BXD Strains Research Articles

Dnajc3 (HSP40) Enhances Axon Regeneration in the Mouse Optic Nerve.

A forward genetics approach was used to identify genomic elements enhancing axon regeneration in the BXD recombinant mouse strains. Axon regeneration was induced by knocking down Pten in retinal ganglion cells (RGCs) using adeno-associated virus (AAV) to deliver an shRNA followed by an intravitreal injection of Zymosan with CPT-cAMP that produced a mild inflammatory response. RGC axons were damaged by optic nerve crush (ONC). Following a 12-day survival period, regenerating axons were labeled by intravitreal injection of Cholera Toxin B (CTB) conjugated with Alexa Fluor 647. Two days later, labeled axons within the optic nerve were examined to determine the number of regenerating axons and the distance they traveled down the optic nerve. The analysis revealed a surprising difference in the amount of axonal regeneration across all 33 BXD strains. There was a 7.5-fold difference in the number of regenerating axons and a 4-fold difference in distance traveled by regenerating axons. These data were used to generate an integral map defining genomic loci modulating the enhanced axonal regeneration. A quantitative trait locus modulating axon regeneration was identified on Chromosome 14 (115 to 119 Mb). Within this locus were 16 annotated genes. Subsequent testing revealed that one candidate gene, Dnajc3 , modulates axonal regeneration. Dnajc3 encodes Heat Shock Protein 40 (HSP40), which is a molecular chaperone. Knocking down Dnajc3 in the high regenerative strain (BXD90) led to a decreased regeneration response, while overexpression of Dnajc3 in a low regenerative strain (BXD34) resulted in an increased regeneration response. These findings suggest that Dnajc3 not only increases the number of regenerating axons, it also increases the distance those axons travel. This may prove to be critical for functional recovery in large mammals, where the distance axons travel to their target is considerably longer than that of the mouse. Thus, Dnajc3 may play a critical role for functional recovery in humans by increasing the number of regenerating axons and the distance the regenerating axons travel.

Abstract Mo009: Systems genetics approach identifies the genetic basis underlying anthracycline-induced cardiotoxicity

Background: Anthracycline-induced cardiotoxicity (ACT) occurs in 57% of treated cancer patients. Effects of genetic variations to individual susceptibility to ACT is emerging. However, diverse lifestyle and environmental factors combined with small sample sizes in human cohorts remain as limitations to effectively clarify the genetic susceptibility to ACT. This study employed a murine genetic reference population (GRP) of BXD mice derived from crosses between DBA/2J and C57BL/6J strains for mapping of quantitative trait loci (QTLs) associated with doxorubicin (DOX)-induced traits using systems genetics methods. Methods: To model the variability in ACT to DOX, 56 BXD strains (N≥3 mice/sex, 3-4 month-old) were injected intraperitonially once with DOX (20 mg/kg). Survival and body weight (BW) were monitored daily for 10 Days (D). Echocardiography was performed before and on D5 after DOX. Genetic mapping was performed using WebQTL (www.genenetwork.org) and multi-criteria scoring was used for filtering candidate genes associated with DOX-induced traits. Results: Varied body weight loss, proportions and median survival rates were found among DOX-treated BXDs. Parental C57BL/6J strain had 100% survival, while 75% of DBA/2J mice survived on D10. Among BXDs tested, 47 strains had median survival of 6±1 D, with the lowest seen in BXD77 (4 D). Echocardiography revealed abnormal small heart and restrictive dysfunction in BXDs, resembling Grinch syndrome seen in ACT patients. A significant QTL (LRS = 17.11) at 86~94 Mb of Chromosome (Chr) 10 was identified (genome wide P < 0.05). Further filtering identified Gnptab , Slc25a3 , Uhrf1bp1l, and Chpt1 as top candidate genes associated with the survival of DOX-treated BXDs. Mapping of QTLs associated with echocardiography values and weight loss are in a progress. Conclusions: Survival, BW loss, and echocardiography parameters significantly varied among DOX-treated BXDs suggesting an influence of genetic background on expression of those traits. Gnptab , Slc25a3, Uhrf1bp1l, and Chpt1 (Chr 10) are associated with DOX-induced survival traits in BXDs of GRP, suggesting that these genes are involved in modulating ACT severity in humans and require further functional and biological validation.

Identification of ethanol analgesia quantitative trait loci and candidate genes in BXD recombinant inbred mouse lines.

