Hypospadias is one of the most common congenital external genital malformations, which is characterized by abnormal urethral meatus (1), and affects approximately 20.9 out of every 10,000 births and has shown significant increases worldwide (2). Over the past decade, an increasing trend in the prevalence of hypospadias has been observed in China (3, 4). The clinical characteristics of hypospadias include proximal urethral opening, ventrally deficient hooded prepuce, and chordee (5). Hypospadias can be classified into two subgroups based on the urethral meatus location: anterior/middle hypospadias and posterior hypospadias (6). The meatus localization is best evaluated during surgery when chordee is corrected.

Although the surgical approach to hypospadias treatment has a great progress over the past decades, its etiology remains incompletely understood (1, 7–9). Individual phenotypic differences, such as disease susceptibility, survival, and treatment response, were identified to be associated with different genetic variants (10). Genetic variants have been observed to be associated with hypospadias risk (11, 12). However, very few studies have focused on variants in potential genes, such as DGKK, MAMLD1, MID1, CYP1A1, GSTM1, and GSTT1, which are associated with susceptibility to hypospadias (9). Some single-nucleotide polymorphisms (SNPs) have been reported in association to hypospadias. Nevertheless, recent studies used small sample sizes and have not been consistently replicated (13, 14).

Geller et al. conducted a genome-wide association study (GWAS) and reported that 17 SNPs were independently associated with hypospadias (15). Yoshiyuki validated these 17 SNPs in a Japanese cohort. However, only HAAO rs3816183 T>C was significantly associated with an increased risk toward hypospadias (16). Considering that ethnic differences exist at some loci, it would prove meaningful to evaluate the effect of SNPs on hypospadias susceptibility in different ethnic groups. Thus, we conducted this study to validate the association of HAAO rs3816183 T>C polymorphism with hypospadias susceptibility.

Hypospadias is a complex, congenital, external genitalia malformation. Genetic factors are important causative reason in the development of hypospadias (11, 12). Kojima et al. replicated rs3816183 of HAAO polymorphism with hypospadias and found that rs3816183 [T] was significantly increased the hypospadias susceptibility toward both posterior and anterior/middle hypospadias (16). However, the HAAO rs3816183 polymorphism was only significantly associated with an increased susceptibility toward anterior/middle hypospadias susceptibility in the present study. Therefore, our study demonstrated that HAAO rs3816183 polymorphism is not equally associated with hypospadias risk in different populations.

The HAAO gene, which is widely distributed in various organs (22–24), encodes a protein that catalyzes the synthesis of quinolinic acid (QUIN) from 3-hydroxyanthranilic acid. Huang et al. showed that hypermethylation of the HAAO gene predicts disease-free survival in patients with endometrioid endometrial cancer (25). Martin et al. reported that hypercholesterolemia and atherosclerosis may be treated and prevented by targeting the HAAO gene (26). Previous studies have demonstrated that the HAAO gene is associated with cancer biomarkers and degenerative diseases. The relationship between the HAAO gene and developmental disorders has also been reported. HAAO has also been correlated with congenital malformations and miscarriage and, when combined with environmental factors, may impair embryo outcomes (27). Pathogenesis of hypospadias has been attributed to the incomplete fusion of the urethra in a portion of the penis and the expression of HAAO in male mouse genital tubercle. Moreover, genetic variants of HAAO may specifically impede the migration and proliferation of normal urethral cells. We hypothesized that the HAAO rs3816183 T>C polymorphism may disrupt the metabolism of its encoded protein leading to disorders of NAD synthesis, which contribute to the pathogenesis of hypospadias (Figure 1). Similar genetic studies have suggested that rs3816183[T] HAAO polymorphisms may result in increased hypospadias susceptibility (16). However, in our study, the association between rs3816183 T>C HAAO polymorphism and hypospadias susceptibility was observed in anterior/middle group but not in posterior hypospadias patients. This discrepancy could be attributed to the sample size and ethnic differences in patients. In addition, causes of hypospadias may be genetic, maternal, environmental, or a combination of all of these factors. Posterior hypospadias have been reported to be associated with maternal factors, such as oligohydramnios, premature birth, and hypertension, suggesting that the underlying placental insufficiency may be an important contributing factor (28). Environmental factors, such as phthalates, have been associated with a toxic effect on the male reproductive system and the development of hypospadias (29). The fact that there may be many complex causes for hypospadias and that the environmental and maternal factors were not accounted for in our study could be the reason that the HAAO rs3816183 variants was found to be associated only with anterior/middle hypospadias.

This is the largest Asian case–control study to investigate the association of HAAO polymorphism rs3816183 T>C with hypospadias susceptibility. Our results demonstrated that the SNPs rs3816183[T] in HAAO may be associated with increased anterior/middle hypospadias but not posterior hypospadias (Table 2), suggesting that HAAO may influence distal part of penile urethral formation.

However, there were some limitations to this study. First, environmental factors, such as difference in diet and geographic locations, were not analyzed. Second, in-depth exploration of HAAO rs3816183T>C and hypospadias sensitivity mechanisms is required. This may have potential implications for hypospadias prevention. Finally, multiple center studies are warranted to confirm our findings.

The HAAO rs3816183[T] is associated with increased risk to anterior/middle hypospadias in Southern Han Chinese population. Our findings support the hypothesis that the mechanism underlying the variations in the HAAO gene may contribute to the pathogenesis of hypospadias and thus requires in-depth research.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The studies involving human participants were reviewed and approved by Ethical Standards of the Institutional Review Board of Guangzhou Women and Children's Medical Center (NO. 39401). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

FD designed experiment. YL, WF, KF, XZ, WJ, NW, GL, and FD collected samples and conducted the study. YZ and XZ analyzed the data. YL and FD wrote the paper. All authors have read and approved the manuscript.

FD thanks the fund from Guangzhou Institute of Pediatrics/Guangzhou Women and Children's Medical Center (Grant No. 0190026) and Science and Technology Project of Guangzhou (Grant No. 202102010238).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.