Audrey E. Hendricks

Intergenerational and striatal CAG repeat instability in Huntington's disease knock-in mice involve different DNA repair genes

Modifying the length of the Huntingtons disease (HD) CAG repeat, the major determinant of age of disease onset, is an attractive therapeutic approach. To explore this we are investigating mechanisms of intergenerational and somatic HD CAG repeat instability. Here, we have crossed HD CAG knock-in mice onto backgrounds deficient in mismatch repair genes, Msh3 and Msh6, to discern the effects on CAG repeat size and disease pathogenesis. We find that different mechanisms predominate in inherited and somatic instability, with Msh6 protecting against intergenerational contractions and Msh3 required both for increasing CAG length and for enhancing an early disease phenotype in striatum. Therefore, attempts to decrease inherited repeat size may entail a full understanding of Msh6 complexes, while attempts to block the age-dependent increases in CAG size in striatal neurons and to slow the disease process will require a full elucidation of Msh3 complexes and their function in CAG repeat instability.

Somatic expansion of the Huntington's disease CAG repeat in the brain is associated with an earlier age of disease onset

The age of onset of Huntingtons disease (HD) is determined primarily by the length of the HD CAG repeat mutation, but is also influenced by other modifying factors. Delineating these modifiers is a critical step towards developing validated therapeutic targets in HD patients. The HD CAG repeat is somatically unstable, undergoing progressive length increases over time, particularly in brain regions that are the targets of neurodegeneration. Here, we have explored the hypothesis that somatic instability of the HD CAG repeat is itself a modifier of disease. Using small-pool PCR, we quantified somatic instability in the cortex region of the brain from a cohort of HD individuals exhibiting phenotypic extremes of young and old disease onset as predicted by the length of their constitutive HD CAG repeat lengths. After accounting for constitutive repeat length, somatic instability was found to be a significant predictor of onset age, with larger repeat length gains associated with earlier disease onset. These data are consistent with the hypothesis that somatic HD CAG repeat length expansions in target tissues contribute to the HD pathogenic process, and support pursuing factors that modify somatic instability as viable therapeutic targets.

KSR2 Mutations Are Associated with Obesity, Insulin Resistance, and Impaired Cellular Fuel Oxidation

Summary Kinase suppressor of Ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Targeted deletion of Ksr2 leads to obesity in mice, suggesting a role in energy homeostasis. We explored the role of KSR2 in humans by sequencing 2,101 individuals with severe early-onset obesity and 1,536 controls. We identified multiple rare variants in KSR2 that disrupt signaling through the Raf-MEK-ERK pathway and impair cellular fatty acid oxidation and glucose oxidation in transfected cells; effects that can be ameliorated by the commonly prescribed antidiabetic drug, metformin. Mutation carriers exhibit hyperphagia in childhood, low heart rate, reduced basal metabolic rate and severe insulin resistance. These data establish KSR2 as an important regulator of energy intake, energy expenditure, and substrate utilization in humans. Modulation of KSR2-mediated effects may represent a novel therapeutic strategy for obesity and type 2 diabetes. PaperFlick

Haplotypes and gene expression implicate the MAPT region for Parkinson disease The GenePD Study

Background: Microtubule-associated protein tau (MAPT) has been associated with several neurodegenerative disorders including forms of parkinsonism and Parkinson disease (PD). We evaluated the association of the MAPT region with PD in a large cohort of familial PD cases recruited by the GenePD Study. In addition, postmortem brain samples from patients with PD and neurologically normal controls were used to evaluate whether the expression of the 3-repeat and 4-repeat isoforms of MAPT, and neighboring genes Saitohin (STH) and KIAA1267, are altered in PD cerebellum. Methods: Twenty-one single-nucleotide polymorphisms (SNPs) in the region of MAPT on chromosome 17q21 were genotyped in the GenePD Study. Single SNPs and haplotypes, including the H1 haplotype, were evaluated for association to PD. Relative quantification of gene expression was performed using real-time RT-PCR. Results: After adjusting for multiple comparisons, SNP rs1800547 was significantly associated with PD affection. While the H1 haplotype was associated with a significantly increased risk for PD, a novel H1 subhaplotype was identified that predicted a greater increased risk for PD. The expression of 4-repeat MAPT, STH, and KIAA1267 was significantly increased in PD brains relative to controls. No difference in expression was observed for 3-repeat MAPT. Conclusions: This study supports a role for MAPT in the pathogenesis of familial and idiopathic Parkinson disease (PD). Interestingly, the results of the gene expression studies suggest that other genes in the vicinity of MAPT, specifically STH and KIAA1267, may also have a role in PD and suggest complex effects for the genes in this region on PD risk.