Alcohol consumption produces acute analgesic effects, and people experiencing pain conditions may drink alcohol to alleviate discomfort. However, tolerance to the analgesic properties of alcohol could prompt escalating consumption and dependence. Both nociception and alcohol-induced analgesia are under significant genetic control. Understanding the genetic architecture of these processes could inform better treatment options for people with pain conditions. This study aims to identify quantitative trait loci (QTL) driving variation in ethanol-induced analgesia across BXD recombinant inbred mouse lines. Male and female mice from 62 BXD strains received ethanol or saline oral gavage for five days and were tested for hot plate (HP) latency at baseline, Day 1, and Day 5. QTL mapping of HP phenotypes identified a significant provisional QTL on chromosome 17 for Day 1 HP latency in mice receiving ethanol. An additional highly suggestive QTL was present on chromosome 9 for the difference in pre- and post-ethanol thermal nociception. Candidate genes within QTL support intervals were provisionally identified using HP phenotypic correlations to transcriptomic database, expression QTL analysis, and other bioinformatics inquiries. The combined behavioral and bioinformatic analyses yielded strong ethanol analgesia candidate genes, specifically Myo6. Thus, the results of this genetic study of ethanol-induced analgesia in BXD mouse strains may contribute significantly to our understanding of the molecular basis for individual variation in the analgesic response to acute ethanol.

Systems genetics analysis reveals the common genetic basis for pain sensitivity and cognitive function.

There is growing evidence of a strong correlation between pain sensitivity and cognitive function under both physiological and pathological conditions. However, the detailed mechanisms remain largely unknown. In the current study, we sought to explore candidate genes and common molecular mechanisms underlying pain sensitivity and cognitive function with a transcriptome-wide association study using recombinant inbred mice from the BXD family. The pain sensitivity determined by Hargreaves' paw withdrawal test and cognition-related phenotypes were systematically analyzed in 60 strains of BXD mice and correlated with hippocampus transcriptomes, followed by quantitative trait locus (QTL) mapping and systems genetics analysis. The pain sensitivity showed significant variability across the BXD strains and co-varies with cognitive traits. Pain sensitivity correlated hippocampual genes showed a significant involvement in cognition-related pathways, including glutamatergic synapse, and PI3K-Akt signaling pathway. Moreover, QTL mapping identified a genomic region on chromosome 4, potentially regulating the variation of pain sensitivity. Integrative analysis of expression QTL mapping, correlation analysis, and Bayesian network modeling identified Ring finger protein 20 (Rnf20) as the best candidate. Further pathway analysis indicated that Rnf20 may regulate the expression of pain sensitivity and cognitive function through the PI3K-Akt signaling pathway, particularly through interactions with genes Ppp2r2b, Ppp2r5c, Col9a3, Met, Rps6, Tnc, and Kras. Our study demonstrated that pain sensitivity is associated with genetic background and Rnf20-mediated PI3K-Akt signaling may involve in the regulation of pain sensitivity and cognitive functions.

GenomeMUSter mouse genetic variation service enables multitrait, multipopulation data integration and analysis.

Hundreds of inbred mouse strains and intercross populations have been used to characterize the function of genetic variants that contribute to disease. Thousands of disease-relevant traits have been characterized in mice and made publicly available. New strains and populations including consomics, the collaborative cross, expanded BXD, and inbred wild-derived strains add to existing complex disease mouse models, mapping populations, and sensitized backgrounds for engineered mutations. The genome sequences of inbred strains, along with dense genotypes from others, enable integrated analysis of trait-variant associations across populations, but these analyses are hampered by the sparsity of genotypes available. Moreover, the data are not readily interoperable with other resources. To address these limitations, we created a uniformly dense variant resource by harmonizing multiple data sets. Missing genotypes were imputed using the Viterbi algorithm with a data-driven technique that incorporates local phylogenetic information, an approach that is extendable to other model organisms. The result is a web- and programmatically accessible data service called GenomeMUSter, comprising single-nucleotide variants covering 657 strains at 106.8 million segregating sites. Interoperation with phenotype databases, analytic tools, and other resources enable a wealth of applications, including multitrait, multipopulation meta-analysis. We show this in cross-species comparisons of type 2 diabetes and substance use disorder meta-analyses, leveraging mouse data to characterize the likely role of human variant effects in disease. Other applications include refinement of mapped loci and prioritization of strain backgrounds for disease modeling to further unlock extant mouse diversity for genetic and genomic studies in health and disease.

