REVIEW URRENT C OPINION
Genetic and environmental origins of hypospadias Jorgen Thorup a,b, Agneta Nordenskjo¨ld c,d, and John M. Hutson e,f,g
Purpose of review The purpose of this study was to review and comment on recent original presentations dealing with genetic and environmental factors in the cause of hypospadias. Recent findings The heritability is definitely high and having an affected family member is the highest identified risk factor so far. Many candidate genes and polymorphisms have been suggested for hypospadias. Some associations with hypospadias were found, and many of these were replicated inconsistently as would be expected in a complex disorder affected by both genes and environment. The consistent association of hypospadias with low birth weight, maternal hypertension, and preeclampsia suggests that placental insufficiency is a major risk factor. Maternal exposure to chemical pollutants or endocrine disruptors in high concentrations related to selected occupations or geographic areas may be additional risk factors for hypospadias, especially in genetically predisposed individuals. So far, however, no environmental chemical pollutants or endocrine disruptor with a general common impact on the risk for hypospadias in most societies has been demonstrated. Summary A major point that should be considered regarding the action of environmental toxicants in inducing hypospadias is the cumulative effects of multiple low-dose exposures. Furthermore, interactions between genetic and environmental factors may help to explain nonreplication in genetic studies of hypospadias. Keywords environment, genes, hypospadias, placenta
INTRODUCTION Hypospadias is one of the most common congenital malformations occurring in male foetuses between gestational weeks 8–16 [1–3]. Hypospadias is considered a complex disorder since both genetic and environmental contributors are involved. Recently, important comprehensive reviews have been published on this specific subject [4–6]. The aim of this study was to review and comment on original presentations dealing with genetic and environmental factors that cause hypospadias published during the last 18 months and therefore not included in the aforementioned reviews. In addition to describing recent trends, we give our opinions of the topics discussed. The terminology in subcategorizations of hypospadias is not ubiquitous in the literature, so the terms used in this review are related to the ones used by the authors of the commented and cited publications.
PATHOGENESIS A well-established risk factor for hypospadias, when corrected for gestational age, is low birth weight
[2,7–10]. In 16 of 18 monozygotic twins discordant for hypospadias, the twin with the lower birth weight had hypospadias [2,11]. As low birth weight and small-for-gestational age (SGA) are associated with suboptimal first trimester growth, the association with hypospadias may be related to early placental malfunction [12]. Accordingly, hypospadias has been correlated with low weight of
a
Department of Pediatric Surgery, Rigshospitalet, bFaculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, c Department of Women’s and Children’s Health, Pediatric Surgery Unit and Center for Molecular Medicine, Karolinska Institutet, dPediatric Surgery, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden, eDepartment of Pediatric Urology, Royal Children’s Hospital, fDouglas Stephens Surgical Research Laboratory, Murdoch Children’s Research Institute, Melbourne and gDepartment of Paediatrics, University of Melbourne, Parkville, Victoria, Australia Correspondence to Jorgen Thorup, MD, PhD, FEBPS, FEAPU, Professor and Chair, Head of Department, 4272 Department of Pediatric Surgery, Rigshospitalet, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. Tel: +45 3545 4868; fax: +45 3545 3888; e-mail: Joergen. [email protected] Curr Opin Endocrinol Diabetes Obes 2014, 21:227–232 DOI:10.1097/MED.0000000000000063
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KEY POINTS
and oligohydramnios among those with severe versus mild hypospadias. Interestingly, in a study of 69 boys with proximal hypospadias, Sekaran et al. [18 ] found that among those boys with proximal hypospadias and undescended testes, there was a higher incidence of premature birth and intrauterine growth restriction than among boys with proximal hypospadias and descended testes. The coexistence of growth restriction and genital anomalies suggested that this association is due to shared causes, such as placental insufficiency with impaired human chorionic gonadotropin (hCG) level and androgen deficiency in early pregnancy [18 ]. There has been described a higher incidence of hypospadias with increasing maternal age, but it was uncertain whether it was a direct consequence of the age or other factors, such as a higher frequency of in-vitro fertilization (IVF) in older women [19]. However, recently van Rooij et al. [16] found no higher incidence of hypospadias with increasing maternal age, but Bang et al. [20] confirmed in a series of 7752 male infants that IVF and IVF/intracytoplasmic sperm injection (ICSI) increase the risk of low birth weight and preterm birth, resulting in increased rates of hypospadias and cryptorchidism, maybe as secondary phenomena. &
Hypospadias is considered a complex disorder because both genetic and environmental contributors are involved. A major risk factor for hypospadias is intrauterine growth restriction suggesting that this association is because of shared causes, such as placental insufficiency with impaired hCG level and androgen deficiency in early pregnancy. The heritability is the highest identified risk factor for hypospadias so many candidate genes and polymorphisms have been suggested for hypospadias, and although some associations with hypospadias were found, many of these were replicated inconsistently as would be expected in a complex disorder affected by both genes and environment. Maternal exposure to chemical pollutants or endocrine disruptors in high concentrations related to selected occupations or geographic areas may be additional risk factors for hypospadias, but a common impact on the risk for hypospadias in most societies caused by chemical pollutants or endocrine disruptors has not been demonstrated so far.
