Genetic Diagnosis of DM1

Although the initial patients in a given family arrive at the clinic as a consequence of some aspects of the multi-systemic consequences of the disease, the accurate determination of trinucleotide repeat length is a key component in establishing a molecular diagnosis and assessing disease prognosis for DM1.

Expansions of existing CTG repeats in germ line and somatic cells contribute to earlier age of onset/greater severity in subsequent generations and increased symptoms as individual age, respectively. Knowledge of CTG repeat length thus has become an important diagnostic and prognostic variable. Molecular diagnosis of DM1 has relied on technology including triplet-repeat primed PCR, small-pool PCR, and a combination of rolling circle amplification and Southern blotting—these approaches are not without drawbacks (arising from both the assays themselves and the inherent instability of the repeat). Thus, there is a clear need for a genetic testing paradigm that assesses repeat length in small samples and with improved accuracy, speed, and cost effectiveness. Likewise, optimized expanded repeat length assays are vital for the development of genome editing as a candidate therapy for DM1.

Development of a Novel Assay for Expanded Repeat Length

Drs. Aurélien Bancaud (Université de Toulouse), Vincent Dion (University of Lausanne), and colleagues have developed a system they designate as μLAS (μLaboratory for DNA Separation), a micro-modular lab-on-chip system to process and characterize expanded repeats in trinucleotide expansion disorders. Their work, reported in Scientific Reports, focuses on quantifying CAG/CTG expanded repeats in Huntington disease (HD) and DM1.

The research team has implemented a microfluidics approach to achieve high efficiency DNA separation/enrichment and high sensitivity detection of expanded repeat alleles in DM1 and HD. The resulting dual-channel microchip assesses test sample and reference in parallel channels. Evaluation of the same non-repetitive sequence samples in both channels showed accuracy/reproducibility within 3 bp. Using μLAS for DNA sizing, μLAS reported out on DNA lengths across a broad range (466 to 3314 bp) within 3% of the known test samples. The system was sufficiently sensitive to determine the length of a 100 bp test sample within a 5 minute run time.

The research team also demonstrated the system’s sensitivity to rapidly (within minutes) detect expanded repeats and alleles of heterogeneous lengths from patient samples, including an ability to quantify the range of expanded repeat lengths reported for HD and DM1. The primary focus of the research group was in evaluation of μLAS for HD, although they established proof of concept for DM1 as well. Some limitations of μLAS were seen for the very large repeat lengths characteristic of CDM.

Reference:

µLAS: Sizing of expanded trinucleotide repeats with femtomolar sensitivity in less than 5 minutes.
Malbec R, Chami B, Aeschbach L, Ruiz Buendía GA, Socol M, Joseph P, Leïchlé T, Trofimenko E, Bancaud A, Dion V.
Sci Rep. 2019 Jan 10;9(1):23. doi: 10.1038/s41598-018-36632-5.