Identification of gene variants in 30 patients from southeastern China with severe hypospadias by whole-exome sequencing

Abstract

Hypospadias is a common congenital malformation of the male external genitalia, with severe cases presenting considerable surgical and long-term challenges. Despite the clinical importance of severe hypospadias demonstrated by prolonged hospital stays, repeated surgeries, and substantial costs, the genetic etiology of severe hypospadias remains incompletely understood, particularly in diverse populations. To determine the molecular basis, we performed whole-exome sequencing (WES) on 30 Chinese patients from southeastern China with confirmed 46,XY karyotypes. Our analysis identified clinically relevant genetic variants, including single-nucleotide variants (SNVs) and copy number variations (CNVs), with subsequent phenotypic correlation. Clinically relevant genetic variants were identified in 33.3% (10/30) of cases, including novel SNVs in gonadal regulators (nuclear receptor subfamily 5 group A member 1 [NR5A1] c.1344dupC/c.244+1G>T and SRY-box 3 [SOX3] c.1273G>C), morphogenetic modulators (GLI family zinc finger 3 [GLI3] c.4731delA and aristaless-related homeobox [mARX] c.644C>G), and syndromic genes (patched domain containing 1 [PTCHD1] c.667G>A and euchromatic histone lysine methyltransferase 1 [EHMT1] c.3081C>T). Additionally, recurrent CNVs at 22q12.3 and a novel CNV exon 18 deletion in myelin regulatory factor (MYRF) and 18q11.2 were identified. Mutation carriers showed a significantly higher frequency of cryptorchidism (40.0% vs 5.0%, P < 0.01) and a higher prevalence of ≥3 associated malformations (80.0% vs 35.0%, P < 0.05) than non-carriers, highlighting genotype–phenotype correlations. The 33.3% diagnostic yield tripled conventional estimates, demonstrating WES efficacy in identifying SNVs and CNVs in severe phenotypes. These findings reveal the genetic heterogeneity of severe hypospadias and support WES utility in uncovering novel variants and structural genomic alterations.

INTRODUCTION

Hypospadias is one of the most common congenital malformations of the male genitalia. Hypospadias is characterized by abnormal ventral placement of the urethral meatus and a dorsal hooded prepuce and is often accompanied by penile curvature (chordee).1,2 Systematic reviews of global surveillance systems have reported that the prevalence of hypospadias ranges from 19.9 to 41.9 per 10 000 male births (0.20%–0.42%) across 42 countries.3 China has a considerably higher burden of hypospadias than other countries, as shown by a recent nationwide surveillance, which demonstrated a prevalence of 112/10 000 male births (1.12%, approximately 1/89).4 However, population-specific estimates for severe forms of hypospadias remain sparse. While mild forms of hypospadias may present with minimal functional impairment, severe cases (particularly posterior hypospadias) pose considerable challenges in surgical correction and long-term outcomes, such as urinary and sexual dysfunction.5 The etiology of hypospadias is multifactorial, involving genetic, hormonal, and environmental factors. However, in severe cases of hypospadias, genetic factors play a predominant role (with an estimated contribution as high as 77%), indicating the critical importance of genetic investigation.6,7 The current incomplete determination of underlying molecular mechanisms of hypospadias severely impedes the clinical diagnosis and development of targeted therapy of this condition.

Advances in genomic technologies, such as whole-exome sequencing (WES), have improved the identification of genetic variants associated with complex congenital disorders. By enabling a comprehensive analysis of coding regions, WES offers a powerful tool to uncover novel genetic contributors and identify molecular pathways in the pathogenesis of severe hypospadias. Mutations in androgen signaling genes, such as androgen receptor (AR), recombinant steroid 5 alpha reductase 2 (SRD5A2), and fibroblast growth factor (FGF), have been implicated in hypospadias. However, these findings account for only a small fraction of cases, highlighting the need for broader genomic investigations to resolve the genetic heterogeneity of hypospadias.7,8,9

Hypospadias, with its complex etiology and challenges of surgical management, has been the focus of recent genomic investigations, and the utility of WES in investigating this condition has been shown in Chinese Han populations. Zhang et al.10 identified novel urethral development-related variants in 130 patients with hypospadias using targeted sequencing. Additionally, Wang et al.5 applied WES to 42 posterior hypospadias cases and showed genotype–phenotype correlations in severe forms. These studies highlight the capacity of WES to detect rare mutations in understudied populations.11,12,13

Despite these advancements, there is a lack of definitions of population-specific genetic architecture of severe hypospadias, particularly in southeastern Chinese cohorts. The heterogeneity of this condition, combined with the interplay of multiple genetic and environmental factors, complicates the identification of causative variants.14 Therefore, we hypothesized that applying WES-driven nucleotide variants (SNV)/copy number variations (CNV) screening would enable the discovery of distinct genetic determinants unique to this population.

