Novel Heat Pulse Extension-PCR–Based Method for Detection of Large CTG-Repeat Expansions in Myotonic Dystrophy Type 1

Abstract

Myotonic dystrophy type 1 (DM1) is an autosomal-dominant disease caused by an expansion of CTG repeats in the 3' untranslated region of the Dystrophia Myotonica Protein Kinase (DMPK) gene. Detection and accurate sizing of the CTG-repeat expansions is clinically important, because the number of CTG repeats correlates with the disease severity. Because difficulties in PCR amplification over large expansions, molecular diagnosis of DM1 is still primarily based on Southern blotting, which is technically demanding and time consuming and requires large amounts of genomic DNA samples. We have recently discovered that the use of multiple heat pulses during Heat Pulse Extension PCR (HPE-PCR) enables efficient amplification over repetitive and GC-rich sequences. Based on this principle, we have developed an assay for efficient amplification of large CTG-repeat expansions seen in DM1 patients. The HPE-PCR method was able to amplify different DMPK1 repeat expansions of up to 1750 CTG repeats in 78 clinical samples with a varying degree of tissue heterogeneity, even in the presence of the short wild-type allele. The CTG-repeat lengths and fragmentation patterns obtained with HPE-PCR were fully concordant with the original diagnostic Southern blotting results. This novel technique provides a PCR-based platform for molecular diagnosis of DM1, and it has been adopted for routine diagnostic use.

Cell Membrane Integrity in Myotonic Dystrophy Type 1: Implications for Therapy

“…We used biopsies from three healthy controls, six DM1 patients and one DMD patient. (CTG)n repeat lengths in the DM1 muscle biopsies ranged between ~80 and >500, as measured by heat pulse extension PCR [ 38 ] ( S6 Fig . ; Table 2 ).…”

“…Genomic DNA was isolated from muscle sections following standard procedures. To amplify expanded (CTG)n repeats a heat pulse extension PCR protocol was used [ 38 ], that allows the generation of DMPK (CTG)n amplicons of up to 1750 CTG repeats. 40 ng DNA was used in a PCR mixture containing 2.25 M betaine (Fluka, Sigma-Aldrich, Germany), 0.2 mM dNTPs (Invitrogen, Carlsbad, CA, USA), 1.33 units DyNAzyme EXT DNA Polymerase (Thermo Scientific, Waltham, MA, USA) and 250 nM of each forward (5´-GCCAGTTCACAACCGCTCCGAGCGTGGGTC-3´) and reverse (5´-ACGCTCCCCAGAGCAGGGCGTCATGC-3´) primers (Biolegio BV, Nijmegen, the Netherlands).…”

“…40 ng DNA was used in a PCR mixture containing 2.25 M betaine (Fluka, Sigma-Aldrich, Germany), 0.2 mM dNTPs (Invitrogen, Carlsbad, CA, USA), 1.33 units DyNAzyme EXT DNA Polymerase (Thermo Scientific, Waltham, MA, USA) and 250 nM of each forward (5´-GCCAGTTCACAACCGCTCCGAGCGTGGGTC-3´) and reverse (5´-ACGCTCCCCAGAGCAGGGCGTCATGC-3´) primers (Biolegio BV, Nijmegen, the Netherlands). Cycling conditions were kept as described [ 38 ], except that an annealing temperature of 66°C followed by ramping to 83°C (0.9°C/sec) was used. PCR was performed in a DNA engine Peltier Thermal Cycler (Bio-Rad, Hercules, CA, USA).…”

Myotonic Dystrophy Type 1 Diagnostics: A Changing Trend

“…This is done to prevent the nonspecific amplification that takes place at lower temperature [24], Heat Pulse Extension (HPE) PCR is another PCR which is done to increase the amplification efficiency over repetitive and GC rich sequences [26] or Nested PCR which is another modified form of PCR which is done to reduce non-specific binding and uses two sets of primers and PCR product of the first reaction being the template for the second reaction [27].…”

The Need for Establishing a Universal CTG Sizing Method in Myotonic Dystrophy Type 1

“…Indeed, the number of CTG repeats can be inversely and directly related with age of disease onset and clinical severity, respectively [9,10]. Although different approaches have been described to assess CTG expansion size in patients with DM1 [5,[11][12][13][14][15], some methodological issues remain to be solved, mainly related to the inherent repeat instability and technical difficulties when amplifying long CTG fragments.…”