Abstract
Tissue remodelling and fibrosis which occur in response to injury play a central role in the development of many diseases. Chymase is a key enzyme believed to mediate these pathological processes. As such, chymase inhibitors have been under active development for the treatment of a number of conditions. To investigate the impact of reduced chymase function, we constructed a genetic score from two pLoF mutations in the gene encoding chymase and tested its association with diseases and biomarkers. Our study found no association between the genetically-predicted reduced chymase function score and heart failure, chronic kidney disease or other predefined conditions. We additionally found no association of the score with any physical measurements or biomarkers. Our results provide no evidence in support of chymase inhibition as a novel therapeutic strategy for the treatment or prevention of heart failure, chronic kidney disease or major cardiovascular events, as previously proposed.
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Chymase is a serine protease secreted from mast cells in response to tissue injury and inflammation. It is encoded in humans by the CMA1 gene. The enzyme contributes, via its role in conversion of Angiotensin I to Angiotensin II and activation of the proteins MMP-9 and TGF-β, towards the processes of fibrosis and tissue remodelling which underlie the pathology of numerous diseases [1]. Thus, chymase inhibition may represent a novel treatment for various chronic conditions.
Animal studies showed that cardiac fibrosis and renal dysfunction can be reduced by chymase inhibition, leading to the hypothesis that chymase inhibitors may be beneficial for treatment of heart failure (HF) and chronic kidney disease (CKD) [1, 2]. However, the relevance of these results to humans is unclear, particularly, as the model animals studied express multiple chymase isoforms [2]. Phase 1 clinical trials have provided evidence on the safety but not efficacy of chymase inhibitors [1]. To inform ongoing and future clinical development programmes, we used predicted loss-of-function (pLoF) mutations within CMA1 to assess the potential clinical utility and safety of chymase inhibition for the treatment/prevention of HF, CKD and other conditions, in a large prospective biobank study.
We identified two variants in CMA1, rs150310098 and rs13306254, that were high confidence pLoF mutations (https://gnomad.broadinstitute.org). It is predicted that rs150310098 causes loss of a splice donor site while rs13306254 introduces an early stop codon, with both predicted to result in reduced levels of functional chymase. The two variants are present at appreciable frequencies (~ 1% minor allele frequency [MAF]) in East Asians but are rare (MAF < 1 × 10−4) in other ancestries. To investigate the impact of these pLoF variants, we used data on up to 100,578 genotyped participants from the China Kadoorie Biobank (CKB) [3] of adults from ten areas of China, with follow-up via linkage to disease and death registries and hospital admission records. The variants are present on different haplotypes (LD R2 = 1.2 × 10−4) and were combined additively into an unweighted score for genetically-predicted reduced chymase function (RCF). Using linear and logistic regression models, we associated the genetic score with a range of physical measurements, biomarkers and disease endpoints. Primary endpoints were selected based on the main indications in clinical development programmes. Secondary endpoints were selected to assess potential adverse effects or alternative indications for chymase inhibition.
In CKB, rs150310098 and rs13306254 had an overall MAF of 0.013 (range by 10 study areas 0.009–0.016) and 0.017 (0.010–0.023), respectively. There was no significant association between genetically predicted RCF and physical measurements or biomarkers (P < 0.002; Bonferroni adjusted threshold) (Fig. 1A). Of note, the RCE score had no association with three quantitative measures of kidney function; cystatin C, creatinine and eGFR (N ~ 17,000). In ~ 5500 participants with creatinine measured using NMR, there was also no association between the score and creatinine levels (Beta = − 0.01 SD, 95% CI (− 0.10, 0.08), P = 0.83). Genetically-predicted RCF had no association with blood pressure or heart rate (Fig. 1A). This is in line with findings from the phase 1 clinical trial, which found chymase inhibitors had no impact on these traits despite the role of angiotensin II in modulation of blood pressure [1]. We also assessed associations of the score with 12 ECG variables, 225 metabolites (Nightingale NMR platform) and 92 proteins (OLINK immune-oncology panel) measured in smaller subsets of participants but found no significant associations (data not shown).