To test this hypothesis, we used WES in patients with hypospadias to identify novel variants through correlations of phenotypes, determine molecular mechanisms causing severe phenotypes, and increase the genetic diagnostic yield (>50% of cases are unresolved by conventional methods), ultimately enabling targeted interventions. Our findings validate this approach by identifying pathogenic variants and bridging the diagnostic gap, which could ultimately improve the outcomes of patients with this challenging condition.

PATIENTS AND METHODS

Patients and methods

This study enrolled male patients with severe hypospadias who were diagnosed at the Department of Urology, Fujian Children’s Hospital (Fuzhou, China) between January 2022 and December 2024. The inclusion criteria comprised the following: (1) confirmed diagnosis of severe hypospadias, which was defined by a urethral meatus located at the penoscrotal or perineal region (type III/IV; classified as posterior hypospadias in accordance with the European Association of the Urology/European Society for Paediatric Urology guidelines),15 accompanied by severe ventral penile curvature (≥20°) requiring surgical correction and/or associated anomalies, such as bifid scrotum or impaired urination; (2) genetic confirmation of a 46,XY karyotype with sex-determining region of Y-chromosome (SRY) gene positivity, excluding disorders of sex development and chromosomal abnormalities; (3) absence of prior hormonal therapy; and (4) verification of urogenital anatomy via renal ultrasound (to rule out renal malformations) and voiding cystourethrography (to assess urethral strictures, fistulas, or bladder neck abnormalities). A prostatic utricle was specifically confirmed by intraoperative urethroscopy or voiding cystourethrography. The exclusion criteria included patients with multisystem malformation syndromes (e.g., vertebral anal cardiac tracheo-esophageal renal and limb [VACTERL] syndrome association with ≥3 core features and coloboma, heart defect, atresia choanae, retarded growth, genital abnormality, and ear anomaly [CHARGE] syndrome), patients with incomplete clinical documentation, or patients who declined to sign informed consent. All participants provided comprehensive clinical documentation, including age, birth history, familial history, associated malformations, and others. The study protocol, which included a plan for data publication, was approved by the Institutional Ethics Committee of Fujian Children’s Hospital (Approval No. 2022ETKLR0121). All parents or legal guardians have signed written informed consent forms for data publication and obtained permission to publicly release the data.

Clinical parameters were assessed under standardized conditions. The penile length was measured dorsally from the pubic symphysis to the glans tip under general anesthesia before surgery using a rigid ruler. The degree of chordee was quantified intraoperatively during artificial erection testing (via saline injection) with a sterile goniometer. The condition of the urethral plate was intraoperatively classified as “good” for adequate thickness (>1 mm) with supple tissue and visible vascularity or “poor” for a thin (<1 mm), fibrotic, scarred, or ischemic appearance.

Whole-exome sequencing

WES was performed using a dual-strategy approach. In a subset of patients, proband-only WES was conducted, followed by a targeted analysis of a predefined 207-gene panel (Supplementary Table 1) to prioritize variants in known hypospadias-associated pathways. In the other patients, family-based trio WES (proband and both parents) was used to identify de novo variants and inheritance patterns, thereby enhancing the discovery of novel candidate genes. In both sequencing strategies, library preparation and sequencing were standardized to ensure cross-group comparability. Genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini Kit (category No. 51104; Qiagen, Hilden, Germany). The extracted DNA was fragmented into 200–300-base pair (bp) segments, and the libraries were prepared using the KAPA HyperPrep Kit (KK8504; Roche, Basel, Switzerland) by following the manufacturer’s protocol. Enrichment used the IDT xGen Exome Research Panel version 2 (Integrated DNA Technologies, Coralville, IA, USA). Captured libraries underwent 150-bp paired-end sequencing on an Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) platform and achieved >95% exome coverage at ≥ 20 × depth. Raw sequencing reads were aligned to the human reference genome (GRCh37/hg19) using the Burrows–Wheeler Aligner (BWA version 0.7.12). Duplicate reads were removed using Picard (version 1.128; Broad Institute, Cambridge, MA, USA). Variant calling was performed using the Genome Analysis Toolkit (GATK 4.1.8.1; Broad Institute) by following established best practices. To minimize batch effects, joint variant calling was conducted using ANNOVAR and the Variant Effect Predictor (VEP, version 105; EMBL-EBI, Hinxton, UK).16

Supplementary Table 1.

