Identifying modifier genes for hypertrophic cardiomyopathy

Abstract

Background: Hypertrophic cardiomyopathy (HCM) severity greatly varies among patients even with the same HCM gene mutations. This variation is largely regulated by modifier gene(s), which, however, remain largely unknown. The current study is aimed to identify modifier genes using BXD strains, a large murine genetic reference population (GRP) derived from crosses between C57BL/6 J (B6) and D2 DBA/2 J (D2) mice. D2 mice natualy carrythe genetic basis and phenotypes of HCM.

Methods: Myocardial hypertrophy, the major phenotype of HCM, was determined by cardiomyocyte size on cardiac sections in 30 BXD strains, and their parental B6 and D2 strains and morphometric analysis was performed. Quantitative Trait Locus (QTL) mapping for cardiomyocyte sizes was conducted with WebQTL in GeneNetwork. Correlation of cardiomyocyte size and cardiac gene expression in BXDs accessed from GeneNetwork were evaluated. QTL candidate genes associated with cardiomyocyte sizes were prioritized based on the score system.

Results: Cardiomyocyte size varied significantly among BXD strains. Interval mapping on cardiomyocyte size data showed a significant QTL on chromosome (Chr) 2 at 66- 73.5 Mb and a suggestive QTL on Chr 5 at 20.9-39.7 Mb. Further score system revealed a high QTL score for Xirp2 in Chr 2. Xirp2 encodes xin actin-binding repeat containing 2, which is highly expressed in cardiac tissue and associate with cardiomyopathy and heart failure. In Chr5 QTL, Nos3, encoding nitric oxide synthase 3, received the highest score, which is significantly correlated with cardiomyocyte size.

Conclusion: These results indicate that Xirp2 and Nos3 serve as novel candidate modifier genes for myocardial hypertrophy in HCM. These candidate genes will be validated in our future studies.

Keywords: Hypertrophic cardiomyopathy; Modifier genes; Murine genetic reference population; Myocyte hypertrophy.

Figure 1:

Cardiomyocyte size in B6, D2 and BXD RI strains. Panel A shows cardiomyocyte diameter in each strain. Panels B and C display cardiac morphology in B6 and BXD169, which present the smallest and largest cardiomyocyte diameter, respectively. Panels B and C: X200

Figure 2:

Genome-wide interval mapping plot for cardiomyocyte size. (A) Interval mapping of cardiomyocyte size was conducted using webQTL in GeneNetwork (www.genenetwork.org). The y axis represents likelihood ratio statistic (LRS) along with the genomic coordinate in x axis. Genome-wide significant (LRS = 16.50, red line) and suggestive (LRS = 10.87, grey line) threshold were determined with 2000 permutation test. One significant and one suggestive QTL were identified on Chr 2 and Chr5, respectively. (B) Interval mapping of cardiomyocyte size was adjusted by combined age + gender + heart weight. Both Chr 2 and Chr 5 QTL are replicated, while another suggestive QTL was mapped to Chr 18.

Figure 3:

Venn diagram of QTLs candidate genes for cardiomyocyte size on Chr 2 (A) and Chr 5 (B). A total of 118 and 411 genes (including pseudogenes) were located in Chr 2 and Chr 5 QTL intervals, respectively. Five criteria were applied to prioritize the candidate genes, including gene average expression in heart tissue, coding sequence variants between parental strain B6 and D2, cis-regulation in heart tissue, correlation between gene expression and cardiomyocyte size, and functional relevance. The venn diagram shows the number of overlapped genes among the five catalogs.

Figure 4:

Xirp2 served as the candidate gene for the cardiomyocyte size QTL on Chr 2. (A) Xirp2 gene expression atlas across mouse and human tissues. Expression levels were represented by Reads Per Kilobase Million (RPKM). Data extracted from NCBI (https://www.ncbi.nlm.nih.gov). (B) Interval mapping of Xirp2 expression demonstrating this gene was cis-regulated (cis-eQTL). The location of Xirp2 was marked with a triangle on x-axis. (C) Abnormal phenotypes were observed in Xirp2 malfunction mouse. Data obtained from Mouse Genome Database (MGI, http://www.informatics.jax.org/).

Figure 5:

Nos3 serves as the candidate gene for the cardiomyocyte size QTL on Chr 5. (A) Nos3 gene expression atlas across mouse and human tissues. Expression levels were represented by Reads Per Kilobase Million. Data extracted from NCBI (https://www.ncbi.nlm.nih.gov). (B) Interval mapping of Nos3 expression demonstrating this gene was cis-regulated (cis-eQTL). The location of Nos3 was marked with a triangle on x-axis. (C) Nos3 expression in the heart correlated with cardiomyocyte size. This analysis was done by Spearman rank order correlations using tool in GeneNetwork (www.genenetwork.org). (D) Abnormal phenotypes were observed in Nos3 malfunction mouse. Data obtained from Mouse Genome Database (MGI, http://www.informatics.jax.org/).

Figure 6:

Correlations between echocardiographic parameters and cardiac expressions of Xirp2 and Nos3. The expression level of Xirp2 was negatively correlated with cardiac output (CO) (panel A). The expression level of Nos3 was negatively correlated with left ventricle posterior wall thickness at systole (LVPW) (panel B).