Simple Repeat-Primed PCR Analysis of the Myotonic Dystrophy Type 1 Gene in a Clinical Diagnostics Environment

Myotonic dystrophy type 1 is an autosomal dominant neuromuscular disorder that is caused by the expansion of a CTG trinucleotide repeat in the DMPK gene. The confirmation of a clinical diagnosis of DM-1 usually involves PCR amplification of the CTG repeat-containing region and subsequent sizing of the amplification products in order to deduce the number of CTG repeats. In the case of repeat hyperexpansions, Southern blotting is also used; however, the latter has largely been superseded by triplet repeat-primed PCR (TP-PCR), which does not yield a CTG repeat number but nevertheless provides a means of stratifying patients regarding their disease severity. We report here a combination of forward and reverse TP-PCR primers that allows for the simple and effective scoring of both the size of smaller alleles and the presence or absence of expanded repeat sequences. In addition, the CTG repeat-containing TP-PCR forward primer can target both the DM-1 and Huntington disease genes, thereby streamlining the work flow for confirmation of clinical diagnoses in a diagnostic laboratory. 1. Introduction Myotonic dystrophy type 1 (DM-1) is a multisystem disorder with a highly variable phenotypic expression. This autosomal dominant neuromuscular disorder has been reported to cause myotonia, progressive muscle weakness, and atrophy in skeletal muscles, as well as cardiomyopathy, cataracts, and dysfunction in the endocrine and central nervous systems [1, 2]. DM-1 is caused by a CTG trinucleotide repeat expansion in the 3′ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene, which is located on chromosome 19q13.3. The number of repeats a patient carries is associated with clinical severity. Repeats of 5–34 are found in unaffected individuals, and those that lie in the range of 35–49 are considered abnormal premutations such that carriers are at risk of having affected offspring with larger repeat lengths. Patients with DM-1 are typically classified into three overlapping groupings based on the number of repeats, the severity of clinical presentation, and the age of onset. These groupings are mild, classical, and congenital with repeat sizes ranging from 50 to 150 repeats, 100 to 1000 repeats, and >2000 repeats, respectively [3, 4]. Despite these repeat ranges, the stratifying of patients into classical and congenital categories can pose difficulties as some congenital patients carry CTG repeats that overlap with the classic range. In addition, the situation is further confounded by the recent identification of a juvenile category with CTG repeats that