Keywords

1 Genes Involved in the Embryology of the Male External Genitalia

Development of the male external genitalia can be divided in three stages:

  • The indifferent stage.

  • The early patterning stage.

  • The masculinization stage.

1.1 Indifferent Stage

Early development of the external genitalia is similar for males and females. The embryonic cloaca, the far end of the hindgut, is separated from the amniotic cavity by the cloacal membrane. Early in the fifth week of development, a swelling develops on both sides of this membrane, the cloacal folds, which meet in the midline anterior to the cloacal membrane, forming the genital tubercle [1]. At the same time, the genital ridges, the precursors of the gonads, develop. Studies in mice showed that this process requires Wilms tumor 1 (Wt1) activity, which activates splicing factor 1 (Sf1) [2], thus preventing degeneration of the developing gonads [3]. During the seventh week of human development, the urorectal septum fuses with the cloacal membrane, dividing the cloaca into the primitive urogenital sinus and the rectum and dividing the cloacal membrane into the urogenital and the anal membrane. The swellings next to the urogenital membrane are then called the urogenital folds, and a new pair of swellings, the labioscrotal swellings, appears on either side of these folds. In addition, the urogenital membrane breaks down [1].

1.2 Early Patterning

Early patterning of the genital tubercle (GT) is androgen-independent. The distal urethral plate epithelium is the signaling center regulating GT outgrowth [4]. Fibroblast growth factor proteins (FGF) play a growth-promoting role in this outgrowth [5], whereas bone morphogenetic proteins (BMP) stimulate apoptosis [6, 7]. Sonic hedgehog (SHH) modulates the balance between proliferation and apoptosis by regulating the expression of the genes encoding these and many other proteins [8]. This way, SHH regulates the initiation of GT outgrowth [4]. Immunohistochemical staining of human fetal penises showed expression of SHH, its receptor, and several of its downstream genes around the time of urethral closure [9].

1.3 Masculinization

Subsequent masculinization relies on hormones produced by the testes. Expression of the sex-determining region Y gene (SRY) induces a cascade of gene interactions, involving SRY-box 9 (SOX9) [1], resulting in differentiation of the gonads into the testes [10]. SRY leads to the differentiation of Sertoli cells [1], which secrete anti-Müllerian hormone. Anti-Müllerian hormone causes regression of the Müllerian ducts that would otherwise form part of the female genital structures [1]. Human chorionic gonadotropin (hCG), produced by the placenta, controls fetal Leydig cell growth and stimulates fetal testicular steroidogenesis, the generation of steroids from cholesterol [11]. The enzymatic steps in steroidogenesis, mainly taking place in the Leydig cell, are well documented, and expression of key genes in this pathway is dependent on expression of NR5A1 (Fig. 11.1) [13]. Testosterone leaves the Leydig cell and is converted into dihydrotestosterone (DHT) by steroid-5-alpha-reductase (SRD5A). Testosterone promotes formation of the internal reproductive structures from the Wolffian ducts, whereas DHT induces development of the external genitalia [1], both through their effects on the androgen receptor (AR). Expression of estrogen receptors (ESR) in male genital tissue during development suggests that the balance between androgens and estrogens is important as well [14].

Fig. 11.1
figure 1

Steroidogenesis in the mitochondrium (top) and smooth endoplasmic reticulum (bottom) of the fetal Leydig cell. Adapted from van der Zanden et al., 2012 [12] (from van der Zanden, L.F.M. and van Rooij, I.A.L.M.: “Aetiology of hypospadias: a systematic review of genes and environment,” Human Reproduction Update, 2012, Vol. 18, Issue 3, by permission of Oxford University Press)

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2 Genes Implicated in the Etiology of Isolated Hypospadias

All genes implicated in one of the three stages mentioned above could play a role in the development of hypospadias. Therefore, much of the genetic research on hypospadias has been focused on these genes.

