Premutation allele pool in myotonic dystrophy type 2

Abstract

Background: The myotonic dystrophies (DM1, DM2) are the most common adult muscle diseases and are characterized by multisystem involvement. DM1 has been described in diverse populations, whereas DM2 seems to occur primarily in European Caucasians. Both are caused by the expression of expanded microsatellite repeats. In DM1, there is a reservoir of premutation alleles; however, there have been no reported premutation alleles for DM2. The (CCTG)(DM2) expansion is part of a complex polymorphic repeat tract of the form (TG)(n)(TCTG)(n)(CCTG)(n)(NCTG)(n)(CCTG)(n). Expansions are as large as 40 kb, with the expanded (CCTG)(n) motif uninterrupted. Reported normal alleles have up to (CCTG)(26) with one or more interruptions.

Methods: To identify and characterize potential DM2 premutation alleles, we cloned and sequenced 43 alleles from 23 individuals. Uninterrupted alleles were identified, and their instability was confirmed by small-pool PCR. We determined the genotype of a nearby single nucleotide polymorphism (rs1871922) known to be in linkage disequilibrium with the DM2 mutation.

Results: We identified three classes of large non-DM2 repeat alleles: 1) up to (CCTG)(24) with two interruptions, 2) up to (CCTG)(32) with up to four interruptions, and 3) uninterrupted (CCTG)(22-33). Large non-DM2 alleles were more common in African Americans than in European Caucasians. Uninterrupted alleles were significantly more unstable than interrupted alleles (p = 10(-4) to 10(-7)). Genotypes at rs1871922 were consistent with the hypothesis that all large alleles occur on the same haplotype as the DM2 expansion.

Conclusions: We conclude that unstable uninterrupted (CCTG)(22-33) alleles represent a premutation allele pool for DM2 full mutations.

Figure 1 Distribution of normal repeat track sizes (A) Alleles with a repeat track ≥160 bp are rare in European Caucasians (17 of 973 non-DM2 chromosomes, 1.8%). (B) Such large alleles occur more frequently in the African American population (8 of 180 chromosomes, 8.3%; p = 1.769 × 10⁻⁶).

Figure 2 Repeat tract structures of the DM2 locus observed in 44 sequenced alleles We observed three classes of repeat tracts at the DM2 locus: 1) interrupted short alleles with up to (CCTG)₂₄ and two interruptions, 2) interrupted long alleles with up to (CCTG)₃₂ and up to four interruptions, and 3) uninterrupted (CCTG)_22–33. Repeat tracts ≥160 bp often showed duplication of the (GCTG)-(CCTG)-(TCTG)-(CCTG)_n portion of the tract. Occasional variations in the interruption sequence were also observed. Repeat tract length in bp and number of alleles observed are indicated for each variant (n = 44). The most common short allele (A) is also found in the Rhesus monkey (NW_001096632), suggesting that it is ancestral. Variants B, D, and F were only seen in the African American samples. Uninterrupted alleles (G and H) were seen only in Caucasians.

Figure 3 Results of small-pool PCR experiment The DM2 locus was constitutively more unstable than other microsatellite loci commonly used to assess instability. Sequencing revealed that this instability is confined to the (TG)_n portion of the tract. Bars represent number of alleles counted in single genome equivalent amplification (small-pool PCR). Solid black and white bars represent the progenitor alleles and striped bars represent novel alleles identified. Both small (A) and large (B) interrupted alleles had average mutation frequencies of 12% to 14%. Uninterrupted alleles (C) had mutation frequencies ranging from 30% to 53%, with an average of 39.4%. These alleles showed instability of the (CCTG)_n tract as well as the (TG)_n. The mutation frequencies for uninterrupted alleles were highly significantly different from interrupted controls. The p values for the four premutation alleles were 6.5 × 10⁻⁷ (896001), 4.3 × 10⁻⁴ (8103001), 1.4 × 10⁻⁴ (8102001), and 3.9 × 10⁻⁷ (106001).

Figure 4 Model for evolution of the DM2 mutation (A) Mis-pairing and unequal crossing over of DM2 repeat tracts (A1) can result in large alleles. Reciprocal crossover products can have either multiple interruptions (A2) or none (A3). These uninterrupted alleles are inherently unstable and eventually expand to pathogenic range (A4). (B) Evolutionary model for the DM2 locus. The ancestral hominid allele probably had a short (CCTG)_n tract with two interruptions in cis with the C allele at rs1871922, the same as the most common modern allele (green line). Based on the higher frequency of large alleles in our African American samples, the first large alleles probably arose in Africa, possibly by unequal crossing over. Long alleles with multiple interruptions (orange line) accumulated variants, whereas uninterrupted alleles (pink line) gradually expanded, eventually reaching a pathogenic threshold (red line). The most parsimonious scenario is that the A allele at rs1871922 arose after the exodus from Africa on the same chromosome as a small two-interruption allele (blue line). The apparent rapid increase in frequency of the A allele could be due to selection at some nearby locus. (C) The Tajima D statistic indicates probable positive selection (D <−2) in Europeans in the region approximately 100 kb centromeric to ZNF9.