Abstract 14573: Dissecting Genetic Determinants Underlying Cardioskeletal Phenotypes Induced by Myopalladin Mutation in Murine Genetic Reference Population of Hybrid BXD Strains

Introduction: Mutations in myopalladin ( MYPN) gene are associated with cardiomyopathies (CM) and skeletal myopathies (SM). This study is aimed to identify Mypn -induced cardioskeletal phenotypes in a murine genetic reference population (GRP) and uncover common genetic mechanisms underlying the development of CM and SM. Hypothesis: Mypn induces common molecular signaling in cardiac and skeletal muscles. Methods: Pearson correlation between cardiac function and expression of Mypn in gastrocnemius muscles was performed in 40 mouse BXD recombinant inbred strains derived from C57BL/6J and DBA/2J parental mice. A causal Mypn-Q526X mutation was introduced to BXDs by crossing with MypnWT/Q526X knock-in mice and this generated F1 hybrid mutant (BXDQ526X) and wild type (BXDWT) strains. Cardiac (morphology, function by echo) and skeletal muscle (muscle mass, strength) phenotypes and transcriptomics (RNA-seq) were assessed in 3- and 6-month-old hybrid mice (N=5). Results: Skeletal muscle expression of Mypn was significantly associated with heart rate (HR) and heart mass (HM) in BXDs, while it was negatively associated with interventricular septum (IVS) thickness, indicating BXD strains with higher skeletal muscle Mypn expression had faster HR, higher HM but thinner IVS. Varied phenotypes in ejection fraction (EF%), ventricular volume and hypertrophy were found among BXD Q526X mice; BXD51 Q526X , BXD48 Q526X and BXD69 Q526X strains had significantly higher EF%, while some mutant mice showed EF% decrease compared to their respective BXD WT littermates. Many mutant lines displayed significantly reduced skeletal muscle strength or mass. Overt SM-related signs (rear limb deformity, restricted movement, waddling) were seen in BXD78 Q526X , while we found overlapping CM/SM traits in BXD51 Q526X mice. Conclusions: In this study, common Mypn -induced molecular signaling in cardiac and skeletal muscles is identified. Introduction of the Mypn -Q526X mutation into the diverse genetic background of BXD GRP exposed CM- and SM-related phenotypes in hybrid lines. The most susceptible lines with overt and overlapping CM/SM phenotypes will be useful to identify common genes, networks and pathways involved in CM and SM using systems genetics methods.

Genetic and dietary modulators of the inflammatory response in the gastrointestinal tract of the BXD mouse genetic reference population.

Inflammatory gut disorders, including inflammatory bowel disease (IBD), can be impacted by dietary, environmental, and genetic factors. While the incidence of IBD is increasing worldwide, we still lack a complete understanding of the gene-by-environment interactions underlying inflammation and IBD. Here, we profiled the colon transcriptome of 52 BXD mouse strains fed with a chow or high-fat diet (HFD) and identified a subset of BXD strains that exhibit an IBD-like transcriptome signature on HFD, indicating that an interplay of genetics and diet can significantly affect intestinal inflammation. Using gene co-expression analyses, we identified modules that are enriched for IBD-dysregulated genes and found that these IBD-related modules share cis-regulatory elements that are responsive to the STAT2, SMAD3, and REL transcription factors. We used module quantitative trait locus analyses to identify genetic loci associated with the expression of these modules. Through a prioritization scheme involving systems genetics in the mouse and integration with external human datasets, we identified Muc4 and Epha6 as the top candidates mediating differences in HFD-driven intestinal inflammation. This work provides insights into the contribution of genetics and diet to IBD risk and identifies two candidate genes, MUC4 and EPHA6, that may mediate IBD susceptibility in humans.

Genetic and dietary modulators of the inflammatory response in the gastrointestinal tract of the BXD mouse genetic reference population

Inflammatory gut disorders, including inflammatory bowel disease (IBD), can be impacted by dietary, environmental, and genetic factors. While the incidence of IBD is increasing worldwide, we still lack a complete understanding of the gene-by-environment interactions underlying inflammation and IBD. Here, we profiled the colon transcriptome of 52 BXD mouse strains fed with a chow or high-fat diet (HFD) and identified a subset of BXD strains that exhibit an IBD-like transcriptome signature on HFD, indicating that an interplay of genetics and diet can significantly affect intestinal inflammation. Using gene co-expression analyses, we identified modules that are enriched for IBD-dysregulated genes and found that these IBD-related modules share cis-regulatory elements that are responsive to the STAT2, SMAD3, and REL transcription factors. We used module quantitative trait locus analyses to identify genetic loci associated with the expression of these modules. Through a prioritization scheme involving systems genetics in the mouse and integration with external human datasets, we identified Muc4 and Epha6 as the top candidates mediating differences in HFD-driven intestinal inflammation. This work provides insights into the contribution of genetics and diet to IBD risk and identifies two candidate genes, MUC4 and EPHA6, that may mediate IBD susceptibility in humans.

A rapid workflow for neuron counting in combined light sheet microscopy and magnetic resonance histology.

