Host genetic variation guides hepacivirus clearance, chronicity, and liver fibrosis in mice.

BACKGROUND AIMS: Human genetic variation is thought to guide the outcome of HCV infection, but model systems within which to dissect these host genetic mechanisms are limited. Norway rat hepacivirus, closely related to HCV, causes chronic liver infection in rats but causes acute self-limiting hepatitis in typical strains of laboratory mice, which resolves in 2 weeks. The Collaborative Cross (CC) is a robust mouse genetics resource comprised of a panel of recombinant inbred strains, which model the complexity of the human genome and provide a system within which to understand diseases driven by complex allelic variation. APPROACH RESULTS: We infected a panel of CC strains with Norway rat hepacivirus and identified several that failed to clear the virus after 4 weeks. Strains displayed an array of virologic phenotypes ranging from delayed clearance (CC046) to chronicity (CC071, CC080) with viremia for at least 10 months. Body weight loss, hepatocyte infection frequency, viral evolution, T-cell recruitment to the liver, liver inflammation, and the capacity to develop liver fibrosis varied among infected CC strains. CONCLUSIONS: These models recapitulate many aspects of HCV infection in humans and demonstrate that host genetic variation affects a multitude of viruses and host phenotypes. These models can be used to better understand the molecular mechanisms that drive hepacivirus clearance and chronicity, the virus and host interactions that promote chronic disease manifestations like liver fibrosis, therapeutic and vaccine performance, and how these factors are affected by host genetic variation.

Forward genetic screen of homeostatic antibody levels in the Collaborative Cross identifies MBD1 as a novel regulator of B cell homeostasis.

Variation in immune homeostasis, the state in which the immune system is maintained in the absence of stimulation, is highly variable across populations. This variation is attributed to both genetic and environmental factors. However, the identity and function of specific regulators have been difficult to identify in humans. We evaluated homeostatic antibody levels in the serum of the Collaborative Cross (CC) mouse genetic reference population. We found heritable variation in all antibody isotypes and subtypes measured. We identified 4 quantitative trait loci (QTL) associated with 3 IgG subtypes: IgG1, IgG2b, and IgG2c. While 3 of these QTL map to genome regions of known immunological significance (major histocompatibility and immunoglobulin heavy chain locus), Qih1 (associated with variation in IgG1) mapped to a novel locus on Chromosome 18. We further associated this locus with B cell proportions in the spleen and identify Methyl-CpG binding domain protein 1 under this locus as a novel regulator of homeostatic IgG1 levels in the serum and marginal zone B cells (MZB) in the spleen, consistent with a role in MZB differentiation to antibody secreting cells.

A mutation in Themis contributes to anaphylaxis severity following oral peanut challenge in CC027 mice.

BACKGROUND: The development of peanut allergy is due to a combination of genetic and environmental factors, although specific genes have proven difficult to identify. Previously, we reported that peanut-sensitized Collaborative Cross strain CC027/GeniUnc (CC027) mice develop anaphylaxis upon oral challenge to peanut, in contrast to C3H/HeJ (C3H) mice. OBJECTIVE: This study aimed to determine the genetic basis of orally induced anaphylaxis to peanut in CC027 mice. METHODS: A genetic mapping population between CC027 and C3H mice was designed to identify the genetic factors that drive oral anaphylaxis. A total of 356 CC027xC3H backcrossed mice were generated, sensitized to peanut, then challenged to peanut by oral gavage. Anaphylaxis and peanut-specific IgE were quantified for all mice. T-cell phenotyping was conducted on CC027 mice and 5 additional Collaborative Cross strains. RESULTS: Anaphylaxis to peanut was absent in 77% of backcrossed mice, with 19% showing moderate anaphylaxis and 4% having severe anaphylaxis. There were 8 genetic loci associated with variation in response to peanut challenge-6 associated with anaphylaxis (temperature decrease) and 2 associated with peanut-specific IgE levels. There were 2 major loci that impacted multiple aspects of the severity of acute anaphylaxis, at which the CC027 allele was associated with worse outcome. At one of these loci, CC027 has a private genetic variant in the Themis gene. Consistent with described functions of Themis, we found that CC027 mice have more immature T cells with fewer CD8+, CD4+, and CD4+CD25+CD127- regulatory T cells. CONCLUSIONS: Our results demonstrate a key role for Themis in the orally reactive CC027 mouse model of peanut allergy.

