Rare Coding Variants in ANGPTL6 Are Associated with Familial Forms of Intracranial Aneurysm

Abstract

Intracranial aneurysms (IAs) are acquired cerebrovascular abnormalities characterized by localized dilation and wall thinning in intracranial arteries, possibly leading to subarachnoid hemorrhage and severe outcome in case of rupture. Here, we identified one rare nonsense variant (c.1378A>T) in the last exon of ANGPTL6 (Angiopoietin-Like 6)-which encodes a circulating pro-angiogenic factor mainly secreted from the liver-shared by the four tested affected members of a large pedigree with multiple IA-affected case subjects. We showed a 50% reduction of ANGPTL6 serum concentration in individuals heterozygous for the c.1378A>T allele (p.Lys460Ter) compared to relatives homozygous for the normal allele, probably due to the non-secretion of the truncated protein produced by the c.1378A>T transcripts. Sequencing ANGPTL6 in a series of 94 additional index case subjects with familial IA identified three other rare coding variants in five case subjects. Overall, we detected a significant enrichment (p = 0.023) in rare coding variants within this gene among the 95 index case subjects with familial IA, compared to a reference population of 404 individuals with French ancestry. Among the 6 recruited families, 12 out of 13 (92%) individuals carrying IA also carry such variants in ANGPTL6, versus 15 out of 41 (37%) unaffected ones. We observed a higher rate of individuals with a history of high blood pressure among affected versus healthy individuals carrying ANGPTL6 variants, suggesting that ANGPTL6 could trigger cerebrovascular lesions when combined with other risk factors such as hypertension. Altogether, our results indicate that rare coding variants in ANGPTL6 are causally related to familial forms of IA.

Trial registration: ClinicalTrials.gov NCT02848495.

Keywords: ANGPTL6; burden test; genetics; intracranial aneurysm; whole-exome sequencing.

Figure 1

Genetic Investigations in a Large Family with Multiple IA-Affected Case Subjects (A) Pedigree of family A showing the segregation pattern of the variant ANGPTL6 c.1378A>T. Filled boxes, empty boxes, and boxes with question marks indicate IA-affected case subjects, non-affected relatives, and individuals with unknown status; plus sign (+) indicates the presence of the ANGPTL6 variant; minus sign (−) indicates its absence; arrow indicates the index case subject; and asterisks indicate the individuals included in WES analysis. (B) Digital substracted angiographies showing two intracranial aneurysms carried by the index case III-1, one on the middle cerebral artery (left) and one on the anterior cerebral artery (right). (C) Summary of the filtering steps applied to genetic variants detected by WES in individuals III-1 and III-5. Abbreviations: MAF, minor allele frequency; IBD, identity by descent; LOF, loss of function.

Figure 2

Familial Case Subjects of IA in the Presence of Rare Coding Variants in ANGPTL6 Filled boxes, empty boxes, and boxes with question marks indicate IA-affected case subjects, non-affected relatives, and individuals with unknown status; plus sign (+) indicates the presence of the ANGPTL6 variant; minus sign (−) indicates its absence; and black arrows indicate the index case subjects.

Figure 3

Analysis of WT and p.Lys460Ter ANGPTL6 in Cultured Cells and Individual Sera (A) Analysis by qPCR of ANGPTL6 transcripts in HEK293 cells encoding WT and p.Lys460Ter ANGPTL6. (B) Analysis by western blot (anti-FLAG Ab) and ELISA of the levels of WT and p.Lys460Ter ANGPTL6 in culture media and lysates from stably transfected HEK293. (C) Immunofluorescence labeling with anti-ANGPTL6 Ab and corresponding quantification in permeabilized HEK293 cells expressing WT and p.Lys460Ter ANGPTL6. (D) Analysis of serum levels of ANGPTL6 in control subjects (WT ANGPTL6) and individuals harboring the p.Lys460Ter ANGPTL6 variant (heterozygous) All results are expressed as means and error bars indicate standard deviation. Significant differences between independent groups were assessed by the Mann-Whitney test (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001).