AUTHOR=Vishnolia Krishan K. , Hoene Celine , Tarhbalouti Karim , Revenstorff Julian , Aherrahrou Zouhair , Erdmann Jeanette 

TITLE=Studies in Zebrafish Demonstrate That CNNM2 and NT5C2 Are Most Likely the Causal Genes at the Blood Pressure-Associated Locus on Human Chromosome 10q24.32

JOURNAL=Frontiers in Cardiovascular Medicine

VOLUME=7

YEAR=2020

URL=https://www.frontiersin.org/journals/cardiovascular-medicine/articles/10.3389/fcvm.2020.00135

DOI=10.3389/fcvm.2020.00135

ISSN=2297-055X

ABSTRACT=<p><bold>Background:</bold> Globally, high blood pressure (BP) is the most important risk factor for cardiovascular disease. Several genome-wide association studies (GWAS) have identified variants associated with BP traits at more than 535 chromosomal loci with genome-wide significance. The post-GWAS challenge is to annotate the most likely causal gene(s) at each locus. Chromosome 10q24.32 is a locus associated with BP that encompasses five genes: <italic>CYP17A1, BORCS7, AS3MT, CNNM2</italic>, and <italic>NT5C2</italic> and warrants investigation to determine the specific gene or genes responsible for the phenotype.</p><p><bold>Aim:</bold> To identify the most likely causal gene(s) associated with BP at the 10q24.32 locus using zebrafish as an animal model.</p><p><bold>Results:</bold> We report significantly higher blood flow, increased arterial pulse, and elevated linear velocity in zebrafish larvae with <italic>cnnm2</italic> and <italic>nt5c2</italic> knocked down using gene-specific splice modification transcriptional morpholinos, compared with controls. No differences in blood-flow parameters were observed after <italic>as3mt, borcs7</italic>, or <italic>cyp17a1</italic> knockdown. There was no effect on vessel diameter in animals with any of the four genes knocked down. At the molecular level, expression of hypertension markers (<italic>crp</italic> and <italic>ace</italic>) was significantly increased in <italic>cnnm2</italic> and <italic>nt5c2</italic> knockdown larvae. Further, the results obtained by morpholino knockdown were validated using zebrafish knockout (KO) lines with <italic>cnnm2</italic> and <italic>nt5c2</italic> deficiency, again resulting in higher blood flow, increased arterial pulse, and elevated linear velocity. Analysis of <italic>nt5c2a</italic> KO larvae demonstrated that lack of this gene resulted in reduced expression of <italic>cnnm2a</italic>, with reciprocal downregulation of <italic>nt5c2a</italic> in <italic>cnnm2a</italic> KO larvae. Staining of whole-blood smears from <italic>nt5c2</italic> mutants revealed that KO of this gene might be associated with an acute lymphoblastic leukemia phenotype, consistent with literature reports. Additional experiments were designed based on previous literature on <italic>cnnm2a</italic> mutant zebrafish revealed impaired renal function, high levels of renin, and significantly increased expression of the <italic>ren</italic> gene, leading us to hypothesize that the observed elevated blood-flow parameters may be attributable to triggering of the renin–angiotensin–aldosterone signaling pathway.</p><p><bold>Conclusion:</bold> Our zebrafish data establish <italic>CNNM2</italic> and <italic>NT5C2</italic> as the most likely causal genes at the 10q24.32 BP locus and indicate that they trigger separate downstream mechanistic pathways.</p>