Genetic basis of thermal nociceptive sensitivity and brain weight in a BALB/c reduced complexity cross.

Thermal nociception involves the transmission of temperature-related noxious information from the periphery to the CNS and is a heritable trait that could predict transition to persistent pain. Rodent forward genetics complement human studies by controlling genetic complexity and environmental factors, analysis of end point tissue, and validation of variants on appropriate genetic backgrounds. Reduced complexity crosses between nearly identical inbred substrains with robust trait differences can greatly facilitate unbiased discovery of novel genes and variants. We found BALB/cByJ mice showed enhanced sensitivity on the 53.5°C hot plate and mechanical stimulation in the von Frey test compared to BALB/cJ mice and replicated decreased gross brain weight in BALB/cByJ versus BALB/cJ. We then identified a quantitative trait locus (QTL) on chromosome 13 for hot plate sensitivity (LOD = 10.7; p < 0.001; peak = 56 Mb) and a QTL for brain weight on chromosome 5 (LOD = 8.7; p < 0.001). Expression QTL mapping of brain tissues identified H2afy (56.07 Mb) as the top transcript with the strongest association at the hot plate locus (FDR = 0.0002) and spliceome analysis identified differential exon usage within H2afy associated with the same locus. Whole brain proteomics further supported decreased H2AFY expression could underlie enhanced hot plate sensitivity, and identified ACADS as a candidate for reduced brain weight. To summarize, a BALB/c reduced complexity cross combined with multiple-omics approaches facilitated identification of candidate genes underlying thermal nociception and brain weight. These substrains provide a powerful, reciprocal platform for future validation of candidate variants.

Zhx2 Is a Candidate Gene Underlying Oxymorphone Metabolite Brain Concentration Associated with State-Dependent Oxycodone Reward.

Understanding the pharmacogenomics of opioid metabolism and behavior is vital to therapeutic success, as mutations can dramatically alter therapeutic efficacy and addiction liability. We found robust, sex-dependent BALB/c substrain differences in oxycodone behaviors and whole brain concentration of oxycodone metabolites. BALB/cJ females showed robust state-dependent oxycodone reward learning as measured via conditioned place preference when compared with the closely related BALB/cByJ substrain. Accordingly, BALB/cJ females also showed a robust increase in brain concentration of the inactive metabolite noroxycodone and the active metabolite oxymorphone compared with BALB/cByJ mice. Oxymorphone is a highly potent, full agonist at the mu opioid receptor that could enhance drug-induced interoception and state-dependent oxycodone reward learning. Quantitative trait locus (QTL) mapping in a BALB/c F2 reduced complexity cross revealed one major QTL on chromosome 15 underlying brain oxymorphone concentration that explained 32% of the female variance. BALB/cJ and BALB/cByJ differ by fewer than 10,000 variants, which can greatly facilitate candidate gene/variant identification. Hippocampal and striatal cis-expression QTL (eQTL) and exon-level eQTL analysis identified Zhx2, a candidate gene coding for a transcriptional repressor with a private BALB/cJ retroviral insertion that reduces Zhx2 expression and sex-dependent dysregulation of cytochrome P450 enzymes. Whole brain proteomics corroborated the Zhx2 eQTL and identified upregulated CYP2D11 that could increase brain oxymorphone in BALB/cJ females. To summarize, Zhx2 is a highly promising candidate gene underlying brain oxycodone metabolite levels. Future studies will validate Zhx2 and its site of action using reciprocal gene editing and tissue-specific viral manipulations in BALB/c substrains. SIGNIFICANCE STATEMENT: Our findings show that genetic variation can result in sex-specific alterations in whole brain concentration of a bioactive opioid metabolite after oxycodone administration, reinforcing the need for sex as a biological factor in pharmacogenomic studies. The cooccurrence of female-specific increased oxymorphone and state-dependent reward learning suggests that this minor yet potent and efficacious metabolite of oxycodone could increase opioid interoception and drug-cue associative learning of opioid reward, which has implications for cue-induced relapse of drug-seeking behavior and for precision pharmacogenetics.

The genetic contributions of SNCA and LRRK2 genes to Lewy Body pathology in Alzheimer's disease.

