
==== Front
Investig Clin Urol
Investig Clin Urol
ICU
Investigative and Clinical Urology
2466-0493
2466-054X
The Korean Urological Association

39249922
10.4111/icu.20230416
Original Article
Pediatric Urology
Identifying infrequent genetic changes in monozygotic twins afflicted with hypospadias via targeted panel sequencing
https://orcid.org/0000-0001-8181-0694
Kim Ja Hye 1*
https://orcid.org/0000-0001-8982-6922
Kim Kun Suk 2*
https://orcid.org/0000-0001-5627-7381
Han Jae Hyeon 3
https://orcid.org/0000-0002-1960-9495
Kim Dongsu 2
https://orcid.org/0000-0001-8396-3061
Kwak Chan Hoon 2
https://orcid.org/0000-0003-1196-7826
Choi Jin-Ho 1
https://orcid.org/0000-0003-4888-483X
Song Sang Hoon 2
1 Department of Pediatrics, Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine, Seoul, Korea.
2 Department of Urology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.
3 Seoul Asan Urology Clinic, Cheongju, Korea.
Corresponding Author: Sang Hoon Song. Department of Urology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea. TEL: +82-2-3010-1554, FAX: +82-2-477-8928, shsong@amc.seoul.kr
*These authors contributed equally to this study and should be considered co-first authors.

9 2024
12 8 2024
65 5 487493
17 12 2023
23 4 2024
17 6 2024
© The Korean Urological Association
2024
The Korean Urological Association
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Purpose

We aimed to identify the genetic causes of hypospadias in children using targeted gene panel sequencing for disorders of sex development (DSD).

Materials and Methods

This study included 18 twin boys with hypospadias: seven and two pairs were monozygotic and dizygotic twins, respectively, and six were discordant and three were concordant twins. Targeted gene panel sequencing for 67 known DSD genes was performed. Sequence variants were classified into five different categories, pathogenic, likely pathogenic, variants of uncertain significance, likely benign, and benign, following the American College of Medical Genetics and Genomics Standards and Guidelines.

Results

The mean gestational age and birth weight were 35.3±2.0 weeks and 1.96±0.61 kg, respectively, with seven patients being small for gestational age. Hypospadias was present in 12 patients, with posterior type in 33.3% and anterior type in 66.7%. In three families with twins, both siblings had hypospadias. In addition, cryptorchidism was observed in one subject. Surgical correction of hypospadias was performed at a mean age of 22.1 months. Molecular analysis identified 12 different genetic variants, including two pathogenic mutations in the AMH (p.E389*) and SRD5A2 (p.R246Q) genes, found in subjects with hypospadias, respectively. However, only heterozygous mutations were detected.

Conclusions

This study did not identify a definitive genetic component contributing to the development of hypospadias; however, the findings suggest that intrauterine growth retardation may play a significant role.

Disorders of sex development
Hypospadias
Twins
Urethra
Asan Institute for Life Sciences, Asan Medical Center https://doi.org/10.13039/501100005006 2016IT0586-1 Korea Health Industry Development Institute https://doi.org/10.13039/501100003710 HR21C0198 Ministry of Science and ICT, South Korea https://doi.org/10.13039/501100014188 2022R1F1A107478211 National Research Foundation of Korea https://doi.org/10.13039/501100003725 2022R1F1A107478211
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pmcINTRODUCTION

Hypospadias is a common urogenital anomaly in boys, with an estimated incidence of 1 to 8/1,000 live births [12]. In hypospadias, the urethral opening may be present anywhere along the ventral aspect of the penis, the scrotum, or even extending to the perineum. Based on severity and location of urethral opening, hypospadias can be classified into three degrees, anterior, penile, and proximal openings [3].