3 Study Types

Different types of studies have been performed to examine whether specific genes have an effect on the occurrence of hypospadias. One example are the mutation analyses, for which researchers sequence candidate genes in hypospadias patients and healthy controls. These studies typically included tens to sometimes a little over a hundred hypospadias patients and healthy controls. Sequencing allows complete coverage of the gene, identifying all genetic variants present in that gene. With this type of studies, researchers were aiming to identify the causal variant for hypospadias in part of their patients. Although several studies identified new and unknown mutations in hypospadias patients that were not present in the healthy controls, it remains unclear whether these mutations truly have functional consequences. Only few studies reported conservation and function of the region in which the mutation is located or predicted a potential influence of the mutation on protein function using bioinformatics. Even if the mutations found are truly causal, they only explain the occurrence of hypospadias in a small part of the patients, and the majority of mutations were found only once and were identified in patients with posterior or penile hypospadias.

Another type of study that has been used to examine whether specific genes have an effect on the occurrence of hypospadias is the association study. In this type of study, known polymorphisms are genotyped in hypospadias patients and healthy controls. Polymorphisms are common genetic variants that occur with a frequency of at least 1%. Due to the haplotype block structure of the human genome, genotyping a specific set of polymorphisms in a gene covers much more of the variation in that gene. Thereby, polymorphisms function as markers for more rare variants with functional consequences. In association studies, typically hundreds to sometimes a thousand hypospadias patients and healthy controls have been genotyped for polymorphisms in candidate genes. If a polymorphism occurs more frequently in the patients compared to the controls, this means that there is an association and the variant may be a marker for a causal variant that increases hypospadias risk. However, due to the large amount of polymorphisms examined, the risk of false-positive results is high. Therefore, replication of results is a major issue in association studies, especially in the genome-wide association studies that aim to cover the whole genome by genotyping hundreds of thousands of polymorphisms.

4 Study Results

Table 11.1

Table 11.1 Result of genetic association studies and mutation studies screening candidate genes in groups of patients with hypospadias and healthy controls. Genes that were only investigated once and did not show mutations or associations in that study are not shown
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shows the candidate genes that were screened for mutations or associations in groups of hypospadias patients and healthy controls. Genes that were only investigated once and did not show mutations or associations in that study are not shown. The table shows that only few studies focused on genes involved in the indifferent and early patterning stage. Although some mutations and associations were found in these genes, there is no gene that shows clear evidence of being involved in hypospadias etiology.

Research on genes involved in the masculinization stage has been much more extensive. Notably, although expression of the SRY gene, located on the Y chromosome, is crucial for development of the testis from the indifferent gonad ([10, 93]), there is not much evidence that this gene plays a role in the development of hypospadias. Research was especially focused on AR and SRD5A2. SRD5A2 converts testosterone to the more potent androgen DHT, and both testosterone and DHT exert their effect through the AR. The AR is expressed in the developing human penis and urethra, and SRD5A2 is expressed during male genital development around the ventral part of the remodeling urethra [94]. Multiple studies found mutations and associations with polymorphisms in these genes (Table 11.1). In addition, there are other studies that indicated the involvement of the AR in hypospadias etiology. For example, some studies indicated different expression levels in patients compared to controls [39, 47, 48, 95,96,97,98], while others suggested decreased DHT binding capacity of the AR in genital skin fibroblasts of patients with hypospadias [34, 99]. Although not all studies confirmed these results [100,101,102,103], evidence that a defect in AR or SRD5A2 may cause or be a risk factor for hypospadias is compelling.

5 Other Genes

Not only steroidogenesis but also the balance between androgens and estrogens appears to be important in the development of the male external genitalia. The estrogen receptors ESR1 and ESR2 are expressed in the developing human male GT [14], and mRNA expression levels seem to be decreased in foreskin of hypospadias patients compared to controls [95]. Although no mutations were found in ESR1 or ESR2, associations have been reported between hypospadias and several polymorphisms in the genes encoding these receptors (Table 11.1), and evidence for these genes to play a role in hypospadias development is quite strong.