Information on regional variation in cell numbers and densities in the CNS provides critical insight into structure, function, and the progression of CNS diseases. However, variability can be real or a consequence of methods that do not account for technical biases, including morphologic deformations, errors in the application of cell type labels and boundaries of regions, errors of counting rules and sampling sites. We address these issues in a mouse model by introducing a workflow that consists of the following steps: 1. Magnetic resonance histology (MRH) to establish the size, shape, and regional morphology of the mouse brain in situ. 2. Light-sheet microscopy (LSM) to selectively label neurons or other cells in the entire brain without sectioning artifacts. 3. Register LSM volumes to MRH volumes to correct for dissection errors and both global and regional deformations. 4. Implement stereological protocols for automated sampling and counting of cells in 3D LSM volumes. This workflow can analyze the cell densities of one brain region in less than 1 min and is highly replicable in cortical and subcortical gray matter regions and structures throughout the brain. This method demonstrates the advantage of not requiring an extensive amount of training data, achieving a F1 score of approximately 0.9 with just 20 training nuclei. We report deformation-corrected neuron (NeuN) counts and neuronal density in 13 representative regions in 5 C57BL/6J cases and 2 BXD strains. The data represent the variability among specimens for the same brain region and across regions within the specimen. Neuronal densities estimated with our workflow are within the range of values in previous classical stereological studies. We demonstrate the application of our workflow to a mouse model of aging. This workflow improves the accuracy of neuron counting and the assessment of neuronal density on a region-by-region basis, with broad applications for studies of how genetics, environment, and development across the lifespan impact cell numbers in the CNS.

The ameliorative effects of choline on ethanol-induced cell death in the neural tube of susceptible BXD strains of mice.

Fetal alcohol spectrum disorders (FASD) are the leading preventable cause of intellectual disability, providing the impetus for evaluating various potential treatments to ameliorate ethanol's teratogenic effects, particularly in the nervous system. One treatment is the dietary supplement choline which has been shown to mitigate at least some of ethanol's teratogenic effects. The present study was designed to investigate the effects of genetics on choline's efficacy in ameliorating cell death in the developing neural tube. Previously, we examined BXD recombinant inbred mice, and their parental C57BL/6 J (B6) and DBA/2 J strains, and identified strains that were sensitive to ethanol's teratogenic actions. Thus, we used these strains to identify response to choline treatment. Timed pregnant mice from 4 strains (B6, BXD51, BXD73, BXD2) were given either ethanol or isocaloric maltose-dextrin (5.8 g/kg in two administrations separated by 2 h) with choline at one of 3 doses: 0, 100 or 250 mg/kg. Subjects were exposed via intragastric gavage on embryonic day 9 and embryos were collected 7 h after the initial ethanol administrations. Cell death was analyzed using TUNEL staining in the developing forebrain and brainstem. Choline ameliorated the ethanol-induced cell death across all 4 strains without causing enhanced cell death in control mice. Choline was effective in both the developing telencephalon and in the brainstem. Both doses diminished cell death, with some differences across strains and brain regions, although the 100 mg/kg dose was most consistent in mitigating ethanol-related cell death. Comparisons across strains showed that there was an effect of strain, particularly in the forebrain at the higher dose. These results show that choline is effective in ameliorating ethanol-induced cell death at this early stage of nervous system development. However, there were some strain differences in its efficacy, especially at the high dose, providing further evidence of the importance of genetics in influencing the ability of choline to protect against prenatal alcohol exposure.

Pleiotropic influence of DNA methylation QTLs on physiological and ageing traits

ABSTRACT DNA methylation is influenced by genetic and non-genetic factors. Here, we chart quantitative trait loci (QTLs) that modulate levels of methylation at highly conserved CpGs using liver methylome data from mouse strains belonging to the BXD family. A regulatory hotspot on chromosome 5 had the highest density of trans-acting methylation QTLs (trans-meQTLs) associated with multiple distant CpGs. We refer to this locus as meQTL.5a. Trans-modulated CpGs showed age-dependent changes and were enriched in developmental genes, including several members of the MODY pathway (maturity onset diabetes of the young). The joint modulation by genotype and ageing resulted in a more ‘aged methylome’ for BXD strains that inherited the DBA/2J parental allele at meQTL.5a. Further, several gene expression traits, body weight, and lipid levels mapped to meQTL.5a, and there was a modest linkage with lifespan. DNA binding motif and protein–protein interaction enrichment analyses identified the hepatic nuclear factor, Hnf1a (MODY3 gene in humans), as a strong candidate. The pleiotropic effects of meQTL.5a could contribute to variations in body size and metabolic traits, and influence CpG methylation and epigenetic ageing that could have an impact on lifespan.

Expression Levels of the Tnni3k Gene in the Heart Are Highly Associated with Cardiac and Glucose Metabolism-Related Phenotypes and Functional Pathways.