The molecular genetic basis that leads to Lewy Body (LB) pathology in 15-20% of Alzheimer disease cases (LBV/AD) was largely unknown. Alpha-synuclein (SNCA) and Leucine-rich repeat kinase2 (LRRK2) have been implicated in the pathogenesis of Parkinson's disease (PD), the prototype of LB spectrum disorders. We tested the association of SNCA variants with LB pathology in AD. We then stratified the SNCA association analyses by LRRK2 genotype. We also investigated the expression regulation of SNCA and LRRK2 in relation to LB pathology. We evaluated the differences in SNCA-mRNA and LRRK2-mRNA levels as a function of LB pathology in the temporal cortex (TC) from autopsy-confirmed LBV/AD cases and AD controls. We further investigated the cis-effect of the LB pathology-associated genetic variants within the SNCA and LRRK2 loci on the mRNA expression of these genes. SNCA SNPs rs3857059 and rs2583988 showed significant associations with increased risk for LB pathology. When the analyses were stratified by LRRK2-rs1491923 genotype, the associations became stronger for both SNPs and an association was also observed with rs2619363. Expression analysis demonstrated that SNCA- and LRRK2-mRNA levels were significantly higher in TC from LBV/AD brains compared with AD controls. Furthermore, SNCA-mRNA expression level in the TC was associated with rs3857059; homozygotes for the minor allele showed significant higher expression. LRRK2-transcript levels were increased in carriers of rs1491923 minor allele. Our findings demonstrated that SNCA contributes to LB pathology in AD patients, possibly via interaction with LRRK2, and suggested that expression regulation of these genes may be the molecular basis underlying the observed LB associations.

A cytosine-thymine (CT)-rich haplotype in intron 4 of SNCA confers risk for Lewy body pathology in Alzheimer's disease and affects SNCA expression.

INTRODUCTION: We recently showed that tagging single-nucleotide polymorphisms across the SNCA locus were significantly associated with increased risk for Lewy body (LB) pathology in Alzheimer's disease (AD) cases. However, the actual genetic variant(s) that underlie the observed associations remain elusive. METHODS: We used a bioinformatics algorithm to catalog structural variants in a region of SNCA intron 4, followed by phased sequencing. We performed a genetic association analysis in autopsy series of LB variant of Alzheimer's disease (LBV/AD) cases compared with AD-only controls. We investigated the biological functions by expression analysis using temporal-cortex samples. RESULTS: We identified four distinct haplotypes within a highly polymorphic low-complexity cytosine-thymine (CT)-rich region. We showed that a specific haplotype conferred risk to develop LBV/AD. We demonstrated that the CT-rich site acts as an enhancer element, where the risk haplotype was significantly associated with elevated levels of SNCA messenger RNA. DISCUSSION: We have discovered a novel haplotype in a CT-rich region in SNCA that contributes to LB pathology in AD patients, possibly via cis-regulation of the gene expression.

The cis-regulatory effect of an Alzheimer's disease-associated poly-T locus on expression of TOMM40 and apolipoprotein E genes.

BACKGROUND: We investigated the genomic region spanning the Translocase of the Outer Mitochondrial Membrane 40-kD (TOMM40) and Apolipoprotein E (APOE) genes, that has been associated with the risk and age of onset of late-onset Alzheimer's disease (LOAD) to determine whether a highly polymorphic, intronic poly-T within this region (rs10524523; hereafter, 523) affects expression of the APOE and TOMM40 genes. Alleles of this locus are classified as S, short; L, long; and VL, very long based on the number of T residues. METHODS: We evaluated differences in APOE messenger RNA (mRNA) and TOMM40 mRNA levels as a function of the 523 genotype in two brain regions from APOE ε3/ε3 white autopsy-confirmed LOAD cases and normal controls. We further investigated the effect of the 523 locus in its native genomic context using a luciferase expression system. RESULTS: The expression of both genes was significantly increased with disease. Mean expression of APOE and TOMM40 mRNA levels were higher in VL homozygotes compared with S homozygotes in the temporal and occipital cortexes from normal and LOAD cases. Results of a luciferase reporter system were consistent with the human brain mRNA analysis; the 523 VL poly-T resulted in significantly higher expression than the S poly-T. Although the effect of poly-T length on reporter expression was the same in HepG2 hepatoma and SH-SY5Y neuroblastoma cells, the magnitude of the effect was greater in the neuroblastoma than in the hepatoma cells, which implies tissue-specific modulation of the 523 poly-T. CONCLUSIONS: These results suggest that the 523 locus may contribute to LOAD susceptibility by modulating the expression of TOMM40 and/or APOE transcription.

