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

Characterization of the Poly-T Variant in the TOMM40 Gene in Diverse Populations

We previously discovered that a polymorphic, deoxythymidine-homopolymer (poly-T, rs10524523) in intron 6 of the TOMM40 gene is associated with age-of-onset of Alzheimer's disease and with cognitive performance in elderly. Three allele groups were defined for rs10524523, hereafter ‘523’, based on the number of ‘T’-residues: ‘Short’ (S, T≤19), ‘Long’ (L, 20≤T≤29) and ‘Very Long’ (VL, T≥30). Homopolymers, particularly long homopolymers like ‘523’, are difficult to genotype because ‘slippage’ occurs during PCR-amplification. We initially genotyped this locus by PCR-amplification followed by Sanger-sequencing. However, we recognized the need to develop a higher-throughput genotyping method that is also accurate and reliable. Here we describe a new ‘523’ genotyping assay that is simple and inexpensive to perform in a standard molecular genetics laboratory. The assay is based on the detection of differences in PCR-fragment length using capillary electrophoresis. We discuss technical problems, solutions, and the steps taken for validation. We employed the novel assay to investigate the ‘523’ allele frequencies in different ethnicities. Whites and Hispanics have similar frequencies of S/L/VL alleles (0.45/0.11/0.44 and 0.43/0.09/0.48, respectively). In African-Americans, the frequency of the L-allele (0.10) is similar to Whites and Hispanics; however, the S-allele is more prevalent (0.65) and the VL-allele is concomitantly less frequent (0.25). The allele frequencies determined using the new methodology are compared to previous reports for Ghanaian, Japanese, Korean and Han Chinese cohorts. Finally, we studied the linkage pattern between TOMM40-‘523’ and APOE alleles. In Whites and Hispanics, consistent with previous reports, the L is primarily linked to ε4, while the majority of the VL and S are linked to ε3. Interestingly, in African-Americans, Ghanaians and Japanese, there is an increased frequency of the ‘523’S-APOEε4 haplotype. These data may be used as references for ‘523’ allele and ‘523’-APOE haplotype frequencies in diverse populations for the design of research studies and clinical trials

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

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

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

<i>APOE</i> allele frequencies by ‘523’ genotypes in different ethnicities in the US.

<p><i>APOE</i> allele frequencies by ‘523’ genotypes in different ethnicities in the US.</p

‘523’ Allele frequencies in non US geographical cohorts (Far Eastern and West Africa).

<p>Genotypes determination was performed by Polymorphic, Inc. using a sequencing based assay.</p

<i>APOE</i> allele frequencies by ‘523’ genotypes in non US geographical cohorts (Far Eastern and West Africa).

<p>Genotypes determination was performed by Polymorphic, Inc.</p