the placenta [2,13,14]. Abnormalities of the foetal– placental–maternal interaction may also explain the finding of an association with dystocia, as women who gave birth to boys with hypospadias had a higher rate of weak contractions during birth, induced deliveries, and cesarean sections [15]. Hypospadias has also been shown to be associated with preeclampsia later during pregnancy [9]. Three recent studies deal with the association of hypospadias, maternal factors, and SGA boys [16,17,18 ]. In a case (n ¼ 405)–control (n ¼ 627) study, van Rooij et al. [16] found that a genetic predisposition plays a role in anterior and middle hypospadias, but preterm delivery and being SGA were strongly associated with posterior hypospadias. SGA was not even associated with anterior hypospadias at all. Maternal obesity (BMI > 30 kg/m2) at conception doubled the risk of having a child with hypospadias. Maternal smoking during the first 4 months of pregnancy increased the risk of hypospadias, but not significantly. In a retrospective study on 88 patients, Huisma et al. [17] confirmed that among boys with hypospadias and SGA significantly more patients (65%) had severe hypospadias compared to 11% with hypospadias and normal birth weights. Other associated abnormalities were more frequent in boys with hypospadias and SGA, but these cases in both groups were excluded before statistical calculation. Within the cohort of SGA boys, there was a higher prevalence of prematurity, maternal hypertension &
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GENETICS Heritability of hypospadias is high. Familial clustering is seen in 4–25% of cases [2,7,14,21–25]. The more severe the malformation of the index patient, the higher the recurrence risk for the next male sibling, ranging between 9 and 17% [2,7,8,14,21]. The distribution of phenotype in sporadic and familial cases differs. Minor hypospadias is more common in familial than sporadic cases, whereas severe variants are more commonly sporadic. [2,16]. It has to be emphasized that a familial history does not equate with a genetic factor. Apparent heritability is usually inflated by including shared early environmental factors (i.e. when twins or siblings grow up in a common environment) as a genetic component. van der Zanden et al. [4] reviewed studies screening groups of patients with hypospadias for single gene defects and found mutations in the genes for WT1, NR5A1(SF1), BMP4, BMP7, HOXA4, HOXB6, FGF8, FGFR2, AR, HSD3B2, SRD5A2, ATF3, MAMLD1, MID1, and BNC2. However, it is uncertain whether these mutations have functional consequences in most cases, as only few studies reported conservation and function of the region in which the mutation is located, or predicted potential influence of the mutation on protein function using bioinformatics [4]. The majority of mutations were found and were identified in posterior or penile Volume 21 Number 3 June 2014
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Genetic and environmental origins of hypospadias Thorup et al.
hypospadias [4]. Other studies focused on investigating associations between hypospadias and genetic polymorphisms in the following genes; FGF8, FGFR2, AR, HSD17B3, SRD5A2, ESR1, ESR2, ATF3, MAMLD1, DGKK, MID1, CYP1A1, GSTM1, and GSTT1 [4]. The androgen receptor gene is located on chromosome Xq12, and the androgen receptor is highly important during the development of the male genitalia. In a case (n ¼ 211)–control ¨ ld [26] (n ¼ 208) study, Adamovic and Nordenskjo found that a higher number of the glutamine codon (CAG) repeat sequence in the androgen receptor gene has a highly significant effect on the risk of hypospadias in whites, thus confirming studies by Lim et al. [27]. These findings were also confirmed in the Chilean population in another case (n ¼ 44)– control (n ¼ 79) study [28]. Additionally the latter study showed that glycine codon (GGN) polymorphisms were similar in cases and controls [28]. Mutations of androgen receptor are generally described as very scarce in isolated hypospadias, but in a study by Kalfa et al. [29], mutations were found in nine (3%) of 292 boys. The mutations were p. Q58L (c.173A>T), four cases of p. P392S (c.1174C>T), two cases of p. A475V (c.1424C>T), one novel mutation p. D551H (c.1651G>C), and finally p. Q799E (c.2395C>G). The p. D551Hmutation was predicted to be damaging based on six in-silico models, and in-vitro functional studies confirmed the lowered transactivation function of the mutated protein. Three mutations had previously only been reported in isolated infertility. No mutations were found in 345 controls [29]. Another new androgen receptor mutation p. 11e738Met (c.2214T>G) was found in a family with multiple components compatible with the testicular dysgenesis syndrome including subfertility, cryptorchidism, hypospadias, and testicular cancer [30]. Pichler et al. [31] found that androgen receptor mRNA and the amount of androgen receptor protein was significantly elevated in the prepuces of 20 boys with hypospadias compared with 20 controls. They concluded that decreased androgen receptor DNA binding and functional capability may result in a compensatory upregulation of both androgen receptor mRNA and protein. Adamovic et al. [32 ] investigated 13 haplotype tagging single nucleotide polymorphisms (SNPs) covering the steroid-5-alpha reductase (SRD5A2) and androgen receptor gene regions, which are known to have an important role in the hormone-dependent stage of sexual development. The study cohort consisted of 260 individuals with mild hypospadias and 77 with severe disease, in addition to 471 healthy male controls. Their study revealed one novel marker located in the androgen &
receptor gene region (rs5919436; g.67024320C>G) to be significantly associated with an increased risk of severe hypospadias [odds ratio (OR): 2.98]. In concordance with this finding, they detected an association of a haplotype tagged by the minor allele of rs5919436. They further detected no association between the investigated disease and the haplotype tagging polymorphisms covering the SRD5A2 gene, which is of importance considering the conflicting results reported previously. Their data implicate that the androgen receptor rs5919436 (g.67024320C>G) polymorphism may act as a novel genetic marker for increased susceptibility to severe hypospadias [32 ]. Carmichael et al. [33] examined the association of 27 diacylglycerol kinase kappa (DGKK) SNPs with hypospadias (665 cases) relative to population-based controls (928 controls). For mild and moderate cases, OR for 15 of the 27 SNPs had P values less than 0.05, with 2 less than 1 and the others ranging from 1.3 to 1.8. Among severe cases, ORs tended to be closer to 1, and none of the P values were less than 0.05. Because of high linkage disequilibrium across the SNPs, haplotype analyses were conducted and two blocks were generated. These analyses identified a set of eight variants associated with a three-fold to four-fold increased risk relative to the most common haplotype, regardless of severity of the phenotype OR 4.1 for mild-to-moderate cases and 3.3 for severe cases. Therefore, the study confirms that DGKK variants are associated with hypospadias. All genes involved in the development of male external genitalia are candidate genes, such as the CTGF, CYR61, and EGF genes identified in expression studies [4]. The importance of single gene defects, polymorphisms, and gene expression in the aforementioned lists of SRD5A2, NR5A1, ESR1, ESR2, and MAMLD1 in relation with hypospadias have recently been confirmed in other studies [34– 36]. Additional evidence for the involvement of genes can be derived from syndromes associated with hypospadias. Two studies published within the last 18 months address this aspect [37,38]. &
