Quantile-quantile plot.

<p>Expected p-values assuming a uniform distribution (X-axis; expected −log10(p-value)) were compared to observed p-values (Y-axis; observed −log10(p-value)) in the continuous analysis (A) and dichotomous analysis (B) to evaluate the levels of inflation in test statistics. Expected p-values were calculated based on the uniform distribution assuming each test was independent of others.</p

Comparison of test statistics.

<p>(A) Pearson’s correlation coefficients in the continuous analysis were compared to fold-changes in the dichotomous analysis to evaluate overall similarity in direction of changes between two analytical methods. The number in each quadrant represents the percentage of probes relative to all probes analyzed. (B) Significance levels in the continuous analysis (−log10(Pearson’s test p-value); X-axis) were compared to those in the dichotomous analysis (−log10(Student’s t-test p-value); Y-axis) in order to evaluate overall similarity in significance between two analytical methods.</p

HD CAGnome.

<p>HD CAGnome is accessible at <a href="http://chgr.partners.org/cgi-bin/cagnome.cgi" target="_blank">http://chgr.partners.org/cgi-bin/cagnome.cgi</a>.</p

Overall characteristics of continuous and dichotomous analysis results.

<p>Pearson’s correlation test was performed for continuous analysis. The distribution of correlation coefficient (A) and correlation coefficient vs. significance (i.e., −log10(p-value)) (B) were plotted. For dichotomous analysis, Student’s-t test was used, and the distribution of fold-changes (C) and fold-change vs. significance (−log10(p-value)) (D) were plotted.</p

Significant probes in continuous and dichotomous analysis.

<p>Pearson’s correlation test and Student’s-t test were used for continuous and dichotomous analysis, respectively. (A) From left to right, numbers in Venn diagram indicate the number of probes significant only in continuous analysis, probes significant in both continuous and dichotomous analysis, and probes significant only in dichotomous analysis by p-value cut-off of 0.01. We did not observe differences between shared genes and specific genes in terms of p-values, correlation coefficients, and fold-changes. (B) Probes significant in both analyses (154 probes) were further categorized based on the correlation coefficient and fold-change.</p

Efficiency of dichotomous analysis in capturing significantly correlated genes.

<p>From 107 samples, we randomly selected equal numbers (n = 3 to 41) of HD and controls to perform dichotomous analysis, repeating 1,000 times for each sample size, and compared to continuous CAG length analysis, as described in the text and methods, plotting the resulting percentages in scatter density plots. (A) Variation in the percentage of CAG-length correlated significant differences from continuous analysis that are detected by dichotomous analysis vs. sample size. (B) Variation in the percentage of differences judged significant by dichotomous analysis that are not CAG-length correlated by continuous analysis vs. sample size. Red lines represent linear regression models describing the relationship between sample size and the corresponding performance metric.</p

Conditional deletion of the <i>floxed Msh2</i> allele in the striatum. A.

<p>Genotyping for the conditional <i>Msh2</i> allele in genomic DNA extracted from striatum of <i>Msh2</i>+/+, <i>Msh2flox</i>/+, <i>Msh2flox</i>/+ <i>D9-Cre</i> and <i>Msh2flox</i>/flox <i>D9-Cre</i> mice. The deleted (Δ) <i>Msh2</i> allele is present only in mice harboring both the <i>Msh2flox</i> allele and the <i>D9-Cre</i> transgene. <b>B.</b> Genotyping for the conditional <i>Msh2</i> allele in genomic DNA extracted from five different tissues from a <i>Msh2flox</i>/+ <i>D9-Cre</i> mouse shows that the deletion is specific for the striatum. Mice were six weeks of age. <i>flox</i>: <i>Msh2</i> allele flanked by <i>loxP</i> sites; Δ:deleted <i>Msh2</i> allele; wt: wild-type <i>Msh2</i> allele.</p

Deletion of <i>Msh2</i> in medium-spiny striatal neurons eliminates the majority of striatal <i>HTT</i> CAG expansions.

<p>GeneMapper traces of PCR-amplified <i>HTT</i> CAG repeats from striatum, cortex, liver and tail DNA of representative five-month <i>HdhQ111</i>/+ mice (<b>A</b>) or from striatum and tail of representative ten month <i>HdhQ111</i>/+ mice (<b>C</b>) with <i>Msh2</i>+/+, <i>Msh2</i>+/−, <i>Msh2Δ</i>/<i>Δ</i>, <i>Msh2Δ</i>/− and <i>Msh2</i>−/− genotypes. Constitutive CAG repeat lengths, as determined in tail DNA, are indicated. Instability indices were quantified from GeneMapper traces of PCR-amplified <i>HTT</i> CAG repeats from five-month striatum, cortex and liver (<b>B</b>) and ten-month striatum (<b>D</b>) of <i>HdhQ111</i>/+ mice with <i>Msh2</i>+/+, <i>Msh2</i>+/−, <i>Msh2Δ</i>/<i>Δ</i>, <i>Msh2Δ</i>/− and <i>Msh2</i>−/−genotypes. Five-month mice: <i>Msh2</i>+/+ (n = 6, CAG 113, 118, 119, 121, 123, 125), <i>Msh2</i>+/− (n = 4, CAG 114, 114, 120, 123), <i>Msh2Δ</i>/<i>Δ</i>(n = 5, CAG 113, 121, 121, 126, 129), <i>Msh2Δ</i>/−(n = 7, CAG 113, 121, 121, 122, 125, 125, 133) and <i>Msh2</i>−/− (n = 3, CAG 112, 120, 123). Ten-month mice: <i>Msh2</i>+/+ (n = 6, CAG 118, 121, 121, 123, 126, 134), <i>Msh2</i>+/− (n = 4, CAG 116, 118, 123, 131), <i>Msh2Δ</i>/<i>Δ</i> (n = 1, CAG 133), <i>Msh2Δ</i>/− (n = 7, CAG 115, 115, 117, 120, 121, 122, 123) and <i>Msh2</i>−/− (n = 1, CAG 132). Bars represent mean ± S.D. *** p<0.0001, * p<0.05 (Student’s t-test).</p

Deletion of Msh2 in medium-spiny neurons delays nuclear huntingtin phenotypes. A, B.