U-CAN-seq: A Universal Competition Assay by Nanopore Sequencing.

RNA viruses quickly evolve subtle genotypic changes that can have major impacts on viral fitness and host range, with potential consequences for human health. It is therefore important to understand the evolutionary fitness of novel viral variants relative to well-studied genotypes of epidemic viruses. Competition assays are an effective and rigorous system with which to assess the relative fitness of viral genotypes. However, it is challenging to quickly and cheaply distinguish and quantify fitness differences between very similar viral genotypes. Here, we describe a protocol for using reverse transcription PCR in combination with commercial nanopore sequencing services to perform competition assays on untagged RNA viruses. Our assay, called the Universal Competition Assay by Nanopore Sequencing (U-CAN-seq), is relatively cheap and highly sensitive. We used a well-studied N24A mutation in the chikungunya virus (CHIKV) nsp3 gene to confirm that we could detect a competitive disadvantage using U-CAN-seq. We also used this approach to show that mutations to the CHIKV 5' conserved sequence element that disrupt sequence but not structure did not affect the fitness of CHIKV. However, similar mutations to an adjacent CHIKV stem loop (SL3) did cause a fitness disadvantage compared to wild-type CHIKV, suggesting that structure-independent, primary sequence determinants in this loop play an important role in CHIKV biology. Our novel findings illustrate the utility of the U-CAN-seq competition assay.

Sarbecovirus disease susceptibility is conserved across viral and host models.

Coronaviruses have caused three severe epidemics since the start of the 21st century: SARS, MERS and COVID-19. The severity of the ongoing COVID-19 pandemic and increasing likelihood of future coronavirus outbreaks motivates greater understanding of factors leading to severe coronavirus disease. We screened ten strains from the Collaborative Cross mouse genetic reference panel and identified strains CC006/TauUnc (CC006) and CC044/Unc (CC044) as coronavirus-susceptible and resistant, respectively, as indicated by variable weight loss and lung congestion scores four days post-infection. We generated a genetic mapping population of 755 CC006xCC044 F2 mice and exposed the mice to one of three genetically distinct mouse-adapted coronaviruses: clade 1a SARS-CoV MA15 (n=391), clade 1b SARS-CoV-2 MA10 (n=274), and clade 2 HKU3-CoV MA (n=90). Quantitative trait loci (QTL) mapping in SARS-CoV MA15- and SARS-CoV-2 MA10-infected F2 mice identified genetic loci associated with disease severity. Specifically, we identified seven loci associated with variation in outcome following infection with either virus, including one, HrS43, that is present in both groups. Three of these QTL, including HrS43, were also associated with HKU3-CoV MA outcome. HrS43 overlaps with a QTL previously reported by our lab that is associated with SARS-CoV MA15 outcome in CC011xCC074 F2 mice and is also syntenic with a human chromosomal region associated with severe COVID-19 outcomes in humans GWAS. The results reported here provide: (a) additional support for the involvement of this locus in SARS-CoV MA15 infection, (b) the first conclusive evidence that this locus is associated with susceptibility across the Sarbecovirus subgenus, and (c) demonstration of the relevance of mouse models in the study of coronavirus disease susceptibility in humans.

Adjuvant-dependent impact of inactivated SARS-CoV-2 vaccines during heterologous infection by a SARS-related coronavirus.

Whole virus-based inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous coronavirus infection, the emergence of novel variants and the presence of large zoonotic reservoirs harboring novel heterologous coronaviruses provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes like vaccine-associated enhanced respiratory disease. Here, we use a female mouse model of coronavirus disease to evaluate inactivated vaccine performance against either homologous challenge with SARS-CoV-2 or heterologous challenge with a bat-derived coronavirus that represents a potential emerging disease threat. We show that inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide can cause enhanced respiratory disease during heterologous infection, while use of an alternative adjuvant does not drive disease and promotes heterologous viral clearance. In this work, we highlight the impact of adjuvant selection on inactivated vaccine safety and efficacy against heterologous coronavirus infection.

