April 21, 2021  |  Rare disease

SMRT Sequencing Detects Clinically Significant Repeat Changes In Triplet Expansion Disorders

When size matters and you need to be able to detect both single nucleotide changes as well as large repeated sequences, SMRT Sequencing on the Sequel II System is the way to go, concluded rare disease researchers at Centre de Recherche en Myologie at Sorbonne Université/INSERM 
Stéphanie Tomé (@TomeStephanie) and colleagues used the highly sensitive, comprehensive long-read sequencing to investigate myotonic dystrophy type 1 (DM1), the most complex and variable trinucleotide repeat disorder, caused by an unstable CTG repeat expansion that can reach up to 4,000 triplets in those affected most severely with the disease.

Myotonic dystrophy type 1 (DM1) is caused by an unstable CTG repeat, the number of copies of this repeat correlate with the severity of symptoms.

As reported previously, the length of these repeated CTG sections and any interruptions in the sequences have been found to correlate with the severity and onset of symptoms of the neuromuscular autosomal disorder, which is the most common form of inherited muscular dystrophy in adults. 
The highly variable clinical presentation of DM1 and current limitations in methods to determine the size and variant repeat interruptions of the large CTG repeat expansions, make genetic counseling for the condition very complex, so Tomé turned to PacBio sequencing to better understand this mutation. She successfully applied for a 2019 Targeted Sequencing SMRT Grant, and the results of her work were recently published in the International Journal of Molecular Sciences.
“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,” Tomé said.
Inherited CTG repeat expansion size and the level of somatic mosaicism are traditionally evaluated by Southern blot and polymerase chain reaction (PCR), which do not provide any information on the sequence of CTG repeat expansion. Triplet-primed PCR testing may detect the presence of interruptions at the 5’ and 3’ ends of the CTG repeat expansion, and short-read sequencing can help identify further interruptions, but the methods give no information about the middle of the sequence.
Using the Sequel II System, Tomé’s team was able to sequence 1,000 CTG triplet-long repeats, detect a single CAG and multiple CCG interruptions, and also estimate somatic mosaicism (the occurrence of two genetically distinct populations of cells within an individual derived from a postzygotic mutation) within two DM1 families—with more accuracy than conventional PCR.
The data enabled them to gain insights into the genetic changes within the families in the study, as well as some observations applicable to the nature of DM1. They revealed the existence of de novo CCG interruptions associated with CTG stabilization/contraction across generations in one of the families. And the heterogeneity of the number and type of interruptions observed in the interrupted expanded alleles suggested new mechanisms leading to base substitution in the sequence and/or duplication of existing interruptions in the repeated sequence. These could be caused by multiple processes, including spontaneous DNA damage, DNA repair and DNA polymerase errors occurring in germ cells and somatic cells throughout embryogenesis and the lifetime of those affected by DM1.

Stéphanie Tomé, Investigator at the Centre de Recherche en Myologie at Sorbonne Université/INSERM.

“Our study reinforced the idea that interrupted alleles do not originate from an ancestral/normal allele, but from unknown mechanisms occurring both in the germline and in somatic cells,” the study concluded. 
“SMRT Sequencing opens new avenues for DM1 disease and will provide a better understanding of the clinical and genetic variability observed in DM1 through global analysis,”  Tomé added. “This new technology is a straightforward way to detect clinically significant repeat changes and estimate the size of the repeat in blood using targeted sequencing.” 

To learn more about how scientists are using highly accurate long-read sequencing in large-scale studies to help identify causative variants, increase solve rates in rare disease research, and support the development of diagnostics for rare and undiagnosed diseases, watch on-demand presentations from PacBio Neuroscience Day, and register for the Rare Disease Week virtual event, April 27-29.

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