The Gly2019Ser Mutation in LRRK2 is not Fully Penetrant in Familial Parkinson's Disease: The GenePD Study

BackgroundWe report age-dependent penetrance estimates for leucine-rich repeat kinase 2 (LRRK2)-related Parkinsons disease (PD) in a large sample of familial PD. The most frequently seen LRRK2 mutation, Gly2019Ser (G2019S), is associated with approximately 5 to 6% of familial PD cases and 1 to 2% of idiopathic cases, making it the most common known genetic cause of PD. Studies of the penetrance of LRRK2 mutations have produced a wide range of estimates, possibly due to differences in study design and recruitment, including in particular differences between samples of familial PD versus sporadic PD.MethodsA sample, including 903 affected and 58 unaffected members from 509 families ascertained for having two or more PD-affected members, 126 randomly ascertained PD patients and 197 controls, was screened for five different LRRK2 mutations. Penetrance was estimated in families of LRRK2 carriers with consideration of the inherent bias towards increased penetrance in a familial sample.ResultsThirty-one out of 509 families with multiple cases of PD (6.1%) were found to have 58 LRRK2 mutation carriers (6.4%). Twenty-nine of the 31 families had G2019S mutations while two had R1441C mutations. No mutations were identified among controls or unaffected relatives of PD cases. Nine PD-affected relatives of G2019S carriers did not carry the LRRK2 mutation themselves. At the maximum observed age range of 90 to 94 years, the unbiased estimated penetrance was 67% for G2019S families, compared with a baseline PD risk of 17% seen in the non-LRRK2-related PD families.ConclusionLifetime penetrance of LRRK2 estimated in the unascertained relatives of multiplex PD families is greater than that reported in studies of sporadically ascertained LRRK2 cases, suggesting that inherited susceptibility factors may modify the penetrance of LRRK2 mutations. In addition, the presence of nine PD phenocopies in the LRRK2 families suggests that these susceptibility factors may also increase the risk of non-LRRK2-related PD. No differences in penetrance were found between men and women, suggesting that the factors that influence penetrance for LRRK2 carriers are independent of the factors which increase PD prevalence in men.

Estimating the probability of de novo HD cases from transmissions of expanded penetrant CAG alleles in the Huntington disease gene from male carriers of high normal alleles (27–35 CAG)

Huntington disease (HD) is a dominantly transmitted neurodegenerative disorder that arises from expansion of a CAG trinucleotide repeat on chromosome 4p16.3. CAG repeat allele lengths are defined as fully penetrant at ≥40, reduced penetrance at 36–39, high normal at 27–35, and normal at ≤26. Fathers, but not mothers, with high normal alleles are at risk of transmitting potentially penetrant HD alleles (≥36) to offspring. We estimated the conditional probability of an offspring inheriting an expanded penetrant allele given a father with a high normal allele by applying probability definitions and rules to estimates of HD incidence, paternal birth rate, frequency of de novo HD, and frequency of high normal alleles in the general population. The estimated probability that a male high normal allele carrier will have an offspring with an expanded penetrant allele ranges from 1/6,241 to 1/951. These estimates may be useful in genetic counseling for male high normal allele carriers.

Assessment of cortical and striatal involvement in 523 Huntington disease brains

Objective: To evaluate the relationship of striatal involvement in Huntington disease (HD) to involvement in other brain regions, CAG repeat size, onset age, and other factors. Methods: We examined patterns of neuropathologic involvement in 664 HD brains submitted to the Harvard Brain Tissue Resource Center. Brains with concomitant Alzheimer or Parkinson changes (n = 82), more than 20% missing data (n = 46), incomplete sample submission (n = 12), or CAG repeat less than 36 (n = 1) were excluded, leaving 523 cases. Standardized ratings from 0 (absent) to 4 (severe) of gross and microscopic involvement were performed for 50 regions. Cluster analysis reduced the data to 2 main measures of involvement: striatal and cortical. Results: The clusters were correlated with each other (r = 0.42) and with disease duration (striatal: r = 0.35; cortical: r = 0.31). The striatal cluster was correlated with HD repeat size (r = 0.50). The cortical cluster showed a stronger correlation with decreased brain weight (r = −0.52) than the striatal cluster (r = −0.33). The striatal cluster was correlated with younger death age (r = −0.31) and onset age (r = −0.46) while the cortical cluster was not (r = 0.09, r = −0.04, respectively). Conclusions: The 2 brain clusters had different relationships to the HD CAG repeat size, onset age, and brain weight, suggesting that neuropathologic involvement does not proceed in a strictly coupled fashion. The pattern and extent of involvement varies substantially from one brain to the next. These results suggest that regional involvement in HD brain is modified by factors which, if identified, may lend insight into novel routes to therapeutics.

