Pms2 suppresses large expansions of the (GAA·TTC)n sequence in neuronal tissues

Abstract

Expanded trinucleotide repeat sequences are the cause of several inherited neurodegenerative diseases. Disease pathogenesis is correlated with several features of somatic instability of these sequences, including further large expansions in postmitotic tissues. The presence of somatic expansions in postmitotic tissues is consistent with DNA repair being a major determinant of somatic instability. Indeed, proteins in the mismatch repair (MMR) pathway are required for instability of the expanded (CAG·CTG)(n) sequence, likely via recognition of intrastrand hairpins by MutSβ. It is not clear if or how MMR would affect instability of disease-causing expanded trinucleotide repeat sequences that adopt secondary structures other than hairpins, such as the triplex/R-loop forming (GAA·TTC)(n) sequence that causes Friedreich ataxia. We analyzed somatic instability in transgenic mice that carry an expanded (GAA·TTC)(n) sequence in the context of the human FXN locus and lack the individual MMR proteins Msh2, Msh6 or Pms2. The absence of Msh2 or Msh6 resulted in a dramatic reduction in somatic mutations, indicating that mammalian MMR promotes instability of the (GAA·TTC)(n) sequence via MutSα. The absence of Pms2 resulted in increased accumulation of large expansions in the nervous system (cerebellum, cerebrum, and dorsal root ganglia) but not in non-neuronal tissues (heart and kidney), without affecting the prevalence of contractions. Pms2 suppressed large expansions specifically in tissues showing MutSα-dependent somatic instability, suggesting that they may act on the same lesion or structure associated with the expanded (GAA·TTC)(n) sequence. We conclude that Pms2 specifically suppresses large expansions of a pathogenic trinucleotide repeat sequence in neuronal tissues, possibly acting independently of the canonical MMR pathway.

Figure 1. Absence of MutSα reduces somatic mutation load of the (GAA·TTC)_n sequence.

Panels A, B, E, and F show representative autoradiographs from SP-PCR analysis of DNA extracted from (A) Msh2^+/+, (B) Msh2^−/−, (E) Msh6^+/+, and (F) Msh6^−/− cerebellum. Progenitor allele lengths determined by conventional PCR are 192, 130, and 89 repeats for Msh2^+/+; 188, 128, and 88 repeats for Msh2^−/−; 186, 133, and 98 repeats for Msh6^+/+; and 187, 129, and 90 repeats for Msh6^−/−, as indicated by arrowheads at the right of each panel. Results from the total analysis of 150 to 282 molecules per genotype are quantified in C and D for Msh2, and G and H for Msh6.

Figure 2. Absence of Pms2 increases somatic expansion load of the (GAA·TTC)_n sequence in cerebrum.

Analysis of instability in cerebrum of Pms2^+/+ and Pms2^−/− mice. Representative autoradiographs from (A) Pms2^+/+ and (B) Pms2^−/− cerebrum are shown. Progenitor allele lengths of 229, 194, 135, and 94 repeats for Pms2^+/+ and 229, 185, 130, and 86 repeats for Pms2^−/− are indicated by arrowheads at the right of each panel. Mutation load and expansion load, quantified from the analysis of ∼550 molecules per genotype, are shown in panels C and D, respectively. Panels E and F indicate the distribution of repeat lengths for all SP-PCR products of (E) Pms2^+/+ and (F) Pms2^−/−. These graphs are therefore an exact, combined representation of all blots analyzed for Pms2^+/+ and Pms2^−/− cerebrum. The number of products measured at each repeat length is on the Y-axis. Note that the actual frequency of products at the progenitor lengths may be slightly higher than the counted frequency, because a single band may represent PCR products amplified from multiple progenitor molecules. The gray bands represent the progenitor allele lengths +/−5%. Lines within the gray bands represent unchanged bands. Lines outside of these ranges represent mutations. Those to the right of the rightmost band represent expansions, those to the left of the leftmost band represent contractions, and those in the middle represent unclassified mutations. Panel G shows the cumulative number of products observed with increasing expansion size. Expansion size was calculated as the percent increase in repeat length above the largest progenitor. An incremental increase is seen in Pms2^−/− versus Pms2^+/+ mice at all expansion sizes, but the increase in magnitude of the difference at higher expansion sizes points to the role of Pms2 in preferentially suppressing large expansions in the cerebrum.

Figure 3. Absence of Pms2 increases somatic expansion load of the (GAA·TTC)_n sequence in cerebellum.

Analysis of instability in cerebellum of Pms2^+/+ and Pms2^−/− mice. Progenitor allele lengths of 232, 199, 143, and 99 repeats for (A) Pms2^+/+, and 224, 195, 152, and 101 repeats for (B) Pms2^−/− are indicated by arrowheads. Mutation load and expansion load, calculated from analysis of ∼400 molecules per genotype, are shown in C and D, respectively. Panels E and F indicate the distribution of repeat lengths for all SP-PCR products of (E) Pms2^+/+ and (F) Pms2^−/−. These graphs are therefore an exact, combined representation of all blots analyzed for Pms2^+/+ and Pms2^−/− cerebrum. The number of products measured at each repeat length is on the Y-axis. Note that the actual frequency of products at the progenitor lengths may be slightly higher than the counted frequency, because a single band may represent PCR products amplified from multiple progenitor molecules. The gray bars represent the progenitor allele lengths +/−5%. Lines within the gray bands represent unchanged bands. Lines outside of these ranges represent mutations. Those to the right of the rightmost band represent expansions, those to the left of the leftmost band represent contractions, and those in the middle represent unclassified mutations. Products outside of these ranges were counted as mutations. Panel G shows the cumulative number of products observed with increasing expansion size. Expansion size was calculated as the percent increase in repeat length above the largest progenitor. An incremental increase is seen in Pms2^−/− versus Pms2^+/+ mice at all expansion sizes, but the increase in magnitude of the difference at higher expansion sizes points to the role of Pms2 in preferentially suppressing large expansions in the cerebellum.

Figure 4. Absence of Pms2 does not affect somatic instability of the (GAA·TTC)_n sequence in non-neuronal tissues.

Panels show the distribution of repeat lengths measured in (A,B) heart and (C,D) kidney from (A,C) Pms2^+/+ and (B,D) Pms2^−/− littermates. The number of products measured at each repeat length is on the Y-axis. Gray bars represent the length of the progenitor alleles, +/−5%. Products that fell outside of these ranges were considered mutations.

Figure 5. Absence of Pms2 results in larger expansions of the (GAA·TTC)_n sequence in cerebellum.

SP-PCR was performed using approximately 80 molecules per reaction to detect rare large expansions. Expansion sizes were calculated as the percent increase, of number of repeats, from the longest progenitor. Expansions of at least 10% were considered large expansions. The size distribution of large expansions was significantly different between Pms2^+/+ and Pms2^−/− cerebellum (P<0.05).

Figure 6. Formation of large expansions due to the absence of Pms2 during double strand break repair.

(A) Repair of a double strand break within the repeat sequence begins with 5′ to 3′ resection of the free ends in the broken molecule (thin solid line). (B) The unpaired end of a strand in the broken molecule anneals to a complementary sequence in the template molecule (thick solid line). (C) In the absence of Pms2, DNA synthesis extends the invading strand (thin arrow). In the presence of Pms2, extension is inhibited. The break may be repaired by non-homologous end joining. (D) Erroneous alignment of the newly synthesized DNA (thick dashed line) during reannealing leaves a gap that is filled by another round of synthesis (thin dashed line), resulting in an expansion. The template molecule remains unchanged.