ENVIRONMENT Despite some inaccurate registers, which underevaluate the number of hypospadias cases and the variable geographical distribution of the malformation, several reports have suggested an increase in hypospadias over the past 20–40 years [3,5]. This observation and the finding of hypospadias being reported in numerous wildlife animal species when the habitant was contaminated by pollutant chemicals such as pesticides were contributing to the hypothesis that environmental factors impacted
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on the pathogenesis of hypospadias [3,5]. Other hypotheses introduced endocrine disruptors, which included environmental toxins that could disrupt the hormonal balance of the foetus and thereby disturb sexual differentiation either by an oestrogenic or an antiandrogenic effect [39]. However, Fisch et al. [40] demonstrated that the rising rates of hypospadias described during the 1960s–1980s trailed off by roughly 1985, after which time there was no rise, or an actual decline. However, so far the comprehensive observational epidemiologic literature falls short on whether or which environmental exposures predispose to hypospadias [3,6]. A recent study [41] from the autumn of 2013, including nationwide Swedish healthcare and demographic registers collected for all boys born during 1973– 2009, showed an increased incidence of boys diagnosed with hypospadias from 1990 to 1999 that was not attributable to previously known risk factors. The increase comprises both mild and severe phenotypes, suggesting that shifts in the diagnostic criteria are not the underlying cause. Boys, born SGA or after IVF, were at specially high risk for hypospadias. The total incidence until 1990 was 4.5 per 1000 increasing to 8 per 1000 during the following decade. The latter present incidence is in accordance with recent data from Denmark [3]. However, one limitation of the Swedish study was that a systematic change in the classification of the diagnosis in registers could not be ruled out [41]. Also in Arkansas, USA, the prevalence of hypospadias in 1998–2007 was 7.4 per 1000 live births. Although the prevalence rates for every degree of hypospadias increased during the period, the prevalence of not-otherwise-specified types decreased. Therefore, improvement in recognition and categorization of degrees of hypospadias may have an impact on the reported prevalence rates [42]. During the last 12–18 months, several studies have dealt with environmental factors possibly having influence on the risk of hypospadias. Carran and Shaw [43] reported increases in the incidences of hypospadias, cryptorchidism, and breast cancer in the children of New Zealand soldiers who served in Malaya in 1948–1960 and were exposed to dibutylphthalate applied daily to their clothing as an acaricide to prevent tick-transmitted bush typhus. The theoretical absorbed dose per day was above the lowest adverse effect level with known potential to inhibit testosterone synthesis and possibly lead to feminization. However, the increases in the aforementioned diseases in the study group have later been questioned [44,45]. In a case–control study of serum samples collected in early pregnancy among 237 women giving birth to boys with hypospadias and 237 controls, it 230
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was demonstrated that high serum levels of hexachlorobenzene (HCB) (>26 ng/ml) and high serum levels of the dichlorodiphenyltrichloroethane (DDT) metabolite 1,1-dichloro-2,2-bis( p-chlorophenyl)ethylene( p,p0 -DDE) (>1.0 ng/ml) ORs of 1.65 and 1.69, respectively, for hypospadias were obtained [46]. Based on studies in rats [47], Rignell-Hydbom et al. [46] claim that the p,p0 -DDE is a strong androgen receptor blocking agent, although this statement may be hard to understand as DDT and its metabolite have extremely weak binding to androgen receptor in molar terms. Both HCB and p,p0 -DDE are organochlorine pollutants and although banned in most counties since the 1970s, the substances are, because of their persistence, still found in some humans [46]. The impact of an endocrine disruptor, bisphenol A (BPA), in hypospadias boys has been studied by Qin et al. [48]. BPA is used extensively in the manufacture of the plastics used to make food and beverage containers. In this study, individual variation in the genetic response to low-dose BPA was investigated in human foreskin fibroblast cells (hFFCs) derived from boys with genital malformations [48]. hFFCs were collected from control boys without genital malformations (n ¼ 5) and boys with cryptorchidism (n ¼ 8) and hypospadias (n ¼ 21). BPA exposure (10 nM) was found significantly to inhibit matrix metalloproteinase-11 expression in the hypospadias group (0.74-fold) but not in the control group (0.93-fold) and cryptorchidism group (0.94-fold). The effect of SNP rs5000770 [guanine/adenine (GA)], located within the aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) locus, on individual sensitivity to low-dose BPA was investigated in the hypospadias group. A significant difference in neurotensin receptor 1 expression in response to 10 nM BPA was observed between adenine/adenine (AA) and adenine/guanine (AG)/guanine/guanine (GG) groups (n ¼ 6 and 15, respectively). However, no significant difference in ARNT2 expression was observed. The results suggest that genetic variability among individuals affects susceptibility to the effects of low-dose BPA as a potential cause of hypospadias [48]. Nutritional factors and the risk for hypospadias have been investigated in two studies reaching somewhat different conclusions [49,50 ]. In a study including 1250 cases with penile shaft, scrotal, or perineal hypospadias and 3118 controls, Carmichael et al. [49] found no association of a vegetarian diet or worse diet quality with hypospadias. However, in another study including 306 boys operated for hypospadias and 306 matched controls, Christensen et al. [50 ] found an association between hypospadias in the offspring and the mother not choosing the organic alternative, and having a high current intake of nonorganic butter &
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Genetic and environmental origins of hypospadias Thorup et al. Table 1. Hypothesis on the action of more than one pathogenic factor necessary for the risk of a hypospadias phenotype when genetic predisposition is weak Placental function
hCG level
Birth weight
Genetic predisposition for hypospadias
Phenotype
Example 1
Insufficient
Low
Low
No
Normal
Example 2
Insufficient
Low
Low
Yes – weak
Hypospadias
Example 3
Normal
Normal
Normal
Yes – weak
Normal
Example 4
Normal
Normal
Normal
Yes – strong
Hypospadias
hCG, human chorionic gonadotropin.
and cheese. This finding could be because of chemical contamination of high-fat dairy products. However, general lifestyle and health behavior related to choosing organic alternatives could also explain the finding.
cumulative effects of multiple low-dose exposures that should be considered [5]. Furthermore, it has been shown that interactions between genetic and environmental factors may help to explain nonreplication in genetic studies of hypospadias [51 ].