The etiology of hypospadias remains indeterminate, although it is hypothesized to result from an interplay of genetic, endocrine, placental, maternal, and environmental factors [45]. Hypospadias has been observed to have a familial association, with up to 10% of children with hypospadias in family segregation analysis [67]. Single gene defects such as MAMLD1, SRD5A2, MBTPS2, NR2F2, and HRAS gene variants are associated with hypospadias [8910]. Next-generation sequencing technologies have been used for the diagnosis of genetic variants in patients with 46, XY disorders of sex development (DSD) [1112]. Srivastava et al. [13], showed pathogenic variants in AR, SRD5A2, NR5A1, WT1, and ARTX in patients with 46, XY severe hypospadias. In addition, polymorphisms of certain genes have been reported to associate with hypospadias [14]. However, comprehensive studies examining a wide spectrum of gene panels implicated in sex hormone biosynthesis, genital tubercle formation, and early urethral development in patients with hypospadias are notably limited [15].

In this study, we assessed the prevalence of genetic variants in twins with or without hypospadias using targeted sequencing of a 67-gene panel specific to DSD genes. By focusing on twin subjects, we mitigated potential confounding effects from maternal factors such as gestational hypertension, progesterone exposure, and contact with environmental toxins—each of which could potentially influence the incidence of hypospadias.

MATERIALS AND METHODS

1. Patient selection

The present study protocol was reviewed and approved by the Institutional Review Board of Asan Medical Center (approval number: 2017-0536). Due to the retrospective nature of the investigation, the requirement for informed consent was waived. Between 2017 and 2020, a total of 18 twin boys, comprising six discordant and three concordant pairs, who underwent targeted gene panel sequencing at Asan Medical Center, were selected. Patients with known genetic etiologies were excluded. The following variables were retrospectively reviewed: birth weight, gestational age, and type of hypospadias. Hypospadias was subdivided by urethral meatus position in anterior (glandular or coronal), penile (distal penile, mid-penile, and proximal penile), and proximal (penoscrotal, scrotal, or perineal) [3].

2. Targeted gene panel sequencing for 67 known DSD genes

DNA was extracted from peripheral blood leukocytes using a Gentra Puregene blood kit (Qiagen) following the manufacturer’s protocol. Targeted gene panel sequencing was performed with 67 known genes involved in sex determination, sex differentiation, and hypogonadism as previously described (Supplementary Table 1). A customized Sure Select Target Enrichment System Kit (Agilent Technologies) was used for capturing exomes. Sequencing was performed using the MiSeq platform (Illumina). Sequence reads were aligned using the Burrows-Wheeler Aligner (BWA version 0.7.5) and the hg19 human reference sequence. The sequence variants with minor allele frequency (≤1%) in the 1000 genomes browser (http://www.internationalgenome.org/1000-genomes-browsers) and gnomAD browser (https://gnomad.broadinstitute.org/) were filtered out. Variant pathogenicity was determined using previously published literature and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and Human Gene Mutation Database (http://www.hgmd.org/). Sequence variants were divided into five categories, pathogenic, likely pathogenic, variants of uncertain significance, likely benign, and benign, based on the American College of Medical Genetics and Genomics Standards and Guidelines [16]. In silico analysis was performed for novel missense variants using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), Sorting Intolerant From Tolerant (http://sift.jcvi.org/), MutationTaster (http://www.mutationtaster.org/), Mutation Assessor (http://mutationassessor.org/r3/), and Protein Variation Effect Analyzer (http://provean.jcvi.org/index.php).

RESULTS

1. Clinical features

A total of 18 patient were born at a mean gestational age of 35.3±2.0 weeks and a mean birth weight of 1.96±0.61 kg. Seven of these were small for gestational age (SGA; less than 3rd percentile for birth weight). The mother of subjects 3 and 4 had high blood pressure during pregnancy with an advanced maternal age of 44 years, while the others did not report any significant medical history during pregnancies. There was no reported family history of hypospadias in our study cohort.

This study included 12 patients with hypospadias. In the cohort, 33.3% (4/12) presented with posterior hypospadias, while 66.7% (8/12) exhibited anterior hypospadias (Table 1): four with posterior hypospadias included two with penoscrotal and two with scrotal hypospadias; eight with anterior hypospadias included six with distal to proximal penile, one with glandular, and one with coronal hypospadias. In three families, both twins had hypospadias: Family 1 and 4 had the anterior type of hypospadias; Family 9 had one twin with anterior and the other with posterior hypospadias. In the remaining six families, one of the twins had hypospadias. Except for three families with hypospadias in both siblings, this condition was found in a twin born with a lower birth weight. Cryptorchidism was found in subject 12, with bilateral testis located in the external ring. subject 12 had multiple anomalies including biliary atresia, patent ductus arteriosus, and bilateral inguinal hernia. Micropenis was evident in subject 11 with stretched penile length of 2.4 cm. All 12 patients who had hypospadias underwent surgical correction at a mean age of 22.1±13.9 months.

