Hypospadias is congenital hypoplasia of the penis, leading to a ventrally displaced urinary meatus, often associated with a dorsal-headed foreskin and chordee (Leung and Robson, 2007). It is the second most common male urogenital abnormality after cryptorchidism (Ságodi et al., 2014). The prevalence of hypospadias varies widely between different populations (0.26–47 per 10,000 births), with reports suggesting higher frequency in white people than in black people and Asians (KŠllŽn et al., 1986; Paulozzi, 1999; Kurahashi et al., 2004; Yang et al., 2004; Carmichael et al., 2007).

Hypospadias is characterized by incomplete fusion of the urethral folds during early embryonic development (Baskin and Ebbers, 2006). From the 8th week onwards, when gonads have differentiated into testes in XY embryonic males, the external genitalia development and urethral modifications are mainly dependent on the secretion and action of androgens (Baskin and Ebbers, 2006; Bouty et al., 2015), in particular, by testosterone produced from the fetal testes and transformed peripherally into dihydrotestosterone that binds to the androgen receptors. The cascades of hormonal activities influenced by numerous genetic factors induce fusion of the urethral folds along the ventral surface of the penis and produce a series of morphological changes in the developing external genitalia (Baskin and Ebbers, 2006). During this hypospadias-critical gestational window (8–14 weeks), the synthesis, concentration, timing, and metabolism of hormones/androgens are important for proper urethral closing and external genitalia development, and any abnormality may result in hypospadias (Bouty et al., 2015).

Numerous lines of evidence suggest a genetic component as an important etiology, with the presence of an affected family member being a high risk factor (van der Zanden et al., 2012; Thorup et al., 2014). First-, second-, or third-degree relatives are affected in 7% of patients with familial clustering of hypospadias (Fredell et al., 2002). In most familial cases, hypospadias shows equal transmission through both maternal and paternal lines with similar recurrence risks between the brothers and sons of patients (Schnack et al., 2008). The chances of hypospadias increase by 9%–17% for the brother of a male with hypospadias. Pedigree and twin studies have reported a higher genetic component of 57%–77% in the heritability of hypospadias (Schnack et al., 2008).

The risk of congenital abnormalities is higher in children born from consanguineous parents. Investigating such affected families provides a higher prospect of genetic findings. Therefore, we investigated a consanguineous family with two hypospadias-affected siblings using whole-exome sequencing. The bioinformatics tools for in silico predictions were applied to assess the structural and functional consequences of the variants observed in the study.

The identified novel homozygous variant (T507A) in HSD3B2 genetically segregates the affected and non-affected individuals of the family. Both the parents and the two non-affected male siblings were heterozygous carriers (T/A), suggesting an autosomal recessive pattern of inheritance.

3β-Hydroxysteroid 2-dehydrogenase (3β-HSD2), a product of the HSD3B2 gene and an isozyme of 3β-hydroxysteroid dehydrogenase (3βHSD), is important for the biosynthesis of steroids, in particular, testosterone (Morrison et al., 1991; Bain et al., 1993). It is expressed predominantly in the adrenal gland, the ovary, and the testis (Lachance et al., 1992). Its role and function are implicated in several studies on hypospadias (Codner et al., 2004a; Carmichael et al., 2014; Kalfa et al., 2019). In embryonic males, the enzymatic steps that contribute to steroidogenesis mainly occur in the fetal Leydig cells via the steroidogenic pathway, which converts cholesterol into sex hormones, especially testosterone and its derivative dihydrotestosterone. The product of HSD3B2 is involved in enzymatic conversion of numerous precursors, such as pregnenolone to progesterone, 17αOH pregnenolone to 17αOH progesterone, dehydroepiandrosterone (DHEA) to androstenedione, and androstenedione to testosterone (Carmichael et al., 2014; Bouty et al., 2015). These are critical for the hormone-dependent development of the male genital tract, including the urethral tract closure and morphogenesis of male genitalia. Abnormalities in this development cause hypospadias (Kotula-Balak et al., 2012). Physiologically, the T507A variant observed in our probands may have affected the normal fetal steroidogenic pathway, leading to hypospadias in the affected siblings.

