Mutations of <i>RNF213</i> are responsible for sporadic cerebral cavernous malformation and lead to a mulberry-like cluster in zebrafish

Abstract

Although familial forms of cerebral cavernous malformation are mainly attributed to three <i>CCM</i> genes (<i>KRIT1</i>, <i>CCM2</i> and <i>PDCD10</i>), no mutation is identified in sporadic cerebral cavernous malformation cases with a unique lesion, indicating additional genes for sporadic cerebral cavernous malformation. To screen the candidate genes, we conducted whole exome sequencing in 31 sporadic cerebral cavernous malformation patients and 32 healthy controls, and identified 5 affected individuals carrying 6 heterozygous deleterious mutations in <i>RNF213</i> but no <i>RNF213</i> mutation in healthy individuals. To further confirm <i>RNF213</i> was associated with cerebral cavernous malformation, we generated <i>rnf213a</i> homozygous knockout zebrafish and found mutation of <i>rnf213a</i> in zebrafish led to a mulberry-like cluster of disordered-flow vascular channels which was reminiscent of human cerebral cavernous malformation. In addition, we revealed <i>kbtbd7</i> and <i>anxa6</i> were significantly downregulated due to <i>rnf213a</i> mutation through transcriptomic sequencing and RT-qPCR analysis. Based on the mulberry-like phenotype partly rescued by mRNA of <i>kbtbd7</i> as well as <i>anxa6</i>, we suggested that <i>rnf213a</i> promoted mulberry-like cluster via downregulation of <i>kbtbd7</i> and <i>anxa6</i>. Altogether, we firstly demonstrate <i>RNF213</i>is a novel candidate gene for sporadic cerebral cavernous malformation and the mutation of <i>rnf213a</i> is responsible for the mulberry-like cluster in zebrafish.