2. Molecular analysis

We found 12 different variants, of which 2 were classified as pathogenic mutations: one is the pathogenic mutation of AMH (p.E389*) in subjects 1 and 2. This mutation was reported likely pathogenic in a patient with cryptorchidism and persistent Mullerian duct remnant syndrome [17]. The other is a known pathogenic mutation of SRD5A2 (p.R246Q) in subject 17 (Table 2) [18]. Despite being dizygotic twins, subjects 17 and 18 had posterior type hypospadias.

DISCUSSION

Hypospadias, a urogenital anomaly with urethral fold closure failure during the 7th to 14th week of gestational age, has been the subject of genetic mutation analysis [19]. In the International Consensus Conference on the definition of DSDs, hypospadias was considered a form of 46, XY DSD [8]. We conducted research to identify the genetic causes of hypospadias in both monozygotic and dizygotic twins. Pathogenic mutations were found in two families. However, the clinical significance of these heterozygous variants is difficult to ascertain. Hypospadias was identified in both siblings in three families, including one with dizygotic twins. Although the number of patients is small, low birth weight could potentially be a risk factor. Since panel testing for known genes has been performed, further studies to discover new genetic causes, such as Whole Exome Sequencing, or to understand epigenetic causes, should be undertaken.

AMH is a glycoprotein dimer and functional marker of fetal Sertoli cells into adulthood. In our study, two monozygotic twins with anterior hypospadias showed an AMH mutation (p.E389*). This heterozygous mutation was previously reported pathogenic mutations, and in a large cohort study, about 65% of homozygous mutations in AMH gene were genetic etiology of Persistent Müllerian duct syndrome [20]. However, research on the clinical significance of heterozygous carriers remains limited. The SRD5A2 gene is crucial in the development and metabolism of sex steroids. It encodes the enzyme, 5-alpha reductase, which plays a pivotal role in converting testosterone to dihydrotestosterone, a potent androgen necessary for the development of male external genitalia during fetal development. Homozygous mutations in the SRD5A2 gene can lead to a diverse range of DSDs. Previous reports have also identified heterozygous mutations in SRD5A2 in patients with hypospadias [21]. However, the impact of these heterozygous variants on disease occurrence is uncertain. To determine whether heterozygous carriers of SRD5A2 and AMH exhibit these phenotypes, a comprehensive large-scale study incorporating statistical analysis is essential.

Previous research has demonstrated a correlation between lower birth weight and increased susceptibility to hypospadias, with the lower-weight twin being more frequently affected [2022]. Studies have also highlighted that twin status, whether monozygotic or dizygotic, increases the risk of hypospadias [23]. Furthermore, a multicenter study conducted in Asia identified several risk factors for hypospadias, including very low birth weight, intrauterine growth retardation, maternal preeclampsia, and being SGA [2425]. These risk factors were observed in our study as well. Placental dysfunction, which can reduce chorionic gonadotropin production, may impair the differentiation of external genitalia, underscoring the importance of in utero environmental factors [5]. Furthermore, there is an elevated risk of hypospadias among offspring of overweight and obese mothers, possibly related to increased levels of free circulating estrogens in adipose tissue [26]. Our findings highlight the significant impact of environmental factors on the development of hypospadias.

