,Sandra Mercier,Pierre Boisseau,Janet Ziegle,Laure De Pontual,Geneviève Gourdon,Cheryl Heiner,John Harting,Stéphanie Tomé,Yu-Chih Tsai

doi:10.3390/ijms22052616

Abstract

Myotonic dystrophy type 1 (DM1) is the most complex and variable trinucleotide repeat disorder caused by an unstable CTG repeat expansion, reaching up to 4000 CTG in the most severe cases. The genetic and clinical variability of DM1 depend on the sex and age of the transmitting parent, but also on the CTG repeat number, presence of repeat interruptions and/or on the degree of somatic instability. Currently, it is difficult to simultaneously and accurately determine these contributing factors in DM1 patients due to the limitations of gold standard methods used in molecular diagnostics and research laboratories. Our study showed the efficiency of the latest PacBio long-read sequencing technology to sequence large CTG trinucleotides, detect multiple and single repeat interruptions and estimate the levels of somatic mosaicism in DM1 patients carrying complex CTG repeat expansions inaccessible to most methods. Using this innovative approach, we revealed the existence of de novo CCG interruptions associated with CTG stabilization/contraction across generations in a new DM1 family. We also demonstrated that our method is suitable to sequence the DM1 locus and measure somatic mosaicism in DM1 families carrying more than 1000 pure CTG repeats. Better characterization of expanded alleles in DM1 patients can significantly improve prognosis and genetic counseling, not only in DM1 but also for other tandem DNA repeat disorders.

Highlights

E2.1 was identified as a DM1 patient carrying interrupted CTG repeat expansion with atypical symptoms
polymerase chain reaction (PCR) amplification of the CTG repeat tracts revealed a decrease in CTG repeat size across two paternal transmissions (Figure 1)
Two distinct gaps were found in both triplet-primed polymerase chain reaction (TP-PCR) traces, whereas a third gap was only observed in individual E.3, introducing the presence of additional interruptions in the fetus E3

Summary

Introduction

More than 40 different human disorders are caused by tri-, tetra-, penta- or hexanucleotide repeat expansions localized either in coding or non-coding regions of the target gene [1]. The pathogenic mechanisms for repeat diseases involve either a loss of protein function or a gain of function at the RNA or protein level, depending on the type and location of the repeat [1]. Among the trinucleotide repeat (TNR) diseases, Fragile X syndrome (FXS (MIM: 300624)), Huntington’s disease (HD (MIM: 143100)), several spinocerebellar ataxias (SCAs) and myotonic dystrophy type 1 (DM1 (MIM: 160900)) have been reported. DM1 is a highly multisystemic disorder caused by an unstable CTG repeat expansion within the 30 -untranslated region (UTR) of the myotonic dystrophy protein kinase (DMPK)

Methods

Results

Conclusion