Highly unstable sequence interruptions of the CTG repeat in the myotonic dystrophy gene

Abstract

Myotonic dystrophy type 1 is caused by the expansion of a CTG repeat in the 3' UTR of the DMPK gene. A length exceeding 50 CTG triplets is pathogenic. Intermediate alleles with 35-49 triplets are not disease-causing but show instability in intergenerational transmissions. We report on the identification of multiple patients with different patterns of CCG and CTC interruptions in the DMPK CTG repeat tract that display unique intergenerational instability. In patients bearing interrupted expanded alleles, the location of the interruptions changed dramatically between generations and the repeats tended to contract. The phenotype for these patients corresponded to the classical form of the disease, but in some cases without muscular dystrophy and possibly with a later onset than expected. Symptomatic patients bearing interrupted intermediate length repeat tracts were also identified, although the role of the interruptions in their phenotype remains unclear. The identification of interruptions in the DMPK repeat has important consequences for molecular genetic testing where they can lead to false negative conclusions.

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).…”

“…11 To investigate the presence of variant repeats, variants of the P4 primer have been used accordingly to published protocols. 4 The characterization of the interruption motifs has been obtained using different approaches, including direct sequencing of: TP-PCR products with primer P1 for 3′ or P2 for 5′ interruptions (patients A2, B1, D, E, F, G, H, I); gel-purified (Gel Extraction Kit, Qiagen, Germany) TP-PCR, using primers P4hex1 or P4-CC, cloned in pCR2.1 vector (TA Cloning, Invitrogen, Carlsbad, CA, USA) using M13 Reverse vector primer (patients A1, A3 and B2); gel-purified LR-PCR products cloned in pCR2.1 vector (TA Cloning, Invitrogen) (patients C1, C2, C3) using M13 Reverse vector primer. In these latter DM1 patients the entire DMPK expanded allele has been characterized.…”

“…4,5 CCG-CTC repeats stabilize the intergenerational variability of the complex DMPK expanded alleles Given the stability of variant repeats in other triplet repeat disorders, [14][15][16] we decided to study the intergenerational dynamics of the complex DMPK alleles in three DM1 families comprising five intergenerational transmissions (4 maternal and 1 paternal) (Figure 2). Interestingly, in all intergenerational events analyzed in this study, we observed a relative stability of the mutated alleles (family A and C) or a tendency to contraction even when the mutation was transmitted from an adult onset affected DM1 mother (from 740-930 to 450-550 CTGs in family B) (Figure 2).…”

“…Although the CTG repeat tract is usually uninterrupted in nonexpanded and expanded alleles, in the last few years, pathological variant expansions containing unstable CCG, CTC and GGC sequence interruptions have been reported in rare instances. [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][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.…”