List of 207 genes in gene panel of hypospadias

Condition	Genes
Sexual development (47)	FSHR, BMP15, PSMC3IP, MCM9, SOHLH1, NUP107, MRPS22, ESR2, SPIDR, ZSWIM7, HSD17B4, LARS2, TWNK, HARS2, ERAL1, CLPP, CFTR, GATA4, INSL3, ANOS1, FGFR1, PROKR2, PROK2, CHD7, FGF8, GNRHR, KISS1R, NSMF, TAC3, TACR3, GNRH1, KISS1, WDR11, HS6ST1, SEMA3A, SPRY4, IL17RD, DUSP6, FGF17, FLRT3, FEZF1, LHB, FSHB, NDNF, TCF12
Sex reversal (14)	SRY, NR0B1, CBX2, MAP3K1, DHH, AKR1C2, AKR1C4, ZFPM2, DHX37, SOX9, AMH, AMHR2, NR5A1, NR2F2
Androgen deficiency (14)	RSPO1, CYP21A2, CYP11B1, HSD3B2, POR, CYP19A1, CYP17A1, STAR, SRD5A2, LHCGR, HSD17B3, DHCR7, AR, ESR1
Other (4)	MAMLD1, NRIP1, TBX18, DSTYK
Syndromic with hypospadias phenotypes (128)	B9D2, B9D1, TMEM231, TXNDC15, TMEM107, TMEM216, NPHP3, MKS1, CEP290, TCTN2, RPGRIP1L, TMEM67, CC2D2A, KIF14, BBS2, ARL6, CFAP418, BBS1, CCDC28B, MKKS, IFT172, BBS4, BBS5, BBS7, TTC8, BBS9, BBS10, TRIM32, BBS12, WDPCP, SDCCAG8, LZTFL1, BBIP1, IFT27, IFT74, NEK1, CEP120, DYNLT2B, WDR19, DYNC2I1, KIAA0586, DYNC2H1, DYNC2I2, WDR34, IFT140, IFT43, IFT81, INTU, KIAA0753, TTC21B, IFT80, DYNC2LI1, IFT52, WDR35, RAD21, SMC3, NIPBL, BRD4, SMC1A, HDAC8, ICR1, PLAG1, HMGA2, IGF2, DVL3, NXN, ROR2, WNT5A, DVL1, MASP1, COLEC11, COLEC10, CUL7, OBSL1, CCDC8, BRAF, KRAS, MAP2K1, FRAS1, FREM2, GRIP1, KMT2D, KDM6A, JAG1, NOTCH2, PTCHD1, SALL1, DACT1, EHMT1, KMT2C, EVC, EVC2, SMG8, MED25, GP1BB, MYMK, AFF4, CDH11, KIFBP, USP7, FAT4, CDKN1C, GPC4, KANSL1, NBN, MED12L, MID1, B3GLCT, CHRM3, NSD2, ZIC3, RPL10, CUL4B, SLC30A7, ARX, OTUD5, CYB5A, FZD2, ROBO1, ABCD1, HOXA13, BNC2, MYRF, TSPYL1, INPP5E, CHRNA3, TBX22, PSMC1

Targeted gene panel

Our hypospadias gene panel comprised 207 genes (Supplementary Table 1) associated with gonadal development, differentiation, sex reversal, and/or related syndromes. The selection of target genes was based on the GeneCards, OMIM, and ClinVar databases, combined with searches in public literature databases (e.g., PubMed, Google Scholar, Web of Science, and China National Knowledge Infrastructure [CNKI]) using keywords, namely “hypospadias”, “disorders of sex development”, and “gonadogenesis”.

Prediction of protein function for novel mutations

The effect of variants on protein structure and function was evaluated using a suite of prediction tools, namely SIFT (https://sift.bii.a-star.edu.sg/; last accessed on 2025 Mar 01), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2; last accessed on 2025 Mar 01), PROVEAN (http://provean.jcvi.org/; last accessed on 2025 Mar 01), ClinPred (https://sites.google.com/site/clinpred/home; last accessed on 2025 Mar 01), Mutation Assessor (http://mutationassessor.org; last accessed on 2025 Mar 01), FATHMM (http://fathmm.biocompute.org.uk; last accessed on 2025 Mar 01), and GERP (http://mendel.stanford.edu/SidowLab/downloads/gerp/; last accessed on 2025 Mar 01). The interpretation of variants was conducted in accordance with the guidelines established by the American College of Medical Genetics and Genomics (ACMG).17 The mutations were classified into the following five categories: pathogenic, likely pathogenic (LP), variants of uncertain significance (VUS), likely benign, or benign. This classification was based on the ACMG standards and by referencing the NCBI, ClinVar, and HGMD databases.

