Hypospadias is a common congenital malformation, caused by failure of fusion of the urethral folds, endodermal differentiation, and ectodermal ingrowth in gestational 8 to 20 weeks, and the severity of hypospadias depends on the time of fusion fails in embryonic period11,12. The etiology of hypospadias is multifactorial and highly heterogeneous, including genetic factors and environmental factors. Environmental factors include early placental malfunction, low birthweight, maternal hypertension, pre-eclampsia, maternal intrauterine diethylstilbestrol exposure, use of intracytoplasmic sperm injection (ICSI), prolonged time-to-pregnancy, high maternal BMI, primiparity, multiple pregnancy, pre-existing maternal diabetes, maternal medication use: anti-epileptic drugs etc13–15. It is generally believed that hypospadias is caused by multiple genetic factors rather than a single gene16. In order to find more suspected risk loci for hypospadias, GWAS and analysis of SNPs are increasingly used over recent years. In 2014, Geller et al7 identified 17 SNPs in European population. Later, Kojima et al8 replicated all of these SNPs in Japanese population found out two SNPs (rs6499755 and rs3816183) associated with hypospadias. Recently, Liu et al9 replicated rs3816183 in Southern Han Chinese population, it was also found that there was a certain correlation between the locus and hypospadias. Our study aimed to replicate the results of rs6499755 and rs3816183 using different races. Firstly, we designed a case-control analysis to assess the relationship between these two SNPs and hypospadias susceptibility in a Northern Han Chinese population. Next, we performed a meta-analysis to evaluate the relationship in multiple races, based on the results of our study and three previous studies.
Through case-control analysis, we found that IRX6 rs6499755 was linked to increased risk of hypospadias (OR = 1.547, p = 0.01), especially anterior (OR = 3.579, p = 0.003) and posterior hypospadias (OR = 1.737, p = 0.005), which probably through recessive models (OR = 1.832, p = 0.026). We also observed HAAO rs3816183 had an associated with increased risk of anterior/middle (OR = 1.775, p = 0.046) hypospadias, especially middle hypospadias (OR = 2.130, p = 0.028). Besides, there was a cumulative effect between the frequency of two risk alleles and the risk of hypospadias (OR = 1.337, p = 0.02). By performing meta-analysis, we found a significant associated between IRX6 rs6499755 / HAAO rs3816183 with the increased risk of hypospadias (ORs = 1.46, p < 0.00001; ORs = 1.23, p < 0.00001, independently). In the subgroup of anterior/middle hypospadias, we observed a mild related with IRX6 rs6499755(ORs = 1.30, p = 0.06) and a significantly associated with HAAO rs3816183 (ORs = 1.39, p = 0.0002). Nevertheless, the further biological mechanisms of IRX6 rs6499755/ HAAO rs3816183 result in the increased hypospadias risks remain unclear.
IRX6 (iroquois homeobox 6) belongs to homeobox genes of the Irx family IrxB cluster17. The Irx family, as regulators of development, plays an important role in embryo proliferation and differentiation, including proliferation of early embryonic cells, directed differentiation of embryonic cells, and organ formation18,19. There has been reported that Irx6 expression was observed in multiple tissues in the process of embryonic development20,21. During external genitalia development of male mouse embryos, IRX6 expresses in ectodermal epithelium, particularly in dorsal ectodermal, and no expression was found in the ventral ectoderm adjacent to the urethral plate8. In both case-controls analysis and meta-analysis, the results revealed that the risk allele [C] of IRX6 rs6499755[C] increase the risk of hypospadias, which greatly increases the reliability of this kind of relationship. It is reported that SNP loci located in other homologous gene box families (such as HOXA cluster, IRX3, IRX5, ZFHX3, etc.) also increase the risk of hypospadias7, which may suggest the focus of the future research. Different from the results of Kojima in Japanese population, we found the association between IRX6 rs6499755 and increased risk of general, anterior and posterior hypospadias, and no significant association with anterior/middle hypospadias in Chinese population. However, the meta-analysis involving data from these two studies found a mild related between the SNPs and anterior/middle hypospadias risk. This may be resulted from ethnic differences, considered the high similarity of origin between the two races, it is more likely to be caused by the small sample size and the low weight of this study in the meta-analysis. In future, studies with larger sample sizes and higher quality are needed.
HAAO (3-hydroxyanthranilate-3,4-dioxygenase) is an enzyme and widely distributed in various organs. The function of HAAO is catalyzing the kynurenine pathway from tryptophan to quinolinic acid22. Hence, it plays a role in disorders associated with altered tissue levels of quinolinic acid, such as neuronal damage23. Although HAAO has been associated with fetal malformation and death in mouse models24, there is no significant upregulation of HAAO expression was observed in the developing male mouse embryos external genitalia8. Up to now, there is still no evidence that HAAO is involved in human male genital development in embryonic period. In our study we found HAAO rs3816183 associated with hypospadias in both of European and Asian population, however, we lacked the data of clinical subgroup analysis in European. In Asian population, we observed the association between HAAO rs3816183 with anterior/middle hypospadias by subgroup analysis. Base on existing data, we doubted HAAO rs3816183 polymorphism may give rise to hinder the process of fusion of the urethral folds with the midline on the ventrum of the penile shaft, especially in middle and advanced stage by changing circulating metabolites in the kynurenine pathway in Asians25.
Although we observed relatively positive results though our work, there are still some limitations, which remain to be improving in the future. First, the samples of the case-controls analysis are relatively small, which may result in the decrease of results veracity. A larger multicenter study is needed to determine the association between SNPs and hypospadias in Chinese population. What’s more, there are few reports on the association of these two loci with hypospadias, as well as the possible existence of unpublished negative results, causing the limitation of meta-analysis. We included only European and Asian populations with unequal distribution of data weights across populations, which may influence the results. It suggests the importance of multinational collaborative to investigate the association between SNPs and hypospadias.
Overall, our study indicates that there are association between variations of IRX6 and HAAO and hypospadias risk in a Chinese population. Furthermore, the results of meta-analysis support identified a strong relationship between the IRX6 and HAAO polymorphisms and hypospadias susceptibility in multiple populations. Further researches with larger sample size involving multiethnic will be needed to validate our results. Besides, more in-depth and comprehensive studies are needed to explore the biological pathogenic mechanism of IRX6 and HAAO leading to hypospadias.