Associations of CYP2B6 genetic polymorphisms with Hirschsprung’s disease in a southern Chinese population

Abstract Background Hirschsprung’s disease (HSCR) is an enteric nervous system birth defect partially caused by a genetic disorder. Single‐nucleotide polymorphisms (SNPs) of the cytochrome P450 family 2 subfamily B member 6 (CYP2B6) gene are reported to be associated with HSCR. Methods We evaluated the association of rs2054675, rs707265, and rs1042389 with HSCR susceptibility in southern Chinese children including 1470 HSCR patients and 1473 controls using the TaqMan SNP Genotyping Assay. Results rs2054675 C allele and the rs707265 G allele were risk SNPs for total colonic aganglionosis (OR = 1.82, 95% CI 1.29 ~ 2.55, P_adj < 0.001 and OR = 0.68, 95% CI 0.48 ~ 0.97, P_adj = 0.034). These results suggested that CYP2B6 rs2054675 and rs707265 polymorphisms were associated with increased susceptibility to the severe HSCR subtype in southern Chinese children. Conclusion We suggest that CYP2B6 rs2054675 and rs707265 polymorphisms are associated with increased susceptibility to the severe HSCR subtype in southern Chinese children.

malformations and chromosomal abnormalities, HSCR can be divided into simple and syndrome types, and the most common comorbidity is trisomy 21. 4 Moreover, there are familial and sporadic types of HSCR according to family inheritance. More than one-fifth of HSCR cases show familial aggregation, whereas most HSCR cases are sporadic. 5 The pathogenesis of HSCR is complex and involves multiple genes and multiple signaling pathways. A genome-wide association study (GWAS) has identified a variety of genes associated with a high risk of HSCR in the RET and EDNRB signaling pathways, which play important roles in the migration of enteric neuron crest cells during the development of the enteric nervous system (ENS). 6 RET intronic enhancer rs2435357 T allele, rs2506004 C>A were associated with a 4-fold increase in risk of HSCR. These variants can alter the binding of transcriptional factors (SOX10, ARNT5/NXF, and HOXB5) and decreased the expression of RET gene. 7 Other common variants of SEMA3C, SEMA3D, NRG1, 19q12, 3p21, VAMP5, and MCC are widely reported to be independently or synergistically associated with the risk of HSCR. 8,9 However, only a small proportion of HSCR cases can be explained by these known factors; for most sporadic cases, missing heritability remains to be identified. Therefore, it is believed that interaction between environmental and genetic factors plays a crucial role in the pathogenesis of HSCR, 10 which remains barely explored.
The human CYP2B6 gene belongs to the human cytochrome P450 enzyme system (CYPs), which are involved in the metabolism of fatty acids, cholesterol, bile acids, vitamin D, retinoids, and eicosanoids. 11 They play a role in some diseases occurring during embryogenesis and infantile development. 12 Notably, P450 family members are environmental responders to dietary components, chemical inducers and signals (i.e., pheromones), drugs, etc. 13 These three SNPs (rs707265, rs1042389, and rs2054675) which were likely to be regulatory variants and satisfied the criteria regarding the minor allele frequency, Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium, were validated using a cohort (262 cases and 290 controls) from eastern Chinese population. 14 Considering distinctive dietary and toxic or drug exposure between eastern and southern Chinese population, we conducted an association study in a southern Chinese population (1470 cases and 1473 controls) to evaluate the association between CYP2B6 polymorphism and susceptibility to HSCR. This study will provide a hint of environmental factors for pathogenesis of HSCR.

| Study subjects
1740 HSCR patients and 1473 controls from southern China were recruited from Guangzhou Women and Children's Medical Center as described previously, 8 and their detailed clinical information is summarized in Table 1

| SNP selection and genotyping
CYP2B6 rs707265, rs1042389, and rs2054675 were selected using criteria as described in our previous study. 15 Briefly, the candidate SNPs that were likely to be regulatory variants and satisfied the criteria regarding the minor allele frequency, Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium were selected for validation.
TIANamp Blood Genomic DNA Kits and TIANquick FFPE DNA Kits (TIANGEN Biotech Co. Ltd.,) were applied to isolate genomic DNA from venous blood and paraffin samples. Subsequently, CYP2B6 SNPs were genotyped using the TaqMan SNP Genotyping Assay on an ABI-7900 real-time quantitative PCR instrument (Applied Biosystem). 16 Three replicates were performed for each sample.