Genetically-predicted RCF was not significantly associated with the pre-defined primary study endpoints: HF, CKD or major cardiovascular events (Fig. 1B). Similarly, there were no associations of the score with the secondary disease endpoints (Fig. 1B). Notably, we found no association with asthma or atrial fibrillation, conditions suggested to be affected by the downstream consequences of chymase’s enzymatic activity and previously reported to show associations with non-functional variants within CMA1 [4, 5].
The low MAF of the variants and low case numbers for HF and CKD meant our study had ~ 80% power to detect an effect size of 1.8 for these conditions, but was underpowered to reject the null hypothesis for smaller effect sizes. To further investigate the impact of RCF, we combined the effect sizes of the pLOF variants from GWAS summary statistics for HF and nephrotic syndrome in Biobank Japan (http://jenger.riken.jp/en/result) into a single effect size for the RCF score using fixed-effects meta-analysis. Neither condition showed a significant association with the genetically-predicted RCF score in Biobank Japan (HF: total cases = 9413, OR = 0.96, 95% CI (0.84–1.09), P = 0.54; Nephrotic syndrome: total cases = 957, OR = 0.82, 95% CI (0.55–1.22), P = 0.33).
These results provide no evidence in support of chymase inhibition as a novel therapeutic option for the treatment or prevention of HF, CKD or major cardiovascular events, as previously proposed [1]. Given that the variants studied were rare in Europeans, this work highlights the importance of large, ancestrally diverse cohorts which widen the available pool of pLoF variants, and supports the use of genetic approaches to provide insights into drug target development.
Abbreviations
- HF:
-
Heart failure
- CKD:
-
Chronic kidney disease
- pLoF:
-
Predicted loss-of-function
- MAF:
-
Minor allele frequency
- CKB:
-
China Kadoorie Biobank
- RCF:
-
Reduced chymase function
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Acknowledgements
The chief acknowledgment is to the participants, the project staff and the China National Centre for Disease Control and Prevention (CDC) and its regional offices for access to death and disease registries. The Chinese National Health Insurance scheme provided electronic linkage to all hospital admission data.
Funding
The CKB baseline survey and the first re-survey were supported by the Kadoorie Charitable Foundation in Hong Kong. The long-term follow-up has been supported by Wellcome grants to Oxford University (212,946/Z/18/Z, 202,922/Z/16/Z, 104,085/Z/14/Z, 088,158/Z/09/Z) and grants from the National Key Research and Development Program of China (2016YFC0900500, 2016YFC0900501, 2016YFC0900504, 2016YFC1303904) and from the National Natural Science Foundation of China (91,843,302). DNA extraction and genotyping was supported by grants from GlaxoSmithKline and the UK Medical Research Council (MC-PC-13049, MC-PC-14135). The UK Medical Research Council (MC_UU_00017/1,MC_UU_12026/2 MC_U137686851), Cancer Research UK (C16077/A29186; C500/A16896) and the British Heart Foundation (CH/1996001/9454) provide core funding to the Clinical Trial Service Unit and Epidemiological Studies Unit at Oxford University for the project. This study was partially funded by a research grant from Bayer AG.
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The China Kadoorie Biobank (CKB) complies with all the required ethical standards for medical research on human subjects. Ethical approvals were granted and have been maintained by the relevant institutional ethical research committees in the UK and China. Informed consent was obtained from all participants included in the study. No animal studies were carried out by the authors for this article.
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Drs. Freitag and Zink are employees of Bayer AG. No other disclosures were reported.
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Fairhurst-Hunter, Z., Walters, R.G., Zink, A. et al. Investigation into the Health Effects of Reduced Chymase Function Using Predicted Loss-of-Function Mutations in CMA1. J. of Cardiovasc. Trans. Res. 15, 1474–1476 (2022). https://doi.org/10.1007/s12265-022-10261-w
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DOI: https://doi.org/10.1007/s12265-022-10261-w