Abstract
Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disease caused by expansion of a CTG trinucleotide repeat in the DMPK gene. Methodology for genetic testing of DM1 is currently not optimal, in particular for the early-onset patients in pediatric populations where large expanded (CTG)n alleles are usually common. Individuals who are homozygous for a normal allele and individuals who are heterozygous for one normal and one large expanded allele are indistinguishable by conventional PCR, as both generate a single product of the normal allele. Thus, reflex Southern blot has often been needed to distinguish these cases. With the aim to decrease the need for reflex Southern blot tests, a novel, single-tube CTG repeat primed PCR technology was designed to distinguish the true homozygous patients from the individuals whose large alleles are missed by conventional PCR. The method utilizes two gene-specific primers that flank the triplet repeat region and a third primer set complementary to the repeated region to detect the large alleles. Compared to traditional PCR, this novel Triplet-repeat Primed PCR can detect the presence of large expanded alleles with demonstrating a ladder pattern. Using this single-step protocol, 45 specimens were tested. The alleles with sizes~í~85 repeats were determined by the gene specific primers. 13 abnormal alleles, which were missed by conventional PCR, were successfully detected by the Triplet-repeat Primed PCR. All the abnormal alleles were confirmed and measured by Southern Blot analysis. In summary, optimized Triplet-Primed PCR (TP-PCR) can accurately detect the presence of the large expanded alleles. With the ability to distinguish the true homozygous patients from the false negative homozygous individuals, the application of the optimized TP-PCR can significantly reduce the need of Southern Blot tests.
Highlights
Mytonic dystrophy type 1(DM1) is the most common adult onset neuromuscular disorder, with a prevalence frequency ranging from 1 to 15 in 100,000, and is inherited in an autosomal dominant pattern (Turner and Hilton-Jones, 2010)
Forty five samples had been previously tested with conventional PCR and reflex Southern blot
The 85 repeat allele is the largest detected by conventional PCR and sizable by Triplet-Primed PCR (TP-PCR). 21 patients had only one peak with the repeat size falling in the normal range
Summary
Mytonic dystrophy type 1(DM1) is the most common adult onset neuromuscular disorder, with a prevalence frequency ranging from 1 to 15 in 100,000, and is inherited in an autosomal dominant pattern (Turner and Hilton-Jones, 2010). DMPK is the only reported gene in which mutations cause DM1. The disease causing mutation is an unstable CTG triplet expansion in the 3 -untranslated region of the DMPK gene (Prior, 2009). The number of CTG repeats ranges from 5 to 34. Individuals with 35 to 49 CTG repeats have not been reported to develop DM1, but the number of the CTG repeats may increase when this allele is passed on to the generation. People are affected with DM1 if the number of CTG repeats is equal to or greater than 50 (Bird, 2013). Patients with congenital or severe DM1 tend to have thousand(s) of CTG repeats in the 3 UTR region of the DMPK gene (Zeesman et al, 2002)
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