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

Abstract

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 In the case of repeat hyperexpansions, Southern blotting is also used; however, the latter has largely been superseded by triplet repeat-primed PCR (TP-PCR), which does not yield a CTG repeat number but nevertheless provides a means of stratifying patients regarding their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.

Identification and characterization of 5′ CCG interruptions in complex DMPK expanded alleles

“…Complex variant repeats were identified at the both ends of the CTG array of DM1 patients of different origin, with an estimated prevalence of 3-5%. 4,5,7,8 In this study we retrospectively revaluated by bi-directional TP-PCR a cohort of 254 Italian DM1 patients estimating a minimum frequency of interrupted expanded alleles of 3.5%, perfectly in line with previous observations. 4,5 Direct sequencing of the entire or partial interrupted alleles, revealed the presence of complex arrays containing Figure 1 TP-PCR analysis of 5′ region of the CTG array in members of family C and in a DM1 patient with an uninterrupted DMPK allele as control (bottom panel, EXP).…”

“…[4][5][6][7] Complex variant repeats were identified at both ends of the CTG array of DM1 patients of different origin, with an estimated prevalence of 3-5% of cases. [4][5][6][7][8] However, the phenotypical consequences of the interrupted alleles on DM1 patients is still controversial leading either to a complex neurological phenotype or to classical/mild DM1 forms. 4,5,7 In the present work, we describe the detailed molecular analysis of multiple patients carrying various patterns of interruptions either at the 3′ or 5′ end of the DMPK repeat identified in our cohort of 254 Italian DM1 patients, including three multi-generation families.…”

Unusual association of a unique CAG interruption in 5′ of DM1 CTG repeats with intergenerational contractions and low somatic mosaicism

“…Recently, multiple CCG interruptions in the 5′ end of the CTG repeat sequence were described in four DM1 patients from two distinct DM1 families. These results show that CCG interruptions are not exclusively located in the 3′end of the CTG repeat expansion (Botta et al., ; Dryland, Doherty, Love, & Love, ). All these interruptions were associated with stabilization or contraction of the expanded CTG repeat tracts through maternal and paternal transmissions (Botta et al., ; Braida et al., ; Musova et al., ).…”

Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells

“…Triplet-repeat primed PCR was performed as described in Philippa et al. ( 46 ). Each 25 μl PCR comprised FastStart Reaction Buffer without MgCl 2 (Roche), 2 mM MgCl 2 (Roche), GC-rich solution (Roche), 10 mM dNTP mix (Roche), 20 μM forward primer (FAM) 5′-AACGGGGCTCGAAGGGTCCTTGTAGC-3′ and reverse primer 5′-GGCGGTGGCGGCTGTTGCCAGCAGCAGCAGCAG-3′ , 1U FastStart Taq DNA Polymerase (Roche) and 50 ng of genomic DNA.…”