Troponin-I interacting kinase encoded by the TNNI3K gene is expressed in nuclei and Z-discs of cardiomyocytes. Mutations in TNNI3K were identified in patients with cardiac conduction diseases, arrhythmias, and cardiomyopathy. We performed cardiac gene expression, whole genome sequencing (WGS), and cardiac function analysis in 40 strains of BXD recombinant inbred mice derived from C57BL/6J (B6) and DBA/2J (D2) strains. Expression quantitative trait loci (eQTLs) mapping and gene enrichment analysis was performed, followed by validation of candidate Tnni3k-regulatory genes. WGS identified compound splicing and missense T659I Tnni3k variants in the D2 parent and some BXD strains (D allele) and these strains had significantly lower Tnni3k expression than those carrying wild-type Tnni3k (B allele). Expression levels of Tnni3k significantly correlated with multiple cardiac (heart rate, wall thickness, PR duration, and T amplitude) and metabolic (glucose levels and insulin resistance) phenotypes in BXDs. A significant cis-eQTL on chromosome 3 was identified for the regulation of Tnni3k expression. Furthermore, Tnni3k-correlated genes were primarily involved in cardiac and glucose metabolism-related functions and pathways. Genes Nodal, Gnas, Nfkb1, Bmpr2, Bmp7, Smad7, Acvr1b, Acvr2b, Chrd, Tgfb3, Irs1, and Ppp1cb were differentially expressed between the B and D alleles. Compound splicing and T659I Tnni3k variants reduce cardiac Tnni3k expression and Tnni3k levels are associated with cardiac and glucose metabolism-related phenotypes.

Developmental coordination disorder: What can we learn from RI mice using motor learning tasks and QTL analysis.

Developmental Coordination Disorder (DCD) is a neurodevelopmental disorder of unknown etiology that affects one in 20 children. There is an indication that DCD has an underlying genetic component due to its high heritability. Therefore, we explored the use of a recombinant inbred family of mice known as the BXD panel to understand the genetic basis of complex traits (i.e., motor learning) through identification of quantitative trait loci (QTLs). The overall aim of this study was to utilize the QTL approach to evaluate the genome-to-phenome correlation in BXD strains of mice in order to better understand the human presentation of DCD. Results of this current study confirm differences in motor learning in selected BXD strains and strains with altered cerebellar volume. Five strains - BXD15, BXD27, BXD28, BXD75, and BXD86 - exhibited the most DCD-like phenotype when compared with other BXD strains of interest. Results indicate that BXD15 and BXD75 struggled primarily with gross motor skills, BXD28 primarily had difficulties with fine motor skills, and BXD27 and BXD86 strains struggled with both fine and gross motor skills. The functional roles of genes within significant QTLs were assessed in relation to DCD-like behavior. Only Rab3a (Ras-related protein Rab-3A) emerged as a high likelihood candidate gene for the horizontal ladder rung task. This gene is associated with brain and skeletal muscle development, but lacked nonsynonymous polymorphisms. This study along with Gill et al. (same issue) is the first studies to specifically examine the genetic linkage of DCD using BXD strains of mice.

Effects of genetics and sex on adolescent behaviors following neonatal ethanol exposure in BXD recombinant inbred strains.

Fetal alcohol spectrum disorders (FASD) are the leading preventable neurodevelopmental disorders and two hallmark symptoms of FASD are abnormal behavior, and cognitive and learning deficits. The severity of alcohol's teratogenic effects on the developing brain is influenced by genetics and sex. We previously identified recombinant inbred BXD mouse strains that show differential vulnerability to ethanol-induced cell death in the developing hippocampus, a brain region important in learning and memory. The present study aimed to test the hypothesis that strains with increased vulnerability to ethanol-induced cell death in the hippocampus have concomitant deficits in multiple hippocampal-related behaviors during adolescence. The current study evaluated the effects of developmental ethanol exposure on adolescent behavior in two BXD strains that show high cell death (BXD48a, BXD100), two that show low cell death (BXD60, BXD71), and the two parental strains (C57BL/6 J (B6), DBA/2 J (D2)). On postnatal day 7, male and female neonatal pups were treated with ethanol (5.0 g/kg) or saline given in two equal doses 2 h apart. Adolescent behavior was assessed across multiple behavioral paradigms including the elevated plus maze, open field, Y-maze, and T-maze. Our results demonstrate that the effects of developmental ethanol exposure on adolescent behavioral responses are highly dependent on strain. The low cell death strains, BXD60 and BXD71, showed minimal effect of ethanol exposure on all behavioral measures but did present sex differences. The parental -B6 and D2-strains and high cell death strains, BXD48a and BXD100, showed ethanol-induced effects on activity-related or anxiety-like behaviors. Interestingly, the high cell death strains were the only strains that showed a significant effect of postnatal ethanol exposure on hippocampal-dependent spatial learning and memory behaviors. Overall, we identified effects of ethanol exposure, strain, and/or sex on multiple behavioral measures. Interestingly, the strains that showed the most effects of postnatal ethanol exposure on adolescent behavior were the BXD strains that show high ethanol-induced cell death in the neonatal hippocampus, consistent with our hypothesis. Additionally, we found evidence for interactions among strain and sex, demonstrating that these factors have a complex effect on alcohol responses and that both are important considerations.