Sanger sequencing validation

All variants suspected of contributing to the phenotypes of hypospadias were validated by Sanger sequencing using proband DNA and parental peripheral blood samples. Additionally, information on the family history was collected to facilitate the interpretation of inheritance patterns (e.g., de novo vs inherited) in the Sanger sequencing results.

Genotype–phenotype association analysis

To systematically examine associations between genetic variants and clinical manifestations, genotype–phenotype associations were evaluated using a multitiered approach. Associated malformations were defined as major congenital anomalies that met all of the following criteria: (1) distinct from the primary diagnosis of hypospadias; (2) requiring medical/surgical intervention; and (3) classifiable into one of the following five categories. These categories were as follows: (1) cryptorchidism (unilateral/bilateral); (2) anorectal anomalies such as imperforate anus; (3) major cardiac defects such as septal defects exceeding 5 mm; (4) major musculoskeletal anomalies, such as polydactyly and syndactyly; and (5) other intervention-requiring conditions, such as symptomatic inguinal hernia and hydrocele. The exclusion criteria for major congenital anomalies were as follows: (1) hypospadias itself; or (2) minor anomalies, such as preauricular pits, cutaneous hemangiomas <2 cm, and skin tags. The patients were classified as having multisystem malformations if they presented with ≥3 distinct associated malformations, which included cryptorchidism as one possible anomaly but did not require its presence. This threshold could be achieved through any combination of qualifying malformations, such as anorectal anomaly, ventricular septal defect, or syndactyly, either individually or in combination.

Statistical analyses

Statistical analyses were performed in R version 4.2.2 (R Foundation for Statistical Computing, Vienna, Austria) using the stats, rstatix, and ggplot2 packages. Continuous variables (e.g., age, penile length, and curvature angle) were shown as the mean ± standard deviation (s.d.) and were compared between groups using Welch’s t-test (assuming unequal variances). Categorical variables (e.g., cryptorchidism and polydactyly) were compared between variant-positive (n = 10) and variant-negative (n = 20) cohorts using Fisher’s exact test, with α = 0.05. The relationships between genetic variants and multisystem malformations (≥3 associated malformations) were analyzed using multivariate logistic regression. P < 0.05 was considered statistically significant.

RESULTS

Clinical features

The overall design of the study is illustrated in Figure 1. Thirty children with severe hypospadias, ranging in age from 6 months to 5 years (mean±s.d.: 1.8 ± 1.3 years), were included in the mutation analysis. All participants presented with severe hypospadias accompanied by varying degrees of penile dysplasia. The urethral opening was predominantly located at the penoscrotal position in 63.3% or scrotal position in 30.0% of patients, and only 6.7% of patients had the urethral opening on the penile shaft. The mean penile length was 2.5 (range: 1.5–3.5) cm, and the degree of penile curvature ranged from 20° to 90°, with a mean value of 42.3°. The urethral plate showed a good condition in 63.3% (19/30) patients and a poor condition in 36.7% (11/30) patients. Among the 11 patients with a poor urethral plate condition, genetic mutations were identified in 5 (45.5%), while 6 (54.5%) showed no detectable mutations. Fisher’s exact test showed no significant difference in the frequency of mutation within this subgroup. In addition to external genital malformations, several children showed other congenital abnormalities. The clinical characteristics of the individual patients are shown in Table 1.

Figure 1.

Overall design of the study. WES: whole-exome sequencing; chr22: chromosome 22; DEL: deletion; SOX3: sex-determining region Y box protein 3; NR5A1: namely nuclear receptor subfamily 5; GLI3: GLI family zinc finger protein 3; EHMT1: euchromatic histone-lysine N-methyltransferase 1; PTCHD1: patched domain-containing protein 1; ARX: automatic retransmission exchange; MYRF: myelin regulatory factor.

Table 1.