<p>Nuclear mutant huntingtin immunostaining is decreased in the striata of five-month old <i>HdhQ111</i>/+ mice with deletion of <i>Msh2</i> in MSNs. <b>A.</b> Fluorescent micrographs of striata double-stained with anti-huntingtin mAb5374 and anti-histone H3 antibodies for three CAG repeat length-matched mice (<i>Msh2</i>+/+ CAG 113, <i>Msh2Δ</i>/<i>Δ</i> CAG 112, <i>Msh2−/−</i> CAG 113). <b>B.</b> Box plot showing upper and lower quartiles, median and range for the normalized mAb5374 immunostaining intensity (total mAb5374 staining intensity normalized to the number of H3-positive nuclei). Outlier (circle) is defined by a standard interquartile method and is included in the analysis. Multiple regression analysis was used to determine the effect of <i>Msh2</i> genotype on mAb5374 staining using normalized mAb5374 intensity (continuous variable) as a dependent variable and <i>Msh2</i> genotype (discrete variable), constitutive CAG length (continuous variable) and position (medial versus lateral, discrete variable) as independent variables. Both constitutive CAG length (P<0.05) and medial versus lateral position (P<0.001) were significantly associated with normalized mAb5374 intensity. Asterisks above the bars indicate a significant difference from <i>Msh2</i>+/+ at a p-value cut-off of p<0.05(*), p<0.01 (**), p<0.001 (***) in the regression analysis. <i>Msh2Δ</i>/− was not significantly different from <i>Msh2</i>+/− (p = 0.18). The five-month mice used in the quantitative analysis are as follows: <i>Msh2</i>+/+ (n = 6, CAG 113, 118, 119, 121, 123, 125), <i>Msh2</i>+/− (n = 4, CAG 114, 114, 120, 123), <i>Msh2Δ</i>/<i>Δ</i> (n = 5, CAG 113, 121, 121, 126, 129), <i>Msh2Δ</i>/− (n = 7, CAG 113, 121, 121, 122, 125, 125, 133) and <i>Msh2</i>−/− (n = 3, CAG 112, 120, 123). Note that the relatively “weak” effect of the <i>Msh2</i>−/− genotype likely reflects the small number of mice of this genotype and hence the least accurate estimate of the relationship of mAb5374 intensity to CAG length in the regression analysis. <b>C, D.</b> Intranuclear inclusions are decreased in the striata of ten-month old <i>HdhQ111</i>/+ mice with deletion of <i>Msh2</i> in MSNs. <b>C.</b> Fluorescent micrographs of striata stained with mAb5374 from mice with <i>Msh2</i>+/+ (CAG 121), <i>Msh2</i>+/− (CAG 123), <i>Msh2Δ</i>/<i>Δ</i> (CAG 133), <i>Msh2Δ</i>/− (CAG 123) and <i>Msh2</i>−/− (CAG 132) genotypes. <b>D.</b> Quantification of the percentage of cells containing an inclusion (more than one inclusion per cell was rarely observed). The total number of cells was determined by co-staining with histone H3 (not shown). The ten-month mice used in the quantitative analysis are as follows: <i>Msh2</i>+/+ (n = 6, CAG 118, 121, 121, 123, 126, 134), <i>Msh2</i>+/− (n = 4, CAG 116, 118, 123, 131), <i>Msh2Δ</i>/<i>Δ</i> (n = 1, CAG 133), <i>Msh2Δ</i>/− (n = 7, CAG 115, 115, 117, 120, 121, 122, 123) and <i>Msh2</i>−/− (n = 1, CAG 132). Bars represent mean ±S.D.</p

Mismatch Repair Genes <i>Mlh1</i> and <i>Mlh3</i> Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches

<div><p>The Huntington's disease gene (<i>HTT</i>) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease <i>Hdh^Q111</i> mice exhibit higher levels of somatic <i>HTT</i> CAG expansion on a C57BL/6 genetic background (B6.<i>Hdh^Q111</i>) than on a 129 background (129.<i>Hdh^Q111</i>). Linkage mapping in (B6x129).<i>Hdh^Q111</i> F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene <i>Mlh1</i> as the most likely candidate modifier. Crossing B6.<i>Hdh^Q111</i> mice onto an <i>Mlh1</i> null background demonstrated that <i>Mlh1</i> is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. <i>Hdh^Q111</i> somatic expansion was also abolished in mice deficient in the <i>Mlh3</i> gene, implicating MutLγ (MLH1–MLH3) complex as a key driver of somatic expansion. Strikingly, <i>Mlh1</i> and <i>Mlh3</i> genes encoding MMR effector proteins were as critical to somatic expansion as <i>Msh2</i> and <i>Msh3</i> genes encoding DNA mismatch recognition complex MutSβ (MSH2–MSH3). The <i>Mlh1</i> locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify <i>Mlh1</i> and <i>Mlh3</i> as novel critical genetic modifiers of <i>HTT</i> CAG instability, point to <i>Mlh1</i> genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.</p></div