MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.体细胞组织中的三核苷酸CAG/CTG重复序列（trinucleotide CAG/CTG repeats）扩增，被认为会在亨廷顿病（Huntington's disease, HD）或肌强直性营养不良患者的一生中推动疾病持续进展。携带扩增重复序列的个体间，重复序列不稳定性的差异范围跨度极大，这提示存在调控重复序列不稳定性的遗传修饰因子。携带扩增CAG/CTG重复序列的小鼠，其重复序列不稳定性水平因小鼠品系不同而存在显著差异，但截至目前，导致这些品系间差异的遗传修饰因子尚未被鉴定。我们的研究表明，在肝脏和纹状体（striatum）中，当R6/1亨廷顿病（HD）(CAG)~100转基因（transgene）处于同类系C57BL/6J（B6）遗传背景时，会发生偏向扩增的重复突变；而当该转基因处于同类系BALB/cByJ（CBy）遗传背景时，重复序列保持稳定。互合同类系小鼠实验证实，Msh3基因是导致重复序列不稳定性差异的决定性因素。在CBy遗传背景下携带纯合B6来源Msh3基因的同类系小鼠中，观察到了扩增偏向性；而在B6遗传背景下携带纯合CBy来源Msh3基因的同类系小鼠中，CAG序列片段则保持稳定。该CAG序列的稳定效果与Msh2基因遗传缺陷时的效果同样显著。B6和CBy来源的Msh3基因启动子序列完全一致，但编码区存在序列差异，且二者的蛋白质表达水平差异极为显著：B6来源的MSH3变异蛋白表达量极高，且与CAG扩增事件相关；而CBy来源的MSH3变异蛋白表达量几乎无法检测到，其对应的CAG序列保持稳定。与Msh3基因共享启动子、反向转录的二氢叶酸还原酶（DHFR）蛋白，在不同小鼠品系间未出现表达水平差异。综上，天然存在的MSH3蛋白质多态性是调控CAG重复序列不稳定性的修饰因子，其作用机制可能与MSH3蛋白稳定性的差异有关。已有研究证据表明，体细胞CAG不稳定性是疾病的修饰因子和预后预测因子，我们的研究结果支持这一假说：DNA修复基因（DNA repair genes）多态性相关的CAG不稳定性水平差异，可能对多种重复序列相关疾病具有预后提示意义。