Clinical features of 30 hypospadias patients

Patient number	Age at diagnosis	Urethral meatus position (GMS grade)	Accompanying malformation	Penis length (cm)	Penile curvature degree (°)	Texture of the urethral plate
Patient 1	1 year	Penoscrotal	Penoscrotal fusion and micropenis	1.5	38	Good
Patient 2	3 years and 9 months	Scrotal	Anal atresia, penoscrotal transposition, and micropenis	3.2	85	Poor
Patient 3	9 months	Scrotal	Penoscrotal transposition, penoscrotal fusion, prostatic utricle, micropenis, and bilateral cryptorchidism	1.8	36	Poor
Patient 4	2 years and 7 months	Penoscrotal	Penoscrotal transposition, penoscrotal fusion, prostatic utricle, micropenis, and bilateral cryptorchidism	2.5	40	Good
Patient 5	2 years	Penoscrotal	Chordee and micropenis	2.7	45	Good
Patient 6	2 years and 11 months	Penoscrotal	Chordee and micropenis	2.6	35	Poor
Patient 7	1 year and 6 months	Penoscrotal	Chordee, syndactyly of the right index finger, micropenis, and hydrocele	2.5	40	Good
Patient 8	2 years	Penoscrotal	Penoscrotal transposition and micropenis	2.0	40	Good
Patient 9	7 months	Penoscrotal	Penoscrotal fusion and micropenis	2.8	42	Poor
Patient 10	5 years	Penis	Indirect inguinal hernia	3.5	20	Good
Patient 11	3 years and 11 months	Penoscrotal	Bilateral cryptorchidism, hydrocele, floating thumb of the left hand, and syndactyly of the right hand	3.5	90	Good
Patient 12	5 months	Penis	Buried penis, atrial septal defect, and polydactyly of the right thumb	3.0	25	Good
Patient 13	8 months	Scrotal	Chordee, penoscrotal fusion, micropenis, and polydactyly of the right thumb	2.0	40	Poor
Patient 14	1 year and 8 months	Penoscrotal	Chordee and micropenis	2.0	35	Good
Patient 15	5 months	Penoscrotal	Chordee, penoscrotal transposition, and micropenis	2.8	45	Poor
Patient 16	9 months	Scrotal	Chordee, penoscrotal transposition, cryptorchidism, micropenis, and atrial septal defect	2.6	41	Good
Patient 17	11 months	Scrotal	Hemangioma of the left scrotum and micropenis	2.8	40	Poor
Patient 18	3 years	Penoscrotal	Chordee	3.5	45	Good
Patient 19	3 years	Penoscrotal	Chordee, micropenis, and penoscrotal fusion	3.0	35	Good
Patient 20	3 days	Penoscrotal	Chordee and micropenis	1.9	50	Good
Patient 21	1 year and 6 months	Scrotal	Chordee and micropenis	2.0	60	Good
Patient 22	3 years	Penile shaft	Chordee and micropenis	2.8	45	Good
Patient 23	1 year and 1 month	Penoscrotal	Chordee and micropenis	2.2	38	Good
Patient 24	7 months	Scrotal	Chordee, penoscrotal transposition and micropenis	2.5	42	Good
Patient 25	2 years	Scrotal	Chordee, penoscrotal transposition, anal atresia, micropenis, and congenital heart disease	2.6	40	Poor
Patient 26	1 year and 8 month	Penoscrotal	Chordee and micropenis	2.4	55	Good
Patient 27	3 years	Penoscrotal	Chordee, penoscrotal fusion, bilateral cryptorchidism, micropenis, and unilateral indirect inguinal hernia	2.5	45	Poor
Patient 28	6 months	Penoscrotal	Chordee and micropenis	2.2	35	Good
Patient 29	3 years	Scrotal	Chordee, penoscrotal transposition, micropenis, and anal atresia	2.1	30	Poor
Patient 30	2 years	Scrotal	Chordee, penoscrotal transposition, micropenis, and anal atresia	2.3	60	Poor