Analysis of electrocardiography parameters in BXD strains and quantitative trait loci for arrhythmia disorders in the mouse BXD family

Introduction: Heart rhythm disorders or cardiac arrhythmias can cause sudden death and heart failure. Risk factors for cardiac arrhythmias vary including genetics, age, and other environmental and lifestyle factors. To sought how genetic background affects an expression of cardiac rhythm phenotypes, we assessed electrocardiography (ECG) traits in BXD family of mice derived from crosses between DBA/2J (D2) and C57BL/6J (B6) strains and explored the quantitative genetic architecture of those traits. Methods: ECG tracings were recorded in 44 BXD male (M) and female (F) strains (N >5 mice/sex) at 4-5 months of age anesthetized with 2% isoflurane. Heart rate (HR), ECG parameters, and frequency of arrhythmias in percentile (‰) were associated with blood pressure, echocardiography, and heart transcriptome values followed by quantitative trait loci (QTLs) mapping. Results: Parental D2 strains had the lowest HR and significantly prolonged QT intervals (62.82 ± 9.42 ms in D2F and 53.42 ± 1.73 ms in D2M) compared to sex matched B6 parental strains. Significantly widened QRS complexes were seen in BXD65F (14.27±2.97 ms) compared to 11.67±1.03 ms in B6F controls. In males, BXD83M had the widest QRS (13.48±1.74 ms) vs 10.89±0.41 ms seen in B6M controls. We found a significant association between QRS duration and increased left ventricular internal diameter (LVID) and reduced ejection fraction and fractional shortening. Further, varied cardiac arrhythmias including bradycardia, atrioventricular block (AVB), premature atrial or ventricular complexes (PAC and PVC, respectively), ventricular tachycardia (VT) and sick sinus syndrome (SSS) were seen among BXD strains. PACs were recorded in BXD73F (14.57 ‰), 40M, 101F, 51M, and 101M strains. Strains, BXD79M (14.97 ‰), 83M, 78M, 69F, and 171F, had frequent PVCs compared to B6 controls. AVB II was recorded in BXD48M and BXD66M had SSS. Both PAC and PVC were positively associated with cardiac phenotype burden. Specifically, PVCs were significantly correlated with echocardiographic pulmonary vein peak pressure, LVID and volumes at end-diastole (P = 0.04) among male BXDs. In females, right ventricular internal diameters (RVID) and cardiac output were significantly correlated with PVC and PAC frequencies. Moreover, PVCs were significantly correlated with systolic blood pressure in both male and female mice. QTL mapping identified a significant locus on Chromosome 3 associated with QTC and JT durations, while the same locus was suggestive for association with QT interval, suggesting that Chromosome 3 loci may be associated with repolarization abnormalities such as long QT syndromes. Conclusions: ECG parameters, heart rate, type and frequency of arrhythmias significantly varied between mouse strains of the BXD family suggesting an influence of genetic background on expression of those traits. Abnormal heart rhythms detected in BXD strains mimic cardiac rhythm and conduction disorders in humans. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cardiac copper content and its relationship with heart physiology: Insights based on quantitative genetic and functional analyses using BXD family mice.

Copper (Cu) is essential for the functioning of various enzymes involved in important cellular and physiological processes. Although critical for normal cardiac function, excessive accumulation, or deficiency of Cu in the myocardium is detrimental to the heart. Fluctuations in cardiac Cu content have been shown to cause cardiac pathologies and imbalance in systemic Cu metabolism. However, the genetic basis underlying cardiac Cu levels and their effects on heart traits remain to be understood. Representing the largest murine genetic reference population, BXD strains have been widely used to explore genotype-phenotype associations and identify quantitative trait loci (QTL) and candidate genes. Cardiac Cu concentration and heart function in BXD strains were measured, followed by QTL mapping. The candidate genes modulating Cu homeostasis in mice hearts were identified using a multi-criteria scoring/filtering approach. Significant correlations were identified between cardiac Cu concentration and left ventricular (LV) internal diameter and volumes at end-diastole and end-systole, demonstrating that the BXDs with higher cardiac Cu levels have larger LV chamber. Conversely, cardiac Cu levels negatively correlated with LV posterior wall thickness, suggesting that lower Cu concentration in the heart is associated with LV hypertrophy. Genetic mapping identified six QTLs containing a total of 217 genes, which were further narrowed down to 21 genes that showed a significant association with cardiac Cu content in mice. Among those, Prex1 and Irx3 are the strongest candidates involved in cardiac Cu modulation. Cardiac Cu level is significantly correlated with heart chamber size and hypertrophy phenotypes in BXD mice, while being regulated by multiple genes in several QTLs. Prex1 and Irx3 may be involved in modulating Cu metabolism and its downstream effects and warrant further experimental and functional validations.

