Background: High blood pressure (BP) is globally the most important risk factor for cardiovascular disease. Recently, several genome-wide association studies (GWAS) have identified variants at more than 270 loci with genome-wide significance. The post-GWAS challenge is to annotate the most likely causal gene at each of these loci. The locus at 10q24.32 encompasses four genes, namely AS3MT, CNNM2, CYP17A1 and NT5C2 and till date, it warrants investigation to pin down the specific gene to be responsible for the phenotype.

Aim: To identify the most likely causal gene at 10q24.32 locus using zebrafish as an animal model.

Methods: Blood flow in zebrafish has been reported to be directly proportional to blood pressure. We performed gene knockdown by injecting target specific translation blocking morpholinos for each of the four genes in freshly fertilized zebrafish embryos. Knockdown was confirmed by quantitative real-time PCR (qPCR).  Different parameters were measured including blood flow, arterial pulse, vessel diameter and linear velocity in 72hrs post hatched larvae. mRNA expression of hypertension specific markers such as glycine amidinotransferase  (GATM) and angiotensin I converting enzyme (ACE) was also measured.  

Results: Using custom-designed morpholinos (MO), we were able to knock down the expression by approximately 50% for each individual gene in zebrafish larvae, as confirmed by qPCR.  Interestingly, we observed a significantly higher blood flow, increased arterial pulse and linear velocity in cnnm2 and nt5c2 knocked down larvae as shown in the table below. No difference was observed for AS3MT and Cyp17a1 genes. The vessel diameter remained unchanged in all knocked down genes.

Table 1: Blood flow parameters data presented for each morpholino knockdown gene in comparison with their controls, presented in the table as control / MO. p values for each gene are mentioned below in each cell. 

 At the molecular level, the gene expression of hypertension markers (GATM and ACE) was found significantly increased in cnnm2 and nt5c2 knockdown larvae. The results obtained by morpholino knockdown were further validated in knockout lines of zebrafish obtained from the European Zebrafish Resource Center (EZRC), where again cnnm2 and nt5c2 deficiency was found to be responsible for higher blood flow, increased arterial pulse and linear velocity.