Sequencing analysis

We identified clinically relevant genetic variants in 10 of 30 severe cases of hypospadias, and these comprised 7 SNVs and 3 CNVs. Among the identified SNVs, six novel mutations were observed, namely nuclear receptor subfamily 5 (NR5A1) c.1344dupC/c.244+1G>T, sex-determining region Y box protein 3 (SOX3) c.1273G>C, GLI family zinc finger protein 3 (GLI3) c.4731delA, euchromatic histone-lysine N-methyltransferase 1 (EHMT1) c.3081C>T, and patched domain-containing protein 1 (PTCHD1) c.667G>A. Additionally, previously reported variant automatic retransmission exchange (ARX) c.644C>G was recurrently detected. The three CNVs comprised two novel deletions (an exon 18 deletion in myelin regulatory factor [MYRF; chr11:61547693-61547762] and a 6.7-Mb chr18:71303262-78016748 deletion spanning the GATA binding protein 6 [GATA6] locus). The other CNV was a pathogenic chr22:21738147-23657724 deletion (22q11.2) associated with 22q11.2 deletion syndrome. A pathogenicity analysis confirmed the 22q11.2 deletion as pathogenic (ACMG Class 5) and classified the NR5A1 variants as LP (PM1+PM2+PP3).13,18 The clinical significance of the other variants remains undetermined (Table 2).

Table 2.

Genotype hypospadias patients with candidate gene abnormalities

Patient number	Type of WES	Gene	Chromosome	Transcript	cDNA	Protein	Exon	Genotype	Origin	ACMG	HGMD
Patient 2	Proband	SOX3	chrX:139585953	NM_005634	c.1273G>C	p.G425R	Exon 1	Hemizygous	Mother	VUS	Not recorded
Patient 3	Proband	NR5A1	chr9:127245078	NM_004959	c.1344dupC	p.N449fs	Exon 7	Heterozygous	De novo	LP	Not recorded
Patient 4	Proband	GLI3	chr7:42003940	NM_000168	c.4731delA	p.V1578fs	Exon 15	Heterozygous	-	VUS	Not recorded
Patient 5	Proband	NR5A1	chr9:127265357	NM_004959	c.244+1G>T	NA	Exon 7	Heterozygous	De novo	LP	Not recorded
Patient 6	Proband	EHMT1	chr9:140707883	NM_024757	c.3081C>T	p.N1027N	Exon 21	Heterozygous	De novo	VUS	Not recorded
Patient 7	Proband	PTCHD1	chrX:23398023	NM_173495	c.667G>A	p.D223N	Exon 2	Hemizygous	Mother	VUS	Not recorded
Patient 11	Trio	22q11.2 deletion	chr22:21738147-23657724	NA	2.0 Mb DEL	Deletion mutation	NA	Heterozygous	De novo	Pathogenic	22q11.2 deletion syndrome
Patient 24	Trio	ARX	chrX:25031468G>C	NM_139058.3	c.644C>G	p.P215R	Exon 2	Hemizygous	Mother	VUS	X-linked hydrocephalus with genital abnormalities
Patient 25	Trio	MYRF exon 18 deletion	chr11:61547693-61547762	NM_001127392.3	Exon 18-DEL	Deletion mutation	Exon 18	Heterozygous	De novo	VUS	Not recorded
Patient 27	Trio	18q11.2 deletion	chr18:71303262-78016748	NA	6.7 Mb DEL	Deletion mutation	NA	Heterozygous	De novo	VUS	Not recorded

SOX3: sex-determining region Y box protein 3; NR5A1: namely nuclear receptor subfamily 5; GLI3: GLI family zinc finger protein 3; EHMT1: euchromatic histone-lysine N-methyltransferase 1; PTCHD1: patched domain-containing protein 1; 22q11.2 deletion: chromosome 22q11.2 deletion; ARX: automatic retransmission exchange; MYRF: myelin regulatory factor; 18q11.2 deletion: chromosome 18q11.2 deletion; VUS: variant of uncertain significance; LP: likely pathogenic; NA: not applicable; -: parental origin unknown; WES: whole-exome sequencing; ACMG: American College of Medical Genetics and Genomics

Clinical features of patients with genetic variants

Among the 30 hypospadias cases, associated anomalies showed marked variability (Figure 2a). A micropenis was the most frequent (86.7%, 26/30), followed by chordee (66.7%, 20/30) and penoscrotal transposition/fusion (33.3%, 10/30). Less common anomalies included cryptorchidism (16.7%, 5/30), anal atresia (13.3%, 4/30), and digital deformities (13.3%, 4/30). Rare associations (<7.0%) comprised indirect inguinal hernia, prostatic utricle, hydrocele, and atrial septal defect (6.7%, 2/30), along with congenital heart disease, a buried penis, and left scrotal hemangioma (3.3%, 1/30). Genetic mutations, specifically pathogenic, LP, and VUS, showed differential association patterns with specific congenital anomalies (Figure 2b).

Figure 2.