Echocardiography phenotyping in murine genetic reference population of BXD strains reveals significant QTLs associated with cardiac function and morphology.

The genetic reference population of recombinant inbred BXD mice has been derived from crosses between C57BL/6J and DBA/2J strains. The DBA/2J parent exhibits cardiomyopathy phenotypes, whereas C57BL/6J has normal heart. BXD mice are sequenced for studying genetic interactions in cardiomyopathies. The study aimed to assess cardiomyopathy traits in BXDs and investigate the quantitative genetic architecture of those traits. Echocardiography, blood pressure, and cardiomyocyte size parameters obtained from 44 strains of BXD family (n > 5/sex) at 4-5 mo of age were associated with heart transcriptomes and expression quantitative trait loci (eQTL) mapping was performed. More than twofold variance in ejection fraction (EF%), fractional shortening (FS%), left ventricular volumes (LVVols), internal dimensions (LVIDs), mass (LVM), and posterior wall (LVPW) thickness was found among BXDs. In male BXDs, eQTL mapping identified Ndrg4 on chromosome 8 QTL to be positively correlated with LVVol and LVID and negatively associated with cardiomyocyte diameter. In female BXDs, significant QTLs were found on chromosomes 7 and 3 to be associated with LVPW and EF% and FS%, respectively, and Josd2, Dap3, and Tpm3 were predicted as strong candidate genes. Our study found variable cardiovascular traits among BXD strains and identified multiple associated QTLs, suggesting an influence of genetic background on expression of echocardiographic and cardiomyocyte diameter traits. Increased LVVol and reduced EF% and FS% represented dilated cardiomyopathy, whereas increased LV mass and wall thickness indicated hypertrophic cardiomyopathy traits. The BXD family is ideal for identifying candidate genes, causal and modifier, that influence cardiovascular phenotypes.NEW & NOTEWORTHY This study aimed to establish a cardiac phenotype-genotype correlation in murine genetic reference population of BXD RI strains by phenotyping the echocardiography, blood pressure, and cardiomyocyte diameter traits and associating each collected phenotype with genetic background. Our study identified several QTLs and candidate genes that have significant association with cardiac hypertrophy, ventricular dilation, and function including systolic hyperfunction and dysfunction.

Abstract 11608: Differences in Genetic Variants and Common Genetic Regulators between Arrhythmogenic Cardiomyopathy and Arrhythmogenic Dilated Cardiomyopathy Identified Using Systems Genetics Approaches

Introduction: Prominent arrhythmias are features of arrhythmogenic cardiomyopathy (ACM) and arrhythmogenic dilated cardiomyopathy (aDCM). Despite clinicopathological differences, these diseases share common genetic causes. Hypothesis: This study aimed to identify variants in ACM and aDCM and map the candidate genetic regulators using a mouse genetic reference population (GRP). Methods: Whole exome sequencing was performed on 226 individuals with ACM and 102 patients with aDCM. Bioinformatics analysis was applied to identify the pathogenic or likely pathogenic variants followed by direct sequencing confirmation. Expression quantitative trait loci (eQTLs) mapping to heart transcriptomes of GRP of BXD recombinant inbred (RI) mice was conducted in GeneNetwork (http://www.genenetwork.org). Results: In the ACM cohort, a total of 82 variants (MAF<0.01) in 24 genes have been confirmed being positive in 42 probands. Depending on the frequency of the identified variants, the top 10 genes (excluding TTN) with the most pathogenic variants were PKP2, MYH6, FLNC, RYR2, MYH7B, PLEC, JPH2, SYNE1, NRAP and POSTN. In the aDCM cohort, 141 pathogenic variants have been identified and 91 patients harbored at least 1 deleterious variant. The top 10 genes were RYR2, FLNC, MYH7, DSP, SCN5A,MYPN, NEBL, CTNNA3, ACAP6 and SLC22A5. We analyzed the distribution of the first principal component (PC1) of mRNA expression levels of those 10 genes (eigentraits) from each cohort in the heart across BXD strains. Interval mapping for PC1 eigentraits for ACM identified a significant eQTL (P<0.05) in the mouse genome at 24-36 Mb of chromosome (Chr) 6. In aDCM, interval mapping for PC1 eigentraits identified 3 suggestive eQTLs at Chr 5 (44-53 Mb), Chr 6 (32-34 Mb) and Chr 15 (82-84 Mb). Further gene exploration of these eQTLs suggested that Mtpn, Lep, Cyb5r3, Tspo, Slit2, and Ppargc1a could be common regulators that interact with those top ACM and aDCM genes. Conclusions: This study identified diverse genes in ACM and aDCM cases. Our joint human and mouse analysis using a systems genetics approach identified genetic regulators that could affected expression of genes with pathogenic variants in ACM and aDCM patients.