CONCLUSION
Acknowledgements This work is supported by National Public Healthcare and National University Institutions.
Heritability of hypospadias is definitely high, so there is no doubt that genetic abnormalities are involved. Many different candidate genes and polymorphisms have been suggested for hypospadias. Although some associations with hypospadias were found, none of these associations have been replicated consistently. Each mutation may explain putative pathogenic factors in only a few percent of hypospadias cases, and most investigators are convinced that the majority of isolated hypospadias cases are a result of several additive low-grade genetic risk factors [4]. The consistent association of hypospadias with low birth weight, maternal hypertension, and preeclampsia suggests that placental insufficiency is a major risk factor for hypospadias, possibly through inadequate provision of hCG to stimulate foetal androgen production [3]. Hypothetically, a genetic predisposition in combination with placental insufficiency may exemplify a strong two-hit risk factor model necessary for a hypospadias phenotype (Table 1). As for environmental factors, maternal exposure to chemical pollutants or endocrine disruptors in high concentrations related to selected occupations or geographic areas may be additional risk factors for hypospadias. The genetic basis for hypospadias is probably at steady state so the rising diagnosis rates related to selected occupations or geographic areas must involve at least some environmental cause – which includes artifacts such as changing diagnostic criteria and surveillance bias as well as genuine environmental causation. A common impact on the risk for hypospadias in most societies caused by chemical pollutants or endocrine disruptors has not been demonstrated so far. However, a major point regarding the action of environmental toxicants in inducing hypospadias is the
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Conflicts of interest There are no conflicts of interest.
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34. Wang R, Dong Z, Wang W, et al. Mutation analysis of the SRD5A2 AR and SF-1 genes in 52 Chinese boys with hypospadias. J Pediatr Endocr Met 2013; 26:887–893. 35. Qiao L, Rodriguez E, Weiss DA, et al. Expression of estrogen receptor alpha and beta is decreased in hypospadias. J Urol 2012; 187:1427–1433. 36. Ogata T, Sano S, Nagata E, et al. MAMLD1 and 46 XY disorders of sex development. Semin Reprod Med 2012; 30:410–416. 37. Heinrich T, Nanda I, Rehn M, et al. Live-born trisomy 22: patient report and review. Mol Syndromol 2012; 3:262–269. 38. Phadke SR, Ranganath P, Boggula VR, et al. Brothers with hypospadias, vertebral segmentation defects, and intellectual disability: new syndrome? Am J Med Genet Part A 2012; 158A:3065–3070. 39. Toppari J, Larsen JC, Christiansen P, et al. Male reproductive health and environmental xenoestrogens. Environ Health Perspect 1996; 104:741– 803. 40. Fisch H, Hyun G, Hensle TW. Rising hypospadias rates: disproving a myth. J Ped Urol 2010; 6:37–39. 41. Nordenvall AS, Frisen L, Nordenstro¨m A, et al. A population-based nationwide study of hypospadias in Sweden 2009: incidence and risk factors. J Urol 2014; 191:783–789. 42. Canon S, Mosley B, Chipollini J, et al. Epidemiological assessment of hypospadias by degree of severity. J Urol 2012; 188:2362–2366. 43. Carran M, Shaw IC. New Zealand Malayan war veterans’ exposure to dibutylphthalate is associated with an increased incidence of cryptorchidism, hypospadias and breast cancer in their children. N Z Med J 2012; 125:52– 63. 44. McBride D, Schep L. Comment on Carran and Shaw’s ‘New Zealand Malayan war veterans’ exposure to dibutylphthalate’ article. N Z Med J 2012; 125:105–106. 45. Elwwod M, Borman B. Increases in disease in Malayan war veteran’s children may be misleading. N Z Med J 2012; 125:145–146. 46. Rignell-Hydbom A, Lindh CH, Dillner J, et al. A nested case–control study of intrauterine exposure to persistent organochlorine pollutants and the risk of hypospadias. PLoS One 2012; 7:e44767. 47. Gray LE, Ostby J, Furr J, et al. Effects of environmental antiandrogens on reproductive development in experimental animals. Human Reprod Update 2001; 7:248–264. 48. Qin XY, Sone H, Kojima Y, et al. Individual variation of the genetic response to bisphenol A in human foreskin fibroblast cells derived from cryptorchidism and hypospadias patients. PLoS One 2012; 7:e52756. 49. Carmichael SL, Ma C, Feldkamp ML, et al. Nutritional factors and hypospadias risks. Paedatr Perinat Epidemiol 2012; 26:353–360. 50. Christensen JS, Asklund C, Skakkebaek NE, et al. Association between & organic dietary choice during pregnancy and hypospadias in offspring: a study of mothers of 306 boys operated on for hypospadias. J Urol 2013; 189:1077–1082. This case–control study shows an association between hypospadias in the offspring and the mother not choosing the organic alternative, and having a high current intake of nonorganic butter and cheese. This finding could be due to chemical contamination of high-fat dairy products. However, general lifestyle and health behavior related to choosing organic alternatives could also explain the finding. 51. van der Zanden LFM, Galesloot TE, Feitz WFJ, et al. Exploration of gene– & environment interactions, maternal effects, and parent-of-origin effects in the etiology of hypospadias. J Urol 2012; 188:2354–2360. This study explains how interactions between genetic and environmental factors may help to understand nonreplication in genetic studies of hypospadias.
