The mismatch repair protein MSH2 is rate limiting for repeat expansion in a fragile X premutation mouse model

Abstract

Fragile X-associated tremor and ataxia syndrome, Fragile X-associated primary ovarian insufficiency, and Fragile X syndrome are Repeat Expansion Diseases caused by expansion of a CGG•CCG-repeat microsatellite in the 5 UTR of the FMR1 gene. To help understand the expansion mechanism responsible for these disorders, we have crossed mice containing∼147 CGG•CCG repeats in the endogenous murine Fmr1 gene with mice containing a null mutation in the gene encoding the mismatch repair protein MSH2. MSH2 mutations are associated with elevated levels of generalized microsatellite instability. However, we show here for the first time that in the FX mouse model, all maternally and paternally transmitted expansions require Msh2. Even the loss of one Msh2 allele reduced the intergenerational expansion frequency significantly. Msh2 is also required for all somatic expansions and loss of even one functional Msh2 allele reduced the extent of somatic expansion in some organs. Tissues with lower levels of MSH2 were more sensitive to the loss of a single Msh2 allele. This suggests that MSH2 is rate limiting for expansion in this mouse model and that MSH2 levels may be a key factor that accounts for tissue-specific differences in expansion risk.

Fig. 1. The frequency of expansions, contractions and unchanged alleles in the offspring of Msh2^+/+, Msh2^+/− and Msh2^−/− mice

The repeat length changes resulting from both maternal and paternal transmissions of the FX PM allele are shown. In all instances the parental repeat number was ~147. All differences between Msh2^+/+ and both Msh2^+/− and Msh2^−/− animals were significant at p<0.05 (Fisher’s exact test), except the difference in the percentage of unchanged alleles in Msh2^+/+ and Msh2^+/− females and the difference in the percentage of contractions in the offspring of Msh2^+/− and Msh2^−/− females. The number above each bar corresponds to the number of animals observed in that category.

Fig. 2. The effect of Msh2 heterozygosity on the repeat length changes seen on intergenerational transmission of the PM allele

Graph depicting the percentage of alleles with the indicated change in repeat length for each genotype for paternal (Panel A) and maternal (Panel B) transmissions. Msh2^+/+ and Msh2^+/− males showed a significant difference in the average number of repeats added when expansions were considered separately from contractions (7.7 vs 5.5 repeats; p=0.009 by t test and p=0.007 by Mann-Whitney). The difference between Msh2^+/+ and Msh2^+/− females was not significant (3.2 vs 3.0 repeats, p=0.762 by t test and p=0.889 by Mann-Whitney). The number of paternal contractions was too small to evaluate differences between genotypes and no statistically significant difference in the number of repeats lost was seen on maternal transmission from Msh2^+/+ and Msh2^+/− and Msh2^−/− animals (average repeat number lost was 2.5 and 2.9 for Msh2^+/+ and Msh2^−/− animals respectively, p=0.707 by t test and p=0.105 by Mann-Whitney).

Fig. 3. The effect of the absence of Msh2 on the PCR product profiles of male PM mice

A) Representative Genemapper profiles from tail DNA of 3 week old Msh2^+/+ and Msh2^−/− mice and from tail DNA of 1 day old Msh2^+/+ animals showing the PCR products corresponding to −5 repeats to +5 repeats from the major allele. Note that the 3 week old Msh2^+/+ mouse inherited a parental allele that was one repeat larger than the parental allele inherited by the other two mice. However, to facilitate comparison of the different peak heights the PCR profiles were aligned so that the major peaks coincide. B) The composite PCR profiles for 3 week old Msh2^+/+ and Msh2^−/− animals and newborn Msh2^+/+ pups were determined as described in the Materials and Methods. The error bars indicate the standard deviation. *different from Msh2^+/+ at p<0.05; **different from Msh2^+/+ at p<0.01.

Fig. 4. The effect of Msh2 gene dosage on somatic expansion in adults

A) GeneMapper profiles of 6 month old Msh2^+/+ and Msh2^−/− mice each with a starting allele having ~147 repeats. The dotted line indicates the size of the original allele based on the allele size in heart, an organ that shows little or no somatic instability (Lokanga, et al., 2013). A LIZ 1200 standard and a ROX 500 standard were used for the Msh2^+/+ and Msh2^−/− mice respectively, but the choice of standard does not affect the profile obtained. Tail 1 refers to the tail DNA sample taken at weaning (3 weeks of age). Tail 2 refers to the tail DNA sample taken at 6 months of age. B) The average somatic instability index (Lee, et al., 2010) was calculated for the indicated organs based on data from 6 Msh2^+/+ and 6 Msh2^−/− animals at 4 and 6 months of age. The error bars indicate the standard deviation. Tail 1 refers to the tail DNA sample taken at weaning (3 weeks of age). Tail 2 refers to the tail DNA sample taken at 6 months of age.

Fig. 5. Somatic expansion in Msh2^+/− mice

A) Superimposed GeneMapper profiles of the repeats in brain, liver, and testis from a 12-month-old Msh2^+/+ and a 12-month-old Msh2^+/− mouse, each with a starting allele having ~147 repeats. The dotted line indicates the size of the original allele based on the allele size in heart, an organ that shows little or no somatic instability (Lokanga, et al., 2013). A ROX 500 standard was used for both the Msh2^+/+ and Msh2^+/− mice. B) The average difference between number of repeats in the original allele (indicated by the dotted line) and the number of repeats in the major derivative allele (repeat number added) for the indicated organs based on data from 6 Msh2^+/+ and 6 Msh2^+/− animals at 12 months of age. The error bars indicate the standard deviation. Differences in the repeat number added to the original allele in Msh2^+/+ and Msh2^+/− mice was significant for brain and liver (p=0.0018 and 0.012 respectively). The differences the repeat number added in the testis of Msh2^+/+ and Msh2^+/− animals was not statistically significant.