Identification of a CCG-enriched expanded allele in DM1 patients using Amplification-free long-read sequencing - Sorbonne Université Access content directly
Conference Papers Year : 2022

Identification of a CCG-enriched expanded allele in DM1 patients using Amplification-free long-read sequencing

Abstract

Background: Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4,000 CTG. The clinical variability depends on CTG repeat number, CNG repeat interruptions and somatic mosaicism. Currently, none of these factors are simultaneously and accurately determined due to the limitations of gold standard methods used in clinical and research laboratories. An amplicon method for targeting DM1 locus using Single-Molecule Real-Time sequencing (Pacific Biosciences) was recently developed to accurately analyze expanded alleles1. However, amplicon-based sequencing still depends on PCR and the inherent bias towards preferential amplification of smaller repeats can be problematic in DM1. Aims: To overcome PCR limitation, we developed a robust amplification free-targeted (No-Amp) long-read sequencing to specifically characterize the DM1 locus in patients. Methods: No-Amp long-read sequencing utilizes the CRISPR/Cas9 system to target and isolate the DNA fragment of interest from genomic DNA, in combination with long-read sequencing. This method was used to sequence the DM1 locus in patients with CTG repeat expansion ranging from 130 to > 1000 CTG (CTG repeat size estimated by Southern blot at diagnosis). Results: We showed that elimination of PCR amplification improves the accuracy of measurement of inherited repeat number and somatic repeat variations, two important key factors in the DM1 severity and age at onset. For the first time, an expansion composed of over 85% CCG repeats was identified in a DM1 family with an atypical clinical profile for whom amplification of the triplet repeat expansion failed by PCR and TP-PCR. Conclusions: This method allows to simultaneously obtain high resolution information on the number of repeats, a complete and accurate sequence and a measure of somatic mosaicism even for long repeats in the same assay. No-amplification targeted sequencing gives us the opportunity to better understand the dynamics of CTG repeat instability and genotype-phenotype association in DM1 but also in other trinucleotide repeat diseases. This work was supported by the Institut National de la Santé et de la Recherche Médicale, Sorbonne Université, Institut de Myologie (Acelerator project) and the 2019 Targeted Sequencing SMRT Grant. 1Mangin et al. (2021). Int J Mol Sci 22, 2616.
No file

Dates and versions

hal-04004408 , version 1 (24-02-2023)

Identifiers

  • HAL Id : hal-04004408 , version 1

Cite

Yu-Chih Tsai, Laure de Pontual, Chéryl Heiner, Tanya Stojkovic, Denis Furling, et al.. Identification of a CCG-enriched expanded allele in DM1 patients using Amplification-free long-read sequencing. FASEB, The dynamic DNA structures in biology conference, Jun 2022, Nova Scotia, Canada. ⟨hal-04004408⟩
15 View
0 Download

Share

Gmail Facebook X LinkedIn More