Approximately 30% of hypospadias cases are thought to be associated with genetic syndromes such as androgen receptor insensitivity or SRD5A type 2 deficiency [27]. However, in such cases, there are often abnormalities in gonadal development, or complete female genitalia may be present. Despite this, a wide range of clinical symptoms can still occur, making differential diagnosis challenging [7]. The prevalence of congenital genital anomalies, including cryptorchidism and hypospadias, may reach as high as 1 in 200 to 1 in 300 [24]. The diagnosis of specific DSDs is generally confined to patients exhibiting proximal hypospadias alongside cryptorchidism [28]. Therefore, in severe hypospadias patients, it is crucial to assess gonadal development and other genitourinary abnormalities. Genetic testing may be necessary for patients who display specific clinical characteristics or anomalies.

Although our study did not investigate epigenetic changes, such changes are known to significantly influence the development of hypospadias [29]. Kaefer et al. [29] have demonstrated a direct link between epigenetic alterations and hypospadias tissue samples. Their study identified numerous differential DNA methylation regions (DMRs) in cases of mild hypospadias, with a decrease in the number of DMRs in moderate cases and even fewer in severe cases. This finding suggests that the increasing prevalence of hypospadias could be related to environmental factors, particularly toxicant exposure, which interferes with epigenetic programming during penile development.

Our study has certain limitations. The sample size was small for investigating genetic variants with a DSD-related genetic panel. The study cohort consisted of twins with low birth weight for their gestational age and shorter gestational age [30]. To investigate genetic effects and rule out the occurrence of hypospadias, a familial aggregation study should be performed to exclude the suspected genetic variant. Finally, our patients had heterozygous for the SRD5A2 and AMH genes, which are known to be inherited in a recessive manner. It is uncertain whether this heterozygosity in recessive inheritance causes hypospadias.

CONCLUSIONS

The role of environmental factors is significant in the development of hypospadias among both monozygotic and dizygotic twins, with SGA is frequently observed. These observations suggest that epigenetic and environmental influences are pivotal in the development of hypospadias. While our study identified a small number of twins with potential pathogenic genetic variants associated with hypospadias, it is premature to concluded that hypospadias is a milder manifestation within the spectrum of DSD. Comprehensive studies with larger cohorts are necessary to fully elucidate the multifactorial etiology of hypospadias, including the identification of additional genetic and environmental contributors.

SUPPLEMENTARY MATERIAL

Supplementary material can be found via https://doi.org/10.4111/icu.20230416.

Supplementary Table 1

DSD genes used to filter exome sequencing variants

Table 1 Subject characteristics

Family No.	Subject	Type of twin	Mother’s age at delivery (y)	Gestational weeks	BW (kg)	SGA	Phenotype	Type	Micropenis	Cryptorchidism	Curvature (°)	Type of urethroplasty	Combined abnormality	
1	1	Monozygotic	35	33+2	2.13	N	Coronal	Ant	N	N	0	TIP		
2	33+2	1.65	N	Distal penile	Ant	N	N	0	TIP		
2	3	Monozygotic	44	32+0	2.00	N	-	-	N	N	-	-		
4	32+0	0.95	Y	Penoscrotal	Post	N	N	0	TIP		
3	5	Monozygotic	32	35+0	1.57	Y	Glandular	Ant	N	N	0	MAGPI		
6	35+0	2.15	N	-	-	N	N	-	-		
4	7	Monozygotic	29	37+0	2.30	N	Proximal penile	Ant	N	N	30	TIP		
8	37+0	2.70	N	Proximal penile	Ant	N	N	60	TPIF		
5	9	Monozygotic	29	34+2	2.00	N	Proximal penile	Ant	N	N	30	TIP		
10	34+2	2.50	N	-	-	N	N	-	-		
6	11	Monozygotic	29	38+0	3.52	N	-	-	Y	N	-	-	Micropenis	
12	38+0	1.77	Y	Scrotal	Post	N	Y	45	Staged urethroplasty	Biliary atresia, PDA, inguinal hernia	
Cryptorchidism	
7	13	Monozygotic	N/A	36+0	1.80	Y	Mid-penile	Ant	N	N	-	TIP		
14	36+0	2.40	N	-	-	N	N	-	-		
8	15	Dizygotic	33	38+0	1.60	Y	-	-	N	N	-	-		
16	38+0	1.40	Y	Mid-penile	Ant	N	N	20	TIP		
9	17	Dizygotic	32	34+4	0.99	Y	Scrotal	Post	N	N	30	Staged urethroplasty		
18	34+4	1.84	N	Penoscrotal	Post	N	N	30	Staged urethroplasty		
BW, birth weight; SGA, small for gestational age; N/A, not available; N, no; Y, yes; Ant, anterior type; Post, posterior type; TIP, tubularized incised plate; MAGPI, meatal advancement and glanduloplasty; TPIF, transverse preputial island flap; PDA, patent ductus arteriosus.