Numerous loci variants for HSD3B2 are reported in earlier studies (Codner et al., 2004a; Wang et al., 2007; Rabbani et al., 2012; Kon et al., 2015). However, the T507A variant (N169K amino acid change) observed in our study is novel and not reported earlier. A ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/) search for the HSD3B2 variant suggests that most genetic changes in HSD3B2 are associated with abnormalities in the urogenital system (Supplementary Table S3). In a systematic mutation screening of 25 known causative/candidate/susceptible genes in Japanese and Vietnamese patients, Kon M et al. suggested that HSD3B2 mutations may potentially lead to non-syndromic hypospadias as a sole clinical manifestation (Kon et al., 2015). In another study, HSD3B2 was genotyped in 90 hypospadias patients and 101 healthy controls. Two missense heterozygous mutations (S213T and S284R) and three heterozygous variants (A238, T259, and T320) with no amino acid change were observed in patients but not in controls. These findings suggested that subtle molecular abnormalities in the HSD3B2 gene may be observed in idiopathic hypospadias patients (Codner et al., 2004a). A 27-bp deletion (687del27) in exon IV, deleting the terminal base pair of codon 229 and the entire codons 230–237, in addition to the first two base pairs of codon 238, is also reported in a normozoospermic man with hypospadias (Donadille et al., 2018). A homozygous missense mutation of A82P in exon 3 of the HSD3B2 gene is also reported in a hypospadias patient (Rabbani et al., 2012). Therefore, mutations of HSD3B2 underlie various types of hypospadias, as shown by the evidence of these earlier studies.

In addition, in silico analysis (using SIFT, PolyPhen-2, MutationTaster, MutationAssessor, FATHMM, and ConSurf) consistently predicted the severely deleterious nature of the observed T507A variant in our patients. We employed six in silico tools to increase the stringency of our analysis because individual tools often disagree, in part because they use different predictive features. Together, these tools encompass a broad range of non-synonymous variant/amino acid classification criteria and methods, collectively making the final predicted outcome more accurate. Three important features that concern the mutant missense variant (N169K) observed in our study are as follows: 1) our variant is in close proximity to one of the two catalytic sites (amino acid 154–158 and 269–273) reported in earlier mutagenesis and structural studies (Morel et al., 1997; Thomas et al., 2002; Simard et al., 2005). The amino acid substitutions located at or near the binding interface or active site could hinder the active site, change the recognition, alter the specificity, or affect the binding affinity (Parker, 2001; Mata et al., 2006). Therefore, the deleterious influence of amino acid substitution on HSD3B2 enzyme activity in our patients cannot be overruled (Figure 2). 2) The N169K variant is located in a locus that is evolutionarily conserved across closely related species (Table 1; Figure 2). The degree of evolutionary conservation of an amino acid reflects the structural and functional importance of the domain. The higher the evolutionary conservation, the more critical is the function of the domain/protein and the need to retain the domain/protein’s structural integrity (Ashkenazy et al., 2016). 3) The T507A mutation substituted the neutral amino acid asparagine with positively charged (basic) lysine.

Although endocrinal disorder has been reported in hypospadias, the percentage of patients with identified endocrinal etiology remains low (Kalfa et al., 2009). Cassorla et al., 2004 reported that isolated patients with hypospadias are more likely to have normal endocrine testicular and androgen end-organ functions (Codner et al., 2004b). In a study by Rey et al. (2005), of 61 patients, only 13 (21.31%) were found to have an androgen synthesis defect (Rey et al., 2005). As development of the urethra occurs during 8–16 weeks gestation, the postnatal hormonal milieu may not reflect early gestation events (Baskin, 2000; Baskin et al., 2001; Holmes et al., 2004). Unfortunately, we could not assess the hormone levels in our patients. However, any relatable clinical feature or symptom associated with any enzyme or hormone imbalance, in particular, adrenal insufficiency, was not observed in our patients.

Inconsistent findings have been reported for the presence of Y-chromosome microdeletions in hypospadias patients (Tateno et al., 2000; Juniarto et al., 2018). We did not observe Y chromosome microdeletions in any of the hypospadias-affected or non-affected siblings in our study.

To the best of our knowledge, this is the second reported familial hypospadias case with HSD3B2 mutations in more than one affected individual from the same family (siblings). The previously published HSD3B2 mutation in hypospadias siblings was published by Ladjouze et al. (2022). In addition, this is the first genetic study on hypospadias from the Yemen ethnicity. Interestingly, an ethnicity-specific difference in gene function is also reported for HSD3B2 variants between Caucasian-American, African-American, Han Chinese-American, and Mexican-American populations (Wang et al., 2007).

In summation, we described a novel variant in the HSD3B2 gene with an autosomal recessive inheritance that probably affected fetal androgen synthesis and caused hypospadias in two siblings. This clinical case re-emphasizes the importance of structurally and functionally intact HSD3B2 in proper urethral folding and external genitalia morphogenesis during fetal life. In silico analysis predicted the variant to be potentially pathogenic to the structure and function of the HSD3B2 gene product. While we have unraveled a single locus for hypospadias, future genetic and functional studies, particularly in patients from the same parents, will provide a better understanding of disease etiology and inheritance. Additionally, it will aid in developing strategies for improved management of future pregnancies in high-risk familial cases.