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Genetic and environmental origins of hypospadias Jorgen Thorup a,b, Agneta Nordenskjo¨ld c,d, and John M. Hutson e,f,g
Purpose of review The purpose of this study was to review and comment on recent original presentations dealing with genetic and environmental factors in the cause of hypospadias. Recent findings The heritability is definitely high and having an affected family member is the highest identified risk factor so far. Many candidate genes and polymorphisms have been suggested for hypospadias. Some associations with hypospadias were found, and many of these were replicated inconsistently as would be expected in a complex disorder affected by both genes and environment. The consistent association of hypospadias with low birth weight, maternal hypertension, and preeclampsia suggests that placental insufficiency is a major risk factor. Maternal exposure to chemical pollutants or endocrine disruptors in high concentrations related to selected occupations or geographic areas may be additional risk factors for hypospadias, especially in genetically predisposed individuals. So far, however, no environmental chemical pollutants or endocrine disruptor with a general common impact on the risk for hypospadias in most societies has been demonstrated. Summary A major point that should be considered regarding the action of environmental toxicants in inducing hypospadias is the cumulative effects of multiple low-dose exposures. Furthermore, interactions between genetic and environmental factors may help to explain nonreplication in genetic studies of hypospadias. Keywords environment, genes, hypospadias, placenta
INTRODUCTION Hypospadias is one of the most common congenital malformations occurring in male foetuses between gestational weeks 8–16 [1–3]. Hypospadias is considered a complex disorder since both genetic and environmental contributors are involved. Recently, important comprehensive reviews have been published on this specific subject [4–6]. The aim of this study was to review and comment on original presentations dealing with genetic and environmental factors that cause hypospadias published during the last 18 months and therefore not included in the aforementioned reviews. In addition to describing recent trends, we give our opinions of the topics discussed. The terminology in subcategorizations of hypospadias is not ubiquitous in the literature, so the terms used in this review are related to the ones used by the authors of the commented and cited publications.
PATHOGENESIS A well-established risk factor for hypospadias, when corrected for gestational age, is low birth weight
[2,7–10]. In 16 of 18 monozygotic twins discordant for hypospadias, the twin with the lower birth weight had hypospadias [2,11]. As low birth weight and small-for-gestational age (SGA) are associated with suboptimal first trimester growth, the association with hypospadias may be related to early placental malfunction [12]. Accordingly, hypospadias has been correlated with low weight of
a
Department of Pediatric Surgery, Rigshospitalet, bFaculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, c Department of Women’s and Children’s Health, Pediatric Surgery Unit and Center for Molecular Medicine, Karolinska Institutet, dPediatric Surgery, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden, eDepartment of Pediatric Urology, Royal Children’s Hospital, fDouglas Stephens Surgical Research Laboratory, Murdoch Children’s Research Institute, Melbourne and gDepartment of Paediatrics, University of Melbourne, Parkville, Victoria, Australia Correspondence to Jorgen Thorup, MD, PhD, FEBPS, FEAPU, Professor and Chair, Head of Department, 4272 Department of Pediatric Surgery, Rigshospitalet, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. Tel: +45 3545 4868; fax: +45 3545 3888; e-mail: Joergen. [email protected] Curr Opin Endocrinol Diabetes Obes 2014, 21:227–232 DOI:10.1097/MED.0000000000000063
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Androgens
KEY POINTS
and oligohydramnios among those with severe versus mild hypospadias. Interestingly, in a study of 69 boys with proximal hypospadias, Sekaran et al. [18 ] found that among those boys with proximal hypospadias and undescended testes, there was a higher incidence of premature birth and intrauterine growth restriction than among boys with proximal hypospadias and descended testes. The coexistence of growth restriction and genital anomalies suggested that this association is due to shared causes, such as placental insufficiency with impaired human chorionic gonadotropin (hCG) level and androgen deficiency in early pregnancy [18 ]. There has been described a higher incidence of hypospadias with increasing maternal age, but it was uncertain whether it was a direct consequence of the age or other factors, such as a higher frequency of in-vitro fertilization (IVF) in older women [19]. However, recently van Rooij et al. [16] found no higher incidence of hypospadias with increasing maternal age, but Bang et al. [20] confirmed in a series of 7752 male infants that IVF and IVF/intracytoplasmic sperm injection (ICSI) increase the risk of low birth weight and preterm birth, resulting in increased rates of hypospadias and cryptorchidism, maybe as secondary phenomena. &
Hypospadias is considered a complex disorder because both genetic and environmental contributors are involved. A major risk factor for hypospadias is intrauterine growth restriction suggesting that this association is because of shared causes, such as placental insufficiency with impaired hCG level and androgen deficiency in early pregnancy. The heritability is the highest identified risk factor for hypospadias so many candidate genes and polymorphisms have been suggested for hypospadias, and although some associations with hypospadias were found, many of these were replicated inconsistently as would be expected in a complex disorder affected by both genes and environment. Maternal exposure to chemical pollutants or endocrine disruptors in high concentrations related to selected occupations or geographic areas may be additional risk factors for hypospadias, but a common impact on the risk for hypospadias in most societies caused by chemical pollutants or endocrine disruptors has not been demonstrated so far.
the placenta [2,13,14]. Abnormalities of the foetal– placental–maternal interaction may also explain the finding of an association with dystocia, as women who gave birth to boys with hypospadias had a higher rate of weak contractions during birth, induced deliveries, and cesarean sections [15]. Hypospadias has also been shown to be associated with preeclampsia later during pregnancy [9]. Three recent studies deal with the association of hypospadias, maternal factors, and SGA boys [16,17,18 ]. In a case (n ¼ 405)–control (n ¼ 627) study, van Rooij et al. [16] found that a genetic predisposition plays a role in anterior and middle hypospadias, but preterm delivery and being SGA were strongly associated with posterior hypospadias. SGA was not even associated with anterior hypospadias at all. Maternal obesity (BMI > 30 kg/m2) at conception doubled the risk of having a child with hypospadias. Maternal smoking during the first 4 months of pregnancy increased the risk of hypospadias, but not significantly. In a retrospective study on 88 patients, Huisma et al. [17] confirmed that among boys with hypospadias and SGA significantly more patients (65%) had severe hypospadias compared to 11% with hypospadias and normal birth weights. Other associated abnormalities were more frequent in boys with hypospadias and SGA, but these cases in both groups were excluded before statistical calculation. Within the cohort of SGA boys, there was a higher prevalence of prematurity, maternal hypertension &
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GENETICS Heritability of hypospadias is high. Familial clustering is seen in 4–25% of cases [2,7,14,21–25]. The more severe the malformation of the index patient, the higher the recurrence risk for the next male sibling, ranging between 9 and 17% [2,7,8,14,21]. The distribution of phenotype in sporadic and familial cases differs. Minor hypospadias is more common in familial than sporadic cases, whereas severe variants are more commonly sporadic. [2,16]. It has to be emphasized that a familial history does not equate with a genetic factor. Apparent heritability is usually inflated by including shared early environmental factors (i.e. when twins or siblings grow up in a common environment) as a genetic component. van der Zanden et al. [4] reviewed studies screening groups of patients with hypospadias for single gene defects and found mutations in the genes for WT1, NR5A1(SF1), BMP4, BMP7, HOXA4, HOXB6, FGF8, FGFR2, AR, HSD3B2, SRD5A2, ATF3, MAMLD1, MID1, and BNC2. However, it is uncertain whether these mutations have functional consequences in most cases, as only few studies reported conservation and function of the region in which the mutation is located, or predicted potential influence of the mutation on protein function using bioinformatics [4]. The majority of mutations were found and were identified in posterior or penile Volume 21 Number 3 June 2014
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Genetic and environmental origins of hypospadias Thorup et al.
hypospadias [4]. Other studies focused on investigating associations between hypospadias and genetic polymorphisms in the following genes; FGF8, FGFR2, AR, HSD17B3, SRD5A2, ESR1, ESR2, ATF3, MAMLD1, DGKK, MID1, CYP1A1, GSTM1, and GSTT1 [4]. The androgen receptor gene is located on chromosome Xq12, and the androgen receptor is highly important during the development of the male genitalia. In a case (n ¼ 211)–control ¨ ld [26] (n ¼ 208) study, Adamovic and Nordenskjo found that a higher number of the glutamine codon (CAG) repeat sequence in the androgen receptor gene has a highly significant effect on the risk of hypospadias in whites, thus confirming studies by Lim et al. [27]. These findings were also confirmed in the Chilean population in another case (n ¼ 44)– control (n ¼ 79) study [28]. Additionally the latter study showed that glycine codon (GGN) polymorphisms were similar in cases and controls [28]. Mutations of androgen receptor are generally described as very scarce in isolated hypospadias, but in a study by Kalfa et al. [29], mutations were found in nine (3%) of 292 boys. The mutations were p. Q58L (c.173A>T), four cases of p. P392S (c.1174C>T), two cases of p. A475V (c.1424C>T), one novel mutation p. D551H (c.1651G>C), and finally p. Q799E (c.2395C>G). The p. D551Hmutation was predicted to be damaging based on six in-silico models, and in-vitro functional studies confirmed the lowered transactivation function of the mutated protein. Three mutations had previously only been reported in isolated infertility. No mutations were found in 345 controls [29]. Another new androgen receptor mutation p. 11e738Met (c.2214T>G) was found in a family with multiple components compatible with the testicular dysgenesis syndrome including subfertility, cryptorchidism, hypospadias, and testicular cancer [30]. Pichler et al. [31] found that androgen receptor mRNA and the amount of androgen receptor protein was significantly elevated in the prepuces of 20 boys with hypospadias compared with 20 controls. They concluded that decreased androgen receptor DNA binding and functional capability may result in a compensatory upregulation of both androgen receptor mRNA and protein. Adamovic et al. [32 ] investigated 13 haplotype tagging single nucleotide polymorphisms (SNPs) covering the steroid-5-alpha reductase (SRD5A2) and androgen receptor gene regions, which are known to have an important role in the hormone-dependent stage of sexual development. The study cohort consisted of 260 individuals with mild hypospadias and 77 with severe disease, in addition to 471 healthy male controls. Their study revealed one novel marker located in the androgen &
receptor gene region (rs5919436; g.67024320C>G) to be significantly associated with an increased risk of severe hypospadias [odds ratio (OR): 2.98]. In concordance with this finding, they detected an association of a haplotype tagged by the minor allele of rs5919436. They further detected no association between the investigated disease and the haplotype tagging polymorphisms covering the SRD5A2 gene, which is of importance considering the conflicting results reported previously. Their data implicate that the androgen receptor rs5919436 (g.67024320C>G) polymorphism may act as a novel genetic marker for increased susceptibility to severe hypospadias [32 ]. Carmichael et al. [33] examined the association of 27 diacylglycerol kinase kappa (DGKK) SNPs with hypospadias (665 cases) relative to population-based controls (928 controls). For mild and moderate cases, OR for 15 of the 27 SNPs had P values less than 0.05, with 2 less than 1 and the others ranging from 1.3 to 1.8. Among severe cases, ORs tended to be closer to 1, and none of the P values were less than 0.05. Because of high linkage disequilibrium across the SNPs, haplotype analyses were conducted and two blocks were generated. These analyses identified a set of eight variants associated with a three-fold to four-fold increased risk relative to the most common haplotype, regardless of severity of the phenotype OR 4.1 for mild-to-moderate cases and 3.3 for severe cases. Therefore, the study confirms that DGKK variants are associated with hypospadias. All genes involved in the development of male external genitalia are candidate genes, such as the CTGF, CYR61, and EGF genes identified in expression studies [4]. The importance of single gene defects, polymorphisms, and gene expression in the aforementioned lists of SRD5A2, NR5A1, ESR1, ESR2, and MAMLD1 in relation with hypospadias have recently been confirmed in other studies [34– 36]. Additional evidence for the involvement of genes can be derived from syndromes associated with hypospadias. Two studies published within the last 18 months address this aspect [37,38]. &
ENVIRONMENT Despite some inaccurate registers, which underevaluate the number of hypospadias cases and the variable geographical distribution of the malformation, several reports have suggested an increase in hypospadias over the past 20–40 years [3,5]. This observation and the finding of hypospadias being reported in numerous wildlife animal species when the habitant was contaminated by pollutant chemicals such as pesticides were contributing to the hypothesis that environmental factors impacted
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on the pathogenesis of hypospadias [3,5]. Other hypotheses introduced endocrine disruptors, which included environmental toxins that could disrupt the hormonal balance of the foetus and thereby disturb sexual differentiation either by an oestrogenic or an antiandrogenic effect [39]. However, Fisch et al. [40] demonstrated that the rising rates of hypospadias described during the 1960s–1980s trailed off by roughly 1985, after which time there was no rise, or an actual decline. However, so far the comprehensive observational epidemiologic literature falls short on whether or which environmental exposures predispose to hypospadias [3,6]. A recent study [41] from the autumn of 2013, including nationwide Swedish healthcare and demographic registers collected for all boys born during 1973– 2009, showed an increased incidence of boys diagnosed with hypospadias from 1990 to 1999 that was not attributable to previously known risk factors. The increase comprises both mild and severe phenotypes, suggesting that shifts in the diagnostic criteria are not the underlying cause. Boys, born SGA or after IVF, were at specially high risk for hypospadias. The total incidence until 1990 was 4.5 per 1000 increasing to 8 per 1000 during the following decade. The latter present incidence is in accordance with recent data from Denmark [3]. However, one limitation of the Swedish study was that a systematic change in the classification of the diagnosis in registers could not be ruled out [41]. Also in Arkansas, USA, the prevalence of hypospadias in 1998–2007 was 7.4 per 1000 live births. Although the prevalence rates for every degree of hypospadias increased during the period, the prevalence of not-otherwise-specified types decreased. Therefore, improvement in recognition and categorization of degrees of hypospadias may have an impact on the reported prevalence rates [42]. During the last 12–18 months, several studies have dealt with environmental factors possibly having influence on the risk of hypospadias. Carran and Shaw [43] reported increases in the incidences of hypospadias, cryptorchidism, and breast cancer in the children of New Zealand soldiers who served in Malaya in 1948–1960 and were exposed to dibutylphthalate applied daily to their clothing as an acaricide to prevent tick-transmitted bush typhus. The theoretical absorbed dose per day was above the lowest adverse effect level with known potential to inhibit testosterone synthesis and possibly lead to feminization. However, the increases in the aforementioned diseases in the study group have later been questioned [44,45]. In a case–control study of serum samples collected in early pregnancy among 237 women giving birth to boys with hypospadias and 237 controls, it 230
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was demonstrated that high serum levels of hexachlorobenzene (HCB) (>26 ng/ml) and high serum levels of the dichlorodiphenyltrichloroethane (DDT) metabolite 1,1-dichloro-2,2-bis( p-chlorophenyl)ethylene( p,p0 -DDE) (>1.0 ng/ml) ORs of 1.65 and 1.69, respectively, for hypospadias were obtained [46]. Based on studies in rats [47], Rignell-Hydbom et al. [46] claim that the p,p0 -DDE is a strong androgen receptor blocking agent, although this statement may be hard to understand as DDT and its metabolite have extremely weak binding to androgen receptor in molar terms. Both HCB and p,p0 -DDE are organochlorine pollutants and although banned in most counties since the 1970s, the substances are, because of their persistence, still found in some humans [46]. The impact of an endocrine disruptor, bisphenol A (BPA), in hypospadias boys has been studied by Qin et al. [48]. BPA is used extensively in the manufacture of the plastics used to make food and beverage containers. In this study, individual variation in the genetic response to low-dose BPA was investigated in human foreskin fibroblast cells (hFFCs) derived from boys with genital malformations [48]. hFFCs were collected from control boys without genital malformations (n ¼ 5) and boys with cryptorchidism (n ¼ 8) and hypospadias (n ¼ 21). BPA exposure (10 nM) was found significantly to inhibit matrix metalloproteinase-11 expression in the hypospadias group (0.74-fold) but not in the control group (0.93-fold) and cryptorchidism group (0.94-fold). The effect of SNP rs5000770 [guanine/adenine (GA)], located within the aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) locus, on individual sensitivity to low-dose BPA was investigated in the hypospadias group. A significant difference in neurotensin receptor 1 expression in response to 10 nM BPA was observed between adenine/adenine (AA) and adenine/guanine (AG)/guanine/guanine (GG) groups (n ¼ 6 and 15, respectively). However, no significant difference in ARNT2 expression was observed. The results suggest that genetic variability among individuals affects susceptibility to the effects of low-dose BPA as a potential cause of hypospadias [48]. Nutritional factors and the risk for hypospadias have been investigated in two studies reaching somewhat different conclusions [49,50 ]. In a study including 1250 cases with penile shaft, scrotal, or perineal hypospadias and 3118 controls, Carmichael et al. [49] found no association of a vegetarian diet or worse diet quality with hypospadias. However, in another study including 306 boys operated for hypospadias and 306 matched controls, Christensen et al. [50 ] found an association between hypospadias in the offspring and the mother not choosing the organic alternative, and having a high current intake of nonorganic butter &
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Genetic and environmental origins of hypospadias Thorup et al. Table 1. Hypothesis on the action of more than one pathogenic factor necessary for the risk of a hypospadias phenotype when genetic predisposition is weak Placental function
hCG level
Birth weight
Genetic predisposition for hypospadias
Phenotype
Example 1
Insufficient
Low
Low
No
Normal
Example 2
Insufficient
Low
Low
Yes – weak
Hypospadias
Example 3
Normal
Normal
Normal
Yes – weak
Normal
Example 4
Normal
Normal
Normal
Yes – strong
Hypospadias
hCG, human chorionic gonadotropin.
and cheese. This finding could be because of chemical contamination of high-fat dairy products. However, general lifestyle and health behavior related to choosing organic alternatives could also explain the finding.
cumulative effects of multiple low-dose exposures that should be considered [5]. Furthermore, it has been shown that interactions between genetic and environmental factors may help to explain nonreplication in genetic studies of hypospadias [51 ].
CONCLUSION
Acknowledgements This work is supported by National Public Healthcare and National University Institutions.
Heritability of hypospadias is definitely high, so there is no doubt that genetic abnormalities are involved. Many different candidate genes and polymorphisms have been suggested for hypospadias. Although some associations with hypospadias were found, none of these associations have been replicated consistently. Each mutation may explain putative pathogenic factors in only a few percent of hypospadias cases, and most investigators are convinced that the majority of isolated hypospadias cases are a result of several additive low-grade genetic risk factors [4]. The consistent association of hypospadias with low birth weight, maternal hypertension, and preeclampsia suggests that placental insufficiency is a major risk factor for hypospadias, possibly through inadequate provision of hCG to stimulate foetal androgen production [3]. Hypothetically, a genetic predisposition in combination with placental insufficiency may exemplify a strong two-hit risk factor model necessary for a hypospadias phenotype (Table 1). As for environmental factors, maternal exposure to chemical pollutants or endocrine disruptors in high concentrations related to selected occupations or geographic areas may be additional risk factors for hypospadias. The genetic basis for hypospadias is probably at steady state so the rising diagnosis rates related to selected occupations or geographic areas must involve at least some environmental cause – which includes artifacts such as changing diagnostic criteria and surveillance bias as well as genuine environmental causation. A common impact on the risk for hypospadias in most societies caused by chemical pollutants or endocrine disruptors has not been demonstrated so far. However, a major point regarding the action of environmental toxicants in inducing hypospadias is the
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Conflicts of interest There are no conflicts of interest.
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34. Wang R, Dong Z, Wang W, et al. Mutation analysis of the SRD5A2 AR and SF-1 genes in 52 Chinese boys with hypospadias. J Pediatr Endocr Met 2013; 26:887–893. 35. Qiao L, Rodriguez E, Weiss DA, et al. Expression of estrogen receptor alpha and beta is decreased in hypospadias. J Urol 2012; 187:1427–1433. 36. Ogata T, Sano S, Nagata E, et al. MAMLD1 and 46 XY disorders of sex development. Semin Reprod Med 2012; 30:410–416. 37. Heinrich T, Nanda I, Rehn M, et al. Live-born trisomy 22: patient report and review. Mol Syndromol 2012; 3:262–269. 38. Phadke SR, Ranganath P, Boggula VR, et al. Brothers with hypospadias, vertebral segmentation defects, and intellectual disability: new syndrome? Am J Med Genet Part A 2012; 158A:3065–3070. 39. Toppari J, Larsen JC, Christiansen P, et al. Male reproductive health and environmental xenoestrogens. Environ Health Perspect 1996; 104:741– 803. 40. Fisch H, Hyun G, Hensle TW. Rising hypospadias rates: disproving a myth. J Ped Urol 2010; 6:37–39. 41. Nordenvall AS, Frisen L, Nordenstro¨m A, et al. A population-based nationwide study of hypospadias in Sweden 2009: incidence and risk factors. J Urol 2014; 191:783–789. 42. Canon S, Mosley B, Chipollini J, et al. Epidemiological assessment of hypospadias by degree of severity. J Urol 2012; 188:2362–2366. 43. Carran M, Shaw IC. New Zealand Malayan war veterans’ exposure to dibutylphthalate is associated with an increased incidence of cryptorchidism, hypospadias and breast cancer in their children. N Z Med J 2012; 125:52– 63. 44. McBride D, Schep L. Comment on Carran and Shaw’s ‘New Zealand Malayan war veterans’ exposure to dibutylphthalate’ article. N Z Med J 2012; 125:105–106. 45. Elwwod M, Borman B. Increases in disease in Malayan war veteran’s children may be misleading. N Z Med J 2012; 125:145–146. 46. Rignell-Hydbom A, Lindh CH, Dillner J, et al. A nested case–control study of intrauterine exposure to persistent organochlorine pollutants and the risk of hypospadias. PLoS One 2012; 7:e44767. 47. Gray LE, Ostby J, Furr J, et al. Effects of environmental antiandrogens on reproductive development in experimental animals. Human Reprod Update 2001; 7:248–264. 48. Qin XY, Sone H, Kojima Y, et al. Individual variation of the genetic response to bisphenol A in human foreskin fibroblast cells derived from cryptorchidism and hypospadias patients. PLoS One 2012; 7:e52756. 49. Carmichael SL, Ma C, Feldkamp ML, et al. Nutritional factors and hypospadias risks. Paedatr Perinat Epidemiol 2012; 26:353–360. 50. Christensen JS, Asklund C, Skakkebaek NE, et al. Association between & organic dietary choice during pregnancy and hypospadias in offspring: a study of mothers of 306 boys operated on for hypospadias. J Urol 2013; 189:1077–1082. This case–control study shows an association between hypospadias in the offspring and the mother not choosing the organic alternative, and having a high current intake of nonorganic butter and cheese. This finding could be due to chemical contamination of high-fat dairy products. However, general lifestyle and health behavior related to choosing organic alternatives could also explain the finding. 51. van der Zanden LFM, Galesloot TE, Feitz WFJ, et al. Exploration of gene– & environment interactions, maternal effects, and parent-of-origin effects in the etiology of hypospadias. J Urol 2012; 188:2354–2360. This study explains how interactions between genetic and environmental factors may help to understand nonreplication in genetic studies of hypospadias.
Volume 21 Number 3 June 2014
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