Genetic associations with expression for genes implicated in GWAS studies for atherosclerotic cardiovascular disease and blood phenotypes

Genome-wide association studies (GWAS) have uncovered many genetic associations for cardiovascular disease (CVD). However, data are limited regarding causal genetic variants within implicated loci. We sought to identify regulatory variants (cis- and trans-eQTLs) affecting expression levels of 93 genes selected by their proximity to SNPs with significant associations in prior GWAS for CVD traits. Expression levels were measured by qRT-PCR in leukocytes from 1846 Framingham Heart Study participants. An additive genetic model was applied to 2.5 million imputed SNPs for each gene. Approximately 45% of genes (N = 38) harbored at least one cis-eSNP after a regional multiple-test adjustment. Applying a more rigorous significance threshold (P < 5 × 10(-8)), we found the expression level of 10 genes was significantly associated with more than one cis-eSNP. The top cis-eSNPs for 7 of these 10 genes exhibited moderate-to-strong association with ≥ 1 CVD clinical phenotypes. Several eSNPs or proxy SNPs (r(2) = 1) were replicated by other eQTL studies. After adjusting for the lead GWAS SNPs for the 10 genes, expression variances explained by top cis-eSNPs were attenuated markedly for LPL, FADS2 and C6orf184, suggesting a shared genetic basis for the GWAS and expression trait. A significant association between cis-eSNPs, gene expression and lipid levels was discovered for LPL and C6orf184. In conclusion, strong cis-acting variants are localized within nearly half of the GWAS loci studied, with particularly strong evidence for a regulatory role of the top GWAS SNP for expression of LPL, FADS2 and C6orf184.

Correction for multiple testing in a gene region

Several methods to correct for multiple testing within a gene region have been proposed. These methods are useful for candidate gene studies, and to fine map gene-regions from GWAs. The Bonferroni correction and permutation are common adjustments, but are overly conservative and computationally intensive, respectively. Other options include calculating the effective number of independent single-nucleotide polymorphisms (SNPs) or using theoretical approximations. Here, we compare a theoretical approximation based on extreme tail theory with four methods for calculating the effective number of independent SNPs. We evaluate the type-I error rates of these methods using single SNP association tests over 10 gene regions simulated using 1000 Genomes data. Overall, we find that the effective number of independent SNP method by Gao et al, as well as extreme tail theory produce type-I error rates at the or close to the chosen significance level. The type-I error rates for the other effective number of independent SNP methods vary by gene region characteristics. We find Gao et al and extreme tail theory to be efficient alternatives to more computationally intensive approaches to control for multiple testing in gene regions.

Progress in methods for rare variant association

Empirical studies and evolutionary theory support a role for rare variants in the etiology of complex traits. Given this motivation and increasing affordability of whole-exome and whole-genome sequencing, methods for rare variant association have been an active area of research for the past decade. Here, we provide a survey of the current literature and developments from the Genetics Analysis Workshop 19 (GAW19) Collapsing Rare Variants working group. In particular, we present the generalized linear regression framework and associated score statistic for the 2 major types of methods: burden and variance components methods. We further show that by simply modifying weights within these frameworks we arrive at many of the popular existing methods, for example, the cohort allelic sums test and sequence kernel association test. Meta-analysis techniques are also described. Next, we describe the 6 contributions from the GAW19 Collapsing Rare Variants working group. These included development of new methods, such as a retrospective likelihood for family data, a method using genomic structure to compare cases and controls, a haplotype-based meta-analysis, and a permutation-based method for combining different statistical tests. In addition, one contribution compared a mega-analysis of family-based and population-based data to meta-analysis. Finally, the power of existing family-based methods for binary traits was compared. We conclude with suggestions for open research questions.