Genome-Wide Association Study of Susceptibility Loci for Radiation-Induced Brain Injury

Abstract

Background: Radiation-induced brain injury is a nonnegligible issue in the management of cancer patients treated by partial or whole brain irradiation. In particular, temporal lobe injury (TLI), a deleterious late complication in nasopharyngeal carcinoma, greatly affects the long-term life quality of these patients. Although genome-wide association studies (GWASs) have successfully identified single nucleotide polymorphisms (SNPs) associated with radiation toxicity, genetic variants contributing to the radiation-induced brain injury have not yet been assessed.

Methods: We recruited and performed follow-up for a prospective observational cohort, Genetic Architecture of Radiotherapy Toxicity and Prognosis, using magnetic resonance imaging for TLI diagnosis. We conducted genome-wide association analysis in 1082 patients and validated the top associations in two independent cohorts of 1119 and 741 patients, respectively. All statistical tests were two-sided.

Results: We identified a promoter variant rs17111237 (A > G, minor allele frequency [MAF] = 0.14) in CEP128 associated with TLI risk (hazard ratio = 1.45, 95% confidence interval = 1.26 to 1.66, Pcombined=3.18 × 10-7) which is in moderate linkage disequilibrium (LD) with rs162171 (MAF = 0.18, R2 = 0.69), the top signal in CEP128 (hazard ratio = 1.46, 95% confidence interval = 1.29-1.66, Pcombined= 6.17 × 10-9). Combining the clinical variables with the top SNP, we divided the patients into different subgroups with varying risk with 5-year TLI-free rates ranging from 33.7% to 95.5%. CEP128, a key component of mother centriole, tightly interacts with multiple radiation-resistant genes and plays an important role in maintaining the functional cilia, which otherwise will lead to a malfunction of the neural network. We found that A > G alteration at rs17111237 impaired the promoter activity of CEP128 and knockdown of CEP128 decreased the clonogenic cell survival of U87 cells under radiation. Noteworthy, 12.7% (27/212) of the GWAS-based associated genes (P < .001) were enriched in the neurogenesis pathway.

Conclusions: This three-stage study is the first GWAS of radiation-induced brain injury that implicates the genetic susceptibility gene CEP128 involved in TLI development and provides the novel insight into the underlying mechanisms of radiation-induced brain injury.

Figure 1.

The flow diagram of the study design. NPC = nasopharyngeal carcinoma; GARTP = Genetic Architecture of the Radiotherapy Toxicity and Prognosis; SNP = single nucleotide polymorphism; GWAS = genome-wide association study; TLI = temporal lobe injury; MRI = magnetic resonance imaging; RT = radiotherapy.

Figure 2.

Genome-wide association results and the estimated curves of TLI development in different stages and subgroups. A) Genome-wide –log₁₀P values from the survival analysis for 445 078 SNPs from the discovery stage are shown. The blue line represents P = 1.0 × 10^–5.B) The –log₁₀P values of the SNPs are shown according to their physical positions. The recombination rates (shown as blue lines) were estimated from the 1000 Genome ASN panel. The genotyped SNPs and the imputed SNPs are shown as circles and diamonds. The two variants rs17111237 and rs17111246 located within 2 kb upstream of CEP128 are in purple outlines. C) The estimated curves of TLI development among patients carrying minor-allele homozygotes, heterozygotes, and major-allele homozygotes at rs162171. HRs and two-sided Wald test P-values were calculated using multivariate Cox proportional hazard regression models assuming additive effect of minor alleles and adjusting age at NPC diagnosis, tumor stage, and radiotherapy technique as covariates. D) The partition trees constructed using the combined samples from the three-stage. Each node was split by the indicated variable that has the best performance in partitioning the samples into two groups. The curves in the terminal nodes reflect the TLI development in different subgroups. T = tumor; IMRT = intensity-modulated radiotherapy; TLI = temporal lobe injury; HR = hazard ratio; CI = confidence interval.

Figure 3.

Expression quantitative trait locus (eQTL) analysis and functional characterizations of the protective role of CEP128 gene in radiosensitivity. A,B) Visualization of association trends for rs162171, rs17111237, and CEP128 mRNA expression in (A) HapMap CHB (Han Chinese in Beijing, China) and JPT (Japanese in Tokyo, Japan) and (B) in the intralobular white matter of human brain in the UK brain expression consortium (UKBEC). Gene expression values observed in each individual are shown by points. The correlation was represented by ρ and P values were calculated by Spearman correlation test. C) The means ± SD (N = 3) of luciferase activity detection. “CEP128 promoter WT” means luciferase vector containing a segment of CEP128 promoter with rs1711237A and rs1711246G, “CEP128 promoter variant” means luciferase vector containing a segment of CEP128 promoter with rs1711237G and rs1711246A. The numbers 1, 2, 3, 4 in the x-axis indicate four independent luciferase vectors. D) The means of relative mRNA expression levels (N = 2) of CEP128 after stable inhibition of endogenous CEP128 using shRNA in the U87 cell line. shCEP128–1, 2, and 3 indicate three shRNAs targeting different regions of CEP128 mRNA. E) The clonogenic survival curve was established on day 10 after stable transfected cells received indicated doses of radiation. Survival fractions were calculated. Data represent means ± SD (N = 3) and P values were calculated by two-sided t-test for shRNA vs sh-control.