Frequency of associated malformations in hypospadias. (a) Frequency of associated malformations in hypospadias in 30 cases. (b) Frequency of genetic mutations in associated malformations of hypospadias. The genetic mutations included pathogenic, likely pathogenic and variant of uncertain significance mutation sites. P: pathogenic; LP: likely pathogenic; VUS: variant of uncertain significance.

A comparative analysis of the groups showed marked disparities in malformation profiles between the genetic mutation (n = 10) and non-mutation (n = 20) groups (Figure 3). The prevalence of cryptorchidism was eight-fold higher in the genetic mutation group than that in the non-mutation group (40.0% vs 5.0%, P < 0.01). Additionally, some mutation-associated anomalies comprising penoscrotal transposition (50.0% vs 25.0%, P < 0.05), digital deformities (20.0% vs 10.0%, P < 0.01), and penoscrotal fusion (30.0% vs 20.0%, P < 0.01) showed significantly higher frequencies in the genetic mutation group than those in the non-mutation group. No significant difference in the frequency of micropenis (90.0% vs 85.0%, P = 0.15) or chordee (60.0% vs 70.0%, P = 0.77) was observed between the genetic mutation and non-mutation groups. Notably, three anomalies, namely prostatic utricle (2/2), congenital heart disease (1/1), and hydrocele (2/2), were exclusively observed in the genetic mutation group. No mutations were detected in cases that involved a buried penis (0/1), atrial septal defect (0/2), or left scrotal hemangioma (0/1), as shown in Figure 3a. Furthermore, 80.0% (8/10) of patients in the genetic mutation group presented with ≥3 associated malformations compared with only 35.0% (7/20) in the non-mutation group (P < 0.05, Figure 3b). Mutations were also identified in patients with few anomalies, such as patients P5 and P6, both presented with two major malformations.

Figure 3.

Associations between gene mutations and associated malformations. (a) Comparative prevalence of specific associated malformations in the genetic mutation group (n = 10) versus the non-mutation group (n = 20). (b) Comparative prevalence of patients with ≥3 associated malformations in the genetic mutation group (n = 10) versus the non-mutation group (n = 20). ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; NS: not significant. Genetic mutation means clinically relevant genetic mutations in hypospadias; non-mutation means without clinically relevant genetic mutations in hypospadias.

DISCUSSION

Research status and genetic landscape

Hypospadias is a common congenital malformation of male external genitalia, with severe cases posing considerable surgical and functional challenges. While genetic studies have identified major contributors to hypospadias, such as NR5A1 and SRD5A2 in androgen signaling pathways, more than 50.0% of cases remain genetically unexplained. Recent advances in WES have begun to unravel the polygenic and structural genomic mechanisms of hypospadias, but critical gaps persist in understanding population-specific variants and the role of CNVs in diverse cohorts.

In this cohort of 30 southeastern Chinese patients with severe hypospadias, WES identified clinically relevant variants in 33.3% (10/30) of patients. These variants included seven SNVs (NR5A1 c.1344dupC/c.244+1G>T, SOX3 c.1273G>C, GLI3 c.4731delA, EHMT1 c.3081C>T, PTCHD1 c.667G>A, and ARX c.644C>G) and three CNVs (novel MYRF exon 18 and chr18:71303262-78016748 deletions and a pathogenic 22q11.2 deletion). The pathogenicity analysis classified the NR5A1 variants as LP (PM1+PM2+PP3) and the 22q11.2 deletion as ACMG Class 5. However, PTCHD1 c.667G>A and EHMT1 c.3081C>T remained VUS because of limited evidence.

Genotype–phenotype associations and clinical actionability

Striking genotype–phenotype associations emerged in mutation carriers. Notably, most of the patients with mutations showed multisystem anomalies (≥3 associated malformations), and this prevalence was more than two-fold higher than that in non-carriers. This finding strongly supports a monogenic/oligogenic basis for syndromic hypospadias. Furthermore, mutation-positive patients showed an eight-fold higher percentage of cryptorchidism than those without mutations. This finding supports global guidelines recommending genetic testing for severe hypospadias (including isolated perineal forms), bilateral cryptorchidism, or hypospadias with cryptorchidism.19 Importantly, syndromic markers, such as prostatic utricle and hydrocele, showed genetic associations in both (2/2) cases, suggesting that they can be used as clinical indicators to prompt genetic evaluation. The condition of the urethral plate, which is a well-established predictor of surgical outcomes, was routinely evaluated in this study. Although no significant association between the condition of the urethral plate and genetic variants was observed, its standardized documentation aids in excluding confounding effects. While these preliminary findings require validation in larger cohorts, they highlight that a poor condition of the urethral plate, such as fibrosis or ischemia, in severe hypospadias likely results from secondary local factors rather than primary genetic defects. This possibility further supports the specificity of identified genotype–phenotype correlations for systemic anomalies, such as cryptorchidism, and highlights the utility of using such anomalies in prioritizing molecular diagnostics for severe hypospadias.