| Correlation analysis of genotype and gene expression
The associations between 3 SNPs (rs2054675, rs707265, and rs1042389) and CYP2B6 gene expression in colon tissues, or the Two-tailed χ2 test of the distribution between HSCR cases and controls.

| Statistical analysis
The statistical analysis of all data in this study was performed by using SAS (version 9.4; SAS Institute). The differences in age or sex between the HSCR and control groups were compared using a two-tailed chisquare test. The Hardy-Weinberg equilibrium test was performed in the control group to assess genotyping quality, and p > 0.05 was considered to indicate a satisfactory goodness-of-fit. Multiple logistic regression analysis was applied to assess the association of CYP2B6 polymorphisms with the risk of HSCR as well as HSCR subtypes (S-HSCR, L-HSCR, and TCA). P_crude and P_adj indicate the association significance without or with adjusting the effects of age and sex. Odds ratios (ORs) were compared between the HSCR and control groups.

| eQTL analysis
To investigate the functional potential of the SNPs, we used the Genotypic Tissue Expression (GTEx) dataset to evaluate the associations of rs2054675, rs707265, and rs1042389 with CYP2B6 expression. 17 We found that rs2054675 and rs707265 were significant splicing quantitative trait loci (eQTLs) (p = 1.3e −6 ) and expression quantitative trait loci (eQTLs) (p = 3.2e −8 ) of the CYP2B6 gene in colon tissues, but rs1042389 was not significant (Figure 1). The results verified regulatory potential of rs2054675 and rs707265.

| Association of CYP2B6 SNPs with HSCR susceptibility
Subsequently, the genotype frequencies of rs2054675, rs707265, and rs1042389 and their associations with HSCR were calculated and are summarized in

| Stratification analysis of CYP2B6 SNPs with HSCR subtypes
Considering the effects of HSCR subtypes in the population, a stratification analysis of the three SNPs with HSCR subtypes was per-  (Table 3).

| DISCUSS ION
The human cytochrome P450 enzyme system (CYP) has been reported to mediate some diseases of neonatal development. CYP2B6 is one of the most important exogenous toxic metabolic enzymes in the CYP family. CYP2B6 is mainly distributed in the human liver, GI tract, pancreas, kidney, and reproductive system and participates in the synthesis and metabolism of a variety of endogenous and exogenous substances. 22,23 The substantial expression of CYP2B6 in the GI tract indicates a potential role in enteric diseases.
CYP2B6 gene polymorphism is related to the occurrence of bronchopulmonary dysplasia (BPD) (especially in infants with extremely low birth weight), 24 acute myeloid leukemia (AML), 25 and breast cancer. 26 Reduced activity of CYP2B6 enzyme due to gene polymorphism is associated with increased response to drug sensitivity and infection. [27][28][29][30] P450 family members, encoding exogenous toxic metabolic enzymes, are environmental responders to dietary components, chemical inducers and signals (i.e., pheromones), drugs, etc., 13  Chinese population, but none of the three SNPs were significantly associated with HSCR subtype. 14 However, in this study, the association of CYP2B6 SNPs with HSCR was not validated, which might reflect different toxic factors from the environment between eastern and southern Chinese population.
Additionally, we found that CYP2B6 rs2054675 and rs707265 polymorphisms are associated with increased susceptibility to the severe HSCR subtype(TCA) in southern Chinese children. TCA is the most severe and rare subtype of HSCR with 1% incidence rate. Previous studies showed that genetic factors were supposed to contribute more to the pathogenesis of TCA than other subtypes, 36

| CON CLUS ION
In conclusion, we suggest that CYP2B6 rs2054675C and rs707265 G alleles are associated with increased susceptibility to the severe HSCR subtype in southern Chinese children. These results indicated different toxic factors from the environment affecting HSCR between eastern and southern Chinese population, which provides a hint of environmental factors for pathogenesis of HSCR. Further study should recruit more TCA patients and include toxic or drug exposure in the association analysis to elucidate environmental factors affecting HSCR.

ACK N OWLED G M ENTS
We acknowledge the Genotype-Tissue Expression (GTEx) Project as the source of eQTL figures.

CO N FLI C T S O F I NTE R E S T
The authors declare that there are no conflicts of interest regarding the publication of this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
All of the data used to support the findings of this study are available from the corresponding author upon request.