Molecular analysis of RNF213 gene for moyamoya disease in the Chinese Han population

Abstract

Background: Moyamoya disease (MMD) is an uncommon cerebrovascular disorder characterized by progressive occlusion of the internal carotid artery causing cerebral ischemia and hemorrhage. Genetic factors in the etiology and pathogenesis of MMD are being increasingly recognized. Previous studies have shown that the RNF213 gene was related to MMD susceptibility in the Japanese population. However, there is no large scale study of the association between this gene and MMD in the Chinese Han population. Thus we designed this case-control study to validate the R4810K mutation and to define the further spectrum of RNF213 mutations in Han Chinese.

Methodology/principal findings: Genotyping of the R4810K mutation in the RNF213 gene was performed in 170 MMD cases and 507 controls from a Chinese Han population. The R4810K mutation was identified in 22 of 170 MMD cases (13%), including 21 heterozygotes and a single familial homozygote. Two of the 507 controls (0.4%) were heterozygous R4810K carriers. The R4810K mutation greatly increased the risk for MMD (OR = 36.7, 95% CI: 8.6~156.6, P = 6.1 E-15). The allele frequency of R4810K was significantly different between patients with ischemia and hemorrhage (OR = 5.4, 95% CI: 1.8~16.1, P = 0.001). Genomic sequencing covering RNF213 exon 40 to exon 68 also identified eight other non-R4810K variants; P4007R, Q4367L, A4399T, T4586P, L4631V, E4950D, A5021V and M5136I. Among them A4399T polymorphism was found in 28/170 cases (16.5%) and 45/507 controls (8.9%) and was associated with MMD (OR = 2.0, 95% CI: 1.2~3.3, P = 0.004), especially with hemorrhage (OR = 2.8, 95% CI: 1.2~6.5, P = 0.014).

Conclusions: RNF213 mutations are associated with MMD susceptibility in Han Chinese. The ischemic type MMD is particularly related to the R4810K mutation. However, A4399T is also a susceptible variant for MMD, primarily associated with hemorrhage. Identification of novel variants in the RNF213 gene further highlights the genetic heterogeneity of MMD.

Figure 1. Genetic variants heat map of RNF213 generated from 1000 genomes database.

(a) variants in whole genome of RNF213. (b) The 35kb highly variable region flanking RNF213 R4810K mutation. The intensity of blue bars indicates the predicted risk of each variation.

Figure 2. High resolution melting analysis for genotyping of RNF213 R4810K.

(a) Derivative melting curves of unlabeled probes and amplicon. Three genotypes (AA, AG, and GG) were discriminated as indicated in the probe region. (b) Difference curves obtained by subtracting each curve from one heterozygote (AG) curve. Three genotypes (AA, AG, and GG) are shown.

Figure 3. Genetic background of the five familial cases.

(a) Family 1, two brothers both harboring a heterozygous R4810K mutation. (b) Family 2, mother and aunt are whole sisters with A4399T heterozygote and R4810K homozygote respectively. The daughter has both the heterozygous A4399T and R4810K variants. All the patients were diagnosed with the ischemia subtype.

Figure 4. Novel mutations found by genomic sequencing of RNF213.

Results from direct sequencing analyzed by Mutation Surveyor 4.06. Mutations are indicated by arrow. (a) g.107149C>G (p.P4007R). (b) g.114926A>T (p.Q4367L). (c) g.115456G>A (p.A4399T). (d) g.120087A>C (p.T4586P). (e) g.120781C>G (p.L4631V). (f) g.125960G>C (p.E4950D). (g) g.128375C>T (p.A5021V). (h) g.129275G>A (p.M5136I).