Genetic loci regulate Sarbecovirus pathogenesis: A comparison across mice and humans.

Coronavirus (CoV) cause considerable morbidity and mortality in humans and other mammals, as evidenced by the emergence of Severe Acute Respiratory CoV (SARS-CoV) in 2003, Middle East Respiratory CoV (MERS-CoV) in 2012, and SARS-CoV-2 in 2019. Although poorly characterized, natural genetic variation in human and other mammals modulate virus pathogenesis, as reflected by the spectrum of clinical outcomes ranging from asymptomatic infections to lethal disease. Using multiple human epidemic and zoonotic Sarbecoviruses, coupled with murine Collaborative Cross genetic reference populations, we identify several dozen quantitative trait loci that regulate SARS-like group-2B CoV pathogenesis and replication. Under a Chr4 QTL, we deleted a candidate interferon stimulated gene, Trim14 which resulted in enhanced SARS-CoV titers and clinical disease, suggesting an antiviral role during infection. Importantly, about 60 % of the murine QTL encode susceptibility genes identified as priority candidates from human genome-wide association studies (GWAS) studies after SARS-CoV-2 infection, suggesting that similar selective forces have targeted analogous genes and pathways to regulate Sarbecovirus disease across diverse mammalian hosts. These studies provide an experimental platform in rodents to investigate the molecular-genetic mechanisms by which potential cross mammalian susceptibility loci and genes regulate type-specific and cross-SARS-like group 2B CoV replication, immunity, and pathogenesis in rodent models. Our study also provides a paradigm for identifying susceptibility loci for other highly heterogeneous and virulent viruses that sporadically emerge from zoonotic reservoirs to plague human and animal populations.

A mutation in Themis contributes to peanut-induced oral anaphylaxis in CC027 mice.

Background: The development of peanut allergy is due to a combination of genetic and environmental factors, although specific genes have proven difficult to identify. Previously, we reported that peanut-sensitized CC027/GeniUnc (CC027) mice develop anaphylaxis upon oral challenge to peanut, unlike C3H/HeJ (C3H) mice. Objective: To determine the genetic basis of orally-induced anaphylaxis to peanut in CC027 mice. Methods: A genetic mapping population between CC027 and C3H mice was designed to identify the genetic factors that drive oral anaphylaxis. A total of 356 CC027xC3H backcrossed mice were generated, sensitized to peanut, then challenged to peanut by oral gavage. Anaphylaxis and peanut-specific IgE were quantified for all mice. T-cell phenotyping was conducted on CC027 and five additional CC strains. Results: Anaphylaxis to peanut was absent in 77% of backcrossed mice, with 19% showing moderate anaphylaxis, and 4% having severe anaphylaxis. A total of eight genetic loci were associated with variation in response to peanut challenge, six associated with anaphylaxis (temperature decrease) and two associated with peanut-specific IgE levels. There were two major loci that impacted multiple aspects of the severity of acute anaphylaxis, at which the CC027 allele was associated with worse outcome. At one of these loci, CC027 has a private genetic variant in the Themis (thymocyte-expressed molecule involved in selection) gene. Consistent with Themis' described functions, we found that CC027 have more immature T cells with fewer CD8+, CD4+, and CD4+CD25+CD127- regulatory T cells. Conclusion: Our results demonstrate a key role for Themis in the orally-reactive CC027 mouse model of peanut allergy.

Adjuvant-dependent effects on the safety and efficacy of inactivated SARS-CoV-2 vaccines during heterologous infection by a SARS-related coronavirus.