These findings are consistent with other data of disorders of sex development in the Chinese population,20 where NR5A1 and AR mutations show pleiotropic effects (cryptorchidism, hypospadias, and multiorgan defects), while GLI3 variants are specifically associated with digital deformity. In our study, the GLI3 p.V1578fs variant (associated with hypospadias10,21) found in patient P4 indicates a critical limitation in the clinical implementation of WES. This limitation is that the absence of parental validation precludes definitive classification of pathogenicity. This case highlights the need for Sanger sequencing and trio-based sequencing in the clinical setting. In addition to the risk of ovarian insufficiency in carriers of NR5A1 variants,22 the established roles of PTCHD1 and EHMT1 variants in autism spectrum disorder and the association between GLI3 variants and hypothalamic hamartoma (Pallister–Hall syndrome) necessitate long-term neurodevelopmental monitoring of these individuals.23,24,25

Ethnogeographic heterogeneity and detection of CNVs

Population-specific genetic architecture occurs throughout the world. Examples of this genetic architecture are that NR5A1 and SRD5A2 are dominant in Asian cohorts,5,10 while AR and hydroxysteroid (17-beta) dehydrogenase 3e (HSD17B3) are prevalent in Europe.26 In China, regional disparities in genetic architecture persist, where the prevalence of SRD5A2 variants is higher in eastern/southeastern provinces (19.7%–22.7%) than that in northern regions (8.0%).27 These findings highlight the requirement for population-specific genetic databases to optimize diagnostic accuracy.

Our study showed the expanded utility of WES by detecting clinically relevant CNVs, including recurrent 18q11.2/22q12.3 deletions and a novel MYRF exon 18 deletion, in 10.0% (3/30) of hypospadias cases. Specifically, the 18q11.2 deletion in patient P27 encompassed the cardiac developmental gene GATA6,28 but presented with urogenital abnormalities without congenital heart disease, clearly demonstrating the remarkable tissue-specific expressivity of CNVs. In contrast, the MYRF exon 18 deletion in P25 manifested with multisystem involvement, including congenital heart disease and anal atresia,29 strongly suggesting potential synergistic effects between structural variants and other genetic modifiers.

Importantly, while CNVs have been previously associated with syndromic hypospadias,30 their detection in our non-syndromic cases indicates the expanded utility of WES in identifying structural variants that are often missed by traditional sequencing approaches.31,32,33 Our findings of variable expressivity and multiorgan involvement further suggest the importance of analyzing gene content and potential modifier interactions within CNV regions. This evidence supports the growing consensus that a comprehensive genetic diagnosis requires integrated SNV/CNV analysis,5,31 particularly in cases with severe or complex phenotypes.

Importantly, WES detected these CNV structural variants even in isolated cases, indicating its superior ability to capture SNVs and CNVs compared with conventional sequencing.31,32,33 This dual detection capability suggests that WES is an essential tool in modern genetic diagnostics for hypospadias.

Clinical translation

The diagnostic yield of WES in this study was 33.3%, which is much higher than the typical 10%–15% yield of conventional methods, primarily because of the dual capacity of WES to detect SNVs and CNVs. Importantly, we identified strong genotype–phenotype correlations to guide clinical decision-making. We found that mutation carriers had higher frequencies of cryptorchidism and the presence of ≥3 associated malformations than non-mutation carriers, although our threshold was conservative because of sample limitations. Following Romano et al.,34 we recommend performing WES for all severe hypospadias cases. Priority should be given to patients with features such as cryptorchidism, anorectal anomalies, cardiac defects, musculoskeletal malformations, or syndromic features. We also emphasize that genetic consultation is indicated even for isolated cases. Pathogenic findings further necessitate tailored management. The 22q11.2 deletion requires cardiac monitoring, while LP NR5A1 variants require the assessment of adrenal function with counseling for familial risks, such as primary ovarian insufficiency, complemented by gonadal tumor screening in disorders of sex development.20 These findings suggest the critical role of WES in identifying underlying genetic etiologies in severe hypospadias, facilitating targeted interventions, and improving long-