Other genes for which evidence is quite strong are activating transcription factor 3 (ATF3), mastermind-like domain containing 1 (MAMLD1), and diacylglycerol kinase κ (DGKK). ATF3 is an estrogen-responsive gene showing strong upregulation in hypospadias [72, 104,105,106,107], and several studies found mutations in this gene in hypospadias patients or polymorphisms in this gene to be associated with hypospadias (Table 11.1). MAMLD1, previously known as CXorf6, contains the NR5A1 target sequence [108] and mutations, and polymorphisms in MAMLD1 were found in patients with hypospadias (Table 11.1). Ogata et al., concluded that MAMLD1 mutations exert their effect primarily via compromised testosterone production around the critical period for sex development [109], and Ratan et al., found lower testosterone levels than the mean for their age in 80% of subjects carrying a polymorphism in MAMLD1 [77]. DGKK was identified as a major risk gene for hypospadias in a genome-wide association study [80], a result that was confirmed by several other studies (Table 11.1). A study in mice revealed that differentiated GT epithelial cells are DGKK positive, while undifferentiated preputial lamina epithelial cells are DGKK negative, suggesting that DGKK is a marker or mediator of squamous cell differentiation [110].

6 Common Clinical Conditions with Gene Defects

6.1 46,XX Disorder of Sex Development (DSD)

The most frequent example of DSD in the 46,XX group is congenital adrenal hyperplasia (CAH), mostly resulting from a deficit of 21-hydoxylase which causes an abnormal growth of the GT and an inappropriate opening of the vagina into the posterior wall of the urethra.

It is important to realize that pubertal and post-pubertal women (46,XX) with CAH develop their GT if they are not compliant to their hormonal substitutive treatment. This shows that GT target tissues remain responsive to steroids until the organ reaches an ultimate size.

In western world, most patients with CAH are raised as female but severely virilized CAH in male-dominant societies or late diagnosis may lead to a male assignment [111].

6.2 46,XY DSD

Mutations in genes implied in testosterone biosynthesis (such as 3-beta-hydroxysteroid-dehydrogenase, 17-beta-hydroxysteroid-deshydrogenase and 17-alpha hydroxylase) can lead to testosterone defects which may present as DSD.

6.3 The Dysgenetic Gonad

The dysgenetic gonad and more specifically the dysgenetic testis is characterized by insufficient production of testosterone and anti-Müllerian hormone (AMH). Although some etiologies are well identified, the histological definition of the gonadal dysgenesis remains unclear [112] as well as the possible underlying genetic anomalies. It is commonly associated with insufficient development of the GT, impaired testicular descent, and persistence of Müllerian structures. It is most likely that gonadal dysgenesis is a dynamic process leading to a progressive loss of testicular functions. The major concern about dysgenetic or underdeveloped gonads is the development of cancers from immature germinal cells [113].

6.4 Partial and Complete Androgen Insensitivity Syndromes

5α-Reductase deficiency is an autosomal recessive syndrome. It is a rare disorder in western countries but frequent in the Dominican Republic, New Guinea, and Gaza strip due to high consanguinity [114]. These 46,XY patients present with quite feminine genitalia at birth which will get virilized after puberty if the testes are left in place. Some newborn babies may present with a slightly more developed hypospadiac micropenis which may grow with topical dihydrotestosterone. These patients represent one of the most difficult situations where gender assignment is an issue as well as the fate of the testes.

Partial and complete androgen insensitivity syndromes (PAIS/CAIS) are related to impaired androgen receptors. CAIS does not raise any gender issue as these individuals have normal female external genitalia and will be raised as females with hormonal substitution from puberty onward. Partial androgen insensitivity syndrome used to raise difficult discussions in terms of gender assignment although most of them are nowadays raised as males [115].