Inactivated whole virus SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide (Alum) are among the most widely used COVID-19 vaccines globally and have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous virus infection in healthy recipients, the emergence of novel SARS-CoV-2 variants and the presence of large zoonotic reservoirs provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes including vaccine-associated enhanced respiratory disease (VAERD). To evaluate this possibility, we tested the performance of an inactivated SARS-CoV-2 vaccine (iCoV2) in combination with Alum against either homologous or heterologous coronavirus challenge in a mouse model of coronavirus-induced pulmonary disease. Consistent with human results, iCoV2 + Alum protected against homologous challenge. However, challenge with a heterologous SARS-related coronavirus, Rs-SHC014-CoV (SHC014), up to at least 10 months post-vaccination, resulted in VAERD in iCoV2 + Alum-vaccinated animals, characterized by pulmonary eosinophilic infiltrates, enhanced pulmonary pathology, delayed viral clearance, and decreased pulmonary function. In contrast, vaccination with iCoV2 in combination with an alternative adjuvant (RIBI) did not induce VAERD and promoted enhanced SHC014 clearance. Further characterization of iCoV2 + Alum-induced immunity suggested that CD4+ T cells were a major driver of VAERD, and these responses were partially reversed by re-boosting with recombinant Spike protein + RIBI adjuvant. These results highlight potential risks associated with vaccine breakthrough in recipients of Alum-adjuvanted inactivated vaccines and provide important insights into factors affecting both the safety and efficacy of coronavirus vaccines in the face of heterologous virus infections.

Pleiotropy and allelic heterogeneity in the TOMM40-APOE genomic region related to clinical and metabolic features of hepatitis C infection.

Hepatitis C virus (HCV) modulates host lipid metabolism as part of its lifecycle and is dependent upon VLDL for co-assembly and secretion. HCV dyslipidemia is associated with steatosis, insulin resistance, IL28B genotype and disease progression. Apolipoprotein E (ApoE) is an important lipid transport protein, a key constituent of VLDL, and is involved in immunomodulation. Our aims were to determine the role of APOE regional polymorphisms on host lipids, IL28B genotype and disease severity in chronic HCV (CHC) patients. The study cohort included 732 CHC patients with available DNA for genotype determination of four polymorphisms in the chromosome 19 region that encompasses the TOMM40, APOE and APOC1 genes. Serum lipid analysis and apolipoproteins levels were measured using an immunoturbidimetric assay. APOE rs7412 polymorphism (capturing the ε2 isoform) was significantly associated with serum ApoE levels in both Caucasians and African-American patients (p = 2.3 × 10(-11)) and explained 7 % of variance in serum ApoE. Among IL28B-CC patients (n = 196), the rs429358 (defines ε4 isoform) and TOMM40 '523' S polymorphisms were associated with 12 % of variance in ApoB levels. Patients homozygous for the APOE ε3 isoform had a greater than twofold increased odds of F2-F4 fibrosis (p = 1.8 × 10(-5)), independent of serum lipid and lipoprotein levels. There were no associations between APOE polymorphisms and serum HDL-C, APO-CIII and triglycerides. In CHC patients, genetic heterogeneity in the APOE/TOMM40 genomic region is significantly associated with variation in serum ApoE and ApoB levels, and also with fibrosis suggesting a pleiotropic attribute of this genomic region.

A homopolymer polymorphism in the TOMM40 gene contributes to cognitive performance in aging.

INTRODUCTION: A highly polymorphic T homopolymer was recently found to be associated with late-onset Alzheimer's disease risk and age of onset. OBJECTIVE: To explore the effects of the polymorphic polyT tract (rs10524523, referred as '523') on cognitive performance in cognitively healthy elderly individuals. METHODS: One hundred eighty-one participants were recruited from local independent-living retirement communities. Informed consent was obtained, and participants completed demographic questionnaires, a conventional paper-and-pencil neuropsychological battery, and the computerized Cambridge Neuropsychological Test Automated Battery (CANTAB). Saliva samples were collected for determination of the TOMM40 '523' (S, L, VL) and the apolipoprotein E (APOE) (ɛ2, 3, 4) genotypes. From the initial sample of 181 individuals, 127 were eligible for the association analysis. Participants were divided into three groups based on '523' genotypes (S/S, S/L-S/VL, and L/L-L/VL-VL/VL). Generalized linear models were used to evaluate the association between the '523' genotypes and neuropsychological test performance. Analyses were adjusted for age, sex, education, depression, and APOE ɛ4 status. A planned subanalysis was undertaken to evaluate the association between '523' genotypes and test performance in a sample restricted to APOE ɛ3 homozygotes. RESULTS: The S homozygotes performed better, although not significantly, than the S/L-S/VL and the VL/L-L/VL-VL/VL genotype groups on measures associated with memory (CANTAB Paired Associates Learning, Verbal Recognition Memory free recall) and executive function (CANTAB measures of Intra-Extra Dimensional Set Shift). Follow-up analysis of APOE ɛ3 homozygotes only showed that the S/S group performed significantly better than the S/VL group on measures of episodic memory (CANTAB Paired Associates Learning and Verbal Recognition Memory free recall), attention (CANTAB Rapid Visual Information Processing latency), and executive function (Digit Symbol Substitution). The S/S group performed marginally better than the VL/VL group on Intra-Extra Dimensional Set Shift. None of the associations remained significant after applying a Bonferroni correction for multiple testing. CONCLUSIONS: Results suggest important APOE-independent associations between the TOMM40 '523' polymorphism and specific cognitive domains of memory and executive control that are preferentially affected in early-stage Alzheimer's disease.

Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice.

The outcome of an encounter with Mycobacterium tuberculosis (Mtb) depends on the pathogen's ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to create a resource for associating bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb transposon mutant fitness (TnSeq) across the CC panel revealed that many virulence pathways are only required in specific host microenvironments, identifying a large fraction of the pathogen's genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits can be associated with genetic variants distributed across the mouse genome, making the CC a unique population for identifying specific host-pathogen genetic interactions that influence pathogenesis.

The effect of SNCA 3' region on the levels of SNCA-112 splicing variant.

Genetic variability at the 3' region of SNCA locus has been repeatedly associated with susceptibility to sporadic Parkinson's disease (PD). Accumulated evidence emphasizes the importance of SNCA dosage and expression levels in PD pathogenesis. However, the mechanism through which the 3' region of SNCA gene modulates the risk to develop sporadic PD remained elusive. We studied the effect of PD risk-associated variants at SNCA 3' regions on SNCA112-mRNA (exon 5 in-frame skipping) levels in vivo in 117 neuropathologically normal, human brain frontal cortex samples. SNPs tagging the SNCA 3' showed significant effects on the relative levels of SNCA112-mRNA from total SNCA transcripts levels. The "risk" alleles were correlated with increased expression ratio of SNCA112-mRNA from total. We provide evidence for functional consequences of PD-associated SNCA gene variants at the 3' region, suggesting that genetic regulation of SNCA splicing plays an important role in the development of the disease. Further studies to determine the definite functional variant/s within SNCA 3'and to establish their association with PD pathology are necessary.

Expansion of the Parkinson disease-associated SNCA-Rep1 allele upregulates human alpha-synuclein in transgenic mouse brain.

Alpha-synuclein (SNCA) gene has been implicated in the development of rare forms of familial Parkinson disease (PD). Recently, it was shown that an increase in SNCA copy numbers leads to elevated levels of wild-type SNCA-mRNA and protein and is sufficient to cause early-onset, familial PD. A critical question concerning the molecular pathogenesis of PD is what contributory role, if any, is played by the SNCA gene in sporadic PD. The expansion of SNCA-Rep1, an upstream, polymorphic microsatellite of the SNCA gene, is associated with elevated risk for sporadic PD. However, whether SNCA-Rep1 is the causal variant and the underlying mechanism with which its effect is mediated by remained elusive. We report here the effects of three distinct SNCA-Rep1 variants in the brains of 72 mice transgenic for the entire human SNCA locus. Human SNCA-mRNA and protein levels were increased 1.7- and 1.25-fold, respectively, in homozygotes for the expanded, PD risk-conferring allele compared with homozygotes for the shorter, protective allele. When adjusting for the total SNCA-protein concentration (endogenous mouse and transgenic human) expressed in each brain, the expanded risk allele contributed 2.6-fold more to the SNCA steady-state than the shorter allele. Furthermore, targeted deletion of Rep1 resulted in the lowest human SNCA-mRNA and protein concentrations in murine brain. In contrast, the Rep1 effect was not observed in blood lysates from the same mice. These results demonstrate that Rep1 regulates human SNCA expression by enhancing its transcription in the adult nervous system and suggest that homozygosity for the expanded Rep1 allele may mimic locus multiplication, thereby elevating PD risk.

Content and Performance of the MiniMUGA Genotyping Array: A New Tool To Improve Rigor and Reproducibility in Mouse Research.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.