Molecular Diagnostics in Myotonic Dystrophy

Myotonic dystrophy (DM) is the most common inherited adult neuromuscular disease. It is inherited as an autosomal dominant disorder with an estimated prevalence of about 1/8,000 in the Caucasian population. The gene is located at 19q13.3 and encodes myotonin kinase. The disorder shows anticipation, that is, the observation that severity increases in later generations. This phenomenon has now been shown to be a consequence of the increase in size in later generations of an unstable CTG trinucleotide repeat located in the 3' untranslated region of the DM gene.

In normal individuals, there are about 5 to 37 CTG repeats in DNA isolated from peripheral blood. In asymptomatic or minimally affected individuals, the CTG repeat is about 40 to 150 repeats. In more severely affected individuals, or cases of congenital myotonic dystrophy, the CTG repeat can range into the thousands of repeats. The number of repeats detected in peripheral blood is typically smaller than the number of repeats detected in muscle tissues. This result indicates significant somatic instability of the expanded DM repeat.

The size of the DM CTG repeat can be evaluated by alternative methods.

  1. Southern blot analysis of Eco RI or Pst I digested genomic DNA for large affected alleles

  2. PCR sizing of the CTG repeat for normal and minimally expanded alleles

Each of these methods are depicted in the figure below. In the left panel an example of an Eco RI Southern blot is shown. Affected individuals are shown as solid red circles and the expanded alleles are indicated by red arrows. Normal alleles are indicated by yellow arrows. In this family, the mother, who was minimally affected as an adult, has transmitted an expanded allele to one of her daughters, who was clinically affected as a child. Her other two children are unaffected and have normal alleles of 9 and/or 10 Kbp. Another child (untested) died neonatally and had clinical features consistent with congenital myotonic dystrophy. Normal individuals have alleles of about 9 or 10 Kbp due to an RFLP detected by the probe.

The expanded alleles depicted in the Southern blot analysis are too large to be evaluated by PCR. However, as shown in the right panel, PCR can be used to accurately size the CTG repeats in normal and asymptomatic individuals. The three samples (from left to right) show DM CTG repeat alleles of 12/14, 5/75, and 5/12 repeats. The patient with the 5/75 repeat alleles is the father of the adult patient shown in the Southern blot analysis. He is reportedly asymptomatic yet is clearly the origin of the DM mutation segregating in this family. Together, the three DM patients depicted here clearly demonstrate the process of anticipation at the molecular level.