Analysis of cardiac function and morphology in genetic reference population of BXD strains reveals associated eQTLs and candidate causal and modifier genes

IntroductionThe murine recombinant inbred (RI) family of BXD strains derived from crosses between C57BL/6J (B6) and DBA/2J (D2) mice are the largest genetic reference population (GRP). The parental D2 mouse is a natural model of hypertrophic cardiomyopathy (HCM). Thus, the BXD family represents an applicable rodent model for phenotype‐genotype studies of complex cardiomyopathy traits.HypothesisGenetic background affects an expression of cardiomyopathy traits.MethodsEchocardiography, electrocardiography (ECG) and blood pressure measurements were performed in 44 BXD strains (N>5/sex) at 4‐5 months of age. Cardiac traits (heart function, mass, rhythm, and arrhythmias) and blood pressure values were then associated with heart transcriptome, and expression quantitative trait loci (eQTL) mapping was performed.ResultsMore than 2‐fold variance in ejection fraction (EF%), fractional shortening (FS%), left ventricular (LV) volumes, internal dimensions (ID) and mass, as well as in thickness of LV posterior wall (LVPW), and interventricular septum (IVS) was found by echocardiography among BXDs. Varied abnormal heart rhythms including bradycardia, atrioventricular block, premature atrial or ventricular complexes, ventricular tachycardia and sick sinus syndrome were recorded among BXD strains using ECG. Parental D2 strains had significantly prolonged QT interval (62.82±9.42 ms and 53.42±1.73 ms, respectively) compared to B6 parental strains. A significant variability in systolic and diastolic BP between BXD strains was also recorded.Among male BXDs, a significant QTL of 91.0 to 97.4 Mb (LRS=18.50) was identified on chromosome (Chr) 8 that is associated with LV volumes and IDs during systole and diastole. eQTL mapping for each of the 131 genes at the QTL interval of Chr 8 identified 6 genes (Coq9, Ndrg4, Crnde, Irx3, Rpgrip1l, and Rbl2) being cis‐regulated. Among those genes, Ndrg4was found to be positively correlated with LV volumes at systole and diastole and ID traits, and negatively correlated with the cardiomyocyte size (r = ‐0.588, p = 0.025). In female BXDs, a significant QTL with LRS=20.1 was found on Chr 7 of 40.2 Mb that is associated with LVPW at diastole, where Josd2is a strong candidate gene that is cis‐regulated and significantly correlated with LVPW trait.ConclusionsOur study found variable traits of echocardiographic, electrocardiographic and blood pressure parameters are segregated among the BXD family. The cardiovascular traits significantly vary between BXD lines suggesting an influence of genetic background on expression of those traits. Traits such as reduced EF%, FS%, and LVPW and increased LV volumes are seen in patients with dilated cardiomyopathy (DCM). Increased LV mass, LV thickness, LVPW, and IVS mimic traits seen in patients with HCM. Varied abnormal heart rhythms detected in BXD strains simulate cardiac conduction abnormalities in humans. Multiple associated eQTLs defined in BXDs demonstrate that the BXD family of RI strains is a suitable model to map gene variants and identify causal genes and genetic modifiers that influence cardiovascular phenotypes including cardiomyopathies.

Genetic loci and metabolic states associated with murine epigenetic aging.

Changes in DNA methylation (DNAm) are linked to aging. Here, we profile highly conserved CpGs in 339 predominantly female mice belonging to the BXD family for which we have deep longevity and genomic data. We use a 'pan-mammalian' microarray that provides a common platform for assaying the methylome across mammalian clades. We computed epigenetic clocks and tested associations with DNAm entropy, diet, weight, metabolic traits, and genetic variation. We describe the multifactorial variance of methylation at these CpGs and show that high-fat diet augments the age-related changes. Entropy increases with age. The progression to disorder, particularly at CpGs that gain methylation over time, was predictive of genotype-dependent life expectancy. The longer-lived BXD strains had comparatively lower entropy at a given age. We identified two genetic loci that modulate epigenetic age acceleration (EAA): one on chromosome (Chr) 11 that encompasses the Erbb2/Her2 oncogenic region, and the other on Chr19 that contains a cytochrome P450 cluster. Both loci harbor genes associated with EAA in humans, including STXBP4, NKX2-3, and CUTC. Transcriptome and proteome analyses revealed correlations with oxidation-reduction, metabolic, and immune response pathways. Our results highlight concordant loci for EAA in humans and mice, and demonstrate a tight coupling between the metabolic state and epigenetic aging.