Table 2 Genetic variants in patients with hypospadias

Type of twin	Subject	Type of hypospadias	Gene	Finding	cDNA change	Amino acid change	Inheritance	Zygosity	Novelty	Pathogenicity	
Monozygotic	1	Ant	AMH	Common	c.1165G>T	p.E389*	AR	Heterozygous	Novel	Pathogenic	
2	Ant	
Monozygotic	3	-	BBS2	Common	c.1120C>T	p.R374W	AR	Heterozygous	Novel	Benign	
4	Post	
Monozygotic	5	Ant	FGFR1	Common	c.230A>G	p.N77S	AD	Heterozygous	Novel	VUS	
6	-	MAMLD1	Common	c.880C>T	p.P294S	XR	Hemizygous	Novel	Likely benign	
Monozygotic	9	Ant	BBS1	Common	c.764G>C	p.G255A	AR	Heterozygous	Novel	Benign	
10	-	
Monozygotic	11	-	WWOX	Common	c.468G>T	p.R156S	AR	Heterozygous	Reported	Benign	
12	Post	
Monozygotic	13	Ant	CHD7	Common	c.7472G>A	p.R2491H	AD	Heterozygous	Reported	Benign	
14	-	
Dizygotic	15	-	BBS1	Only in 15	c.723+7G>A	-	AR	Heterozygous	Novel	Likely benign	
15	-	AR	Common	c.170T>A	p.L57Q	XR	Hemizygous	Novel	Likely benign	
16	Ant	
Dizygotic	17	Post	SRD5A2	Common	c.736G>A	p.R246Q	AR	Heterozygous	Reported	Pathogenic	
18	Post	BBS12	Common	c.79A>G	p.R27G	AR	Heterozygous	Novel	VUS	
18	Post	MKKS	Only in 18	c.757T>C	p.S253P	AR	Heterozygous	Reported	Benign	
Ant, anterior type; Post, posterior type; AR, autosomal recessive; AD, autosomal dominant; XR, X-linked recessive; VUS, variant of uncertain clinical significance.

CONFLICTS OF INTEREST: The authors have nothing to disclose.

FUNDING: This research was partially supported by a grant (2016IT0586-1) from the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea, a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HR21C0198) and a grant funded by the Ministry of Science and ICT of the Republic of Korea and the National Research Foundation of Korea (grant number: 2022R1F1A107478211).

AUTHORS’ CONTRIBUTIONS: Research conception and design: Jin-Ho Choi, Sang Hoon Song, and Kun Suk Kim.

Data acquisition: Chan Hoon Kwak, Jae Hyeon Han, Jin-Ho Choi, Kun Suk Kim, and Sang Hoon Song.

Statistical analysis: Chan Hoon Kwak, Jin-Ho Choi, Kun Suk Kim, and Sang Hoon Song.

Data analysis and interpretation: Chan Hoon Kwak, Jin-Ho Choi, Ja Hye Kim, Kun Suk Kim, Sang Hoon Song, and Dongsu Kim.

Drafting of the manuscript: Ja Hye Kim, Chan Hoon Kwak, Jae Hyeon Han, Jin-Ho Choi, Kun Suk Kim, and Sang Hoon Song.

Critical revision of the manuscript: Ja Hye Kim, Jin-Ho Choi, Kun Suk Kim, Sang Hoon Song, and Dongsu Kim.

Obtaining funding: Sang Hoon Song and Kun Suk Kim.

Administrative, technical, or material support: Jin-Ho Choi, Kun Suk Kim, and Sang Hoon Song.

Supervision: Ja Hye Kim and Sang Hoon Song.

Approval of the final manuscript: all authors.
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