Best practice guidelines and recommendations on the molecular diagnosis of myotonic dystrophy types 1 and 2

Abstract

Myotonic dystrophy is an autosomal dominant, multisystem disorder that is characterized by myotonic myopathy. The symptoms and severity of myotonic dystrophy type l (DM1) ranges from severe and congenital forms, which frequently result in death because of respiratory deficiency, through to late-onset baldness and cataract. In adult patients, cardiac conduction abnormalities may occur and cause a shorter life span. In subsequent generations, the symptoms in DM1 may present at an earlier age and have a more severe course (anticipation). In myotonic dystrophy type 2 (DM2), no anticipation is described, but cardiac conduction abnormalities as in DM1 are observed and patients with DM2 additionally have muscle pain and stiffness. Both DM1 and DM2 are caused by unstable DNA repeats in untranslated regions of different genes: A (CTG)n repeat in the 3'-UTR of the DMPK gene and a (CCTG)n repeat in intron 1 of the CNBP (formerly ZNF9) gene, respectively. The length of the (CTG)n repeat expansion in DM1 correlates with disease severity and age of onset. Nevertheless, these repeat sizes have limited predictive values on individual bases. Because of the disease characteristics in DM1 and DM2, appropriate molecular testing and reporting is very important for the optimal counseling in myotonic dystrophy. Here, we describe best practice guidelines for clinical molecular genetic analysis and reporting in DM1 and DM2, including presymptomatic and prenatal testing.

Figure 1

Molecular diagnostic tests in myotonic dystrophy type 1. (a) Fragment-length analysis of TP-PCR products of the CTG repeat in the DMPK gene. Fluorescently-labeled PCR products of a healthy individual having two normal alleles (5 and 13 CTG repeats; top panel) and an affected individual (bottom panel) with one normal allele (5 repeats) and one expanded allele were separated by capillary electrophoresis. (b) Southern blotting of long-range PCR-products of the CTG repeat in the DMPK gene. Long-range PCR fragments were subjected to to agarose gel electophoresis and capillary transfer to a nylon membrane. Subsequently, the membrane was hybridized with a labeled (CAG)₅ probe. Visualized repeats were from a healthy control (lane 2), a patient with a heterozygous expansion in the size range of 51–150 repeats (lane 1), and a patient with a heterozygous expansion in the size range over 150 repeats (lane 3). (c) Southern blotting of genomic DNA probed for the DMPK gene. Genomic DNA samples were fragmented using BglI and subjected to electophoresis and capillary blotting to a nylon membrane. Subsequently, the membrane was hybridized with a P-labeled probe for the DMPK gene. Visualized fragments were from a healthy control (1), and patients with one normal and one expanded allele (2 and 3). Estimation of the lengths of the repeat expansions can be done using the size markers (M). Normal sized alleles and expansions are indicated.

Figure 2

Molecular diagnostic tests in myotonic dystrophy type 2. (a) Fragment-length analysis of QP-PCR products of the CCTG repeat in the CNBP gene. Fluorescently-labeled PCR products of a healthy individual (top panel) and an affected individual (bottom panel) with one normal allele and one expanded allele (>100 repeats) were separated by capillary electrophoresis. (b) Southern blotting of long-range PCR products of the CCTG repeat in the CNBP gene. Long-range PCR fragments were subjected to agarose gel electophoresis and capillary transfer to a nylon membrane. Subsequently, the membrane was hybridized with an end-labeled (CAGG)₅ probe. Visualized repeats were from a healthy compound heterozygous individual (lane 1) and a patients (lane 2) with one normal and one expanded allele. (c) Southern blotting of genomic DNA probed for the CNBP gene. Genomic DNA samples were fragmented using EcoRI and subjected to electophoresis and capillary blotting to a nylon membrane. Subsequently, the membrane was hybridized with a mixture of the CL3N58 (within the CNBP gene) and a reference probe (both P-labeled). Visualized fragments were from a healthy control (lane 1), and patients with one normal and one expanded allele (lanes 2 and 3). Note the decrease in intensity of the normal allele in the patients with an expansion, when compared with the control probe signal. Normal sized CNPB alleles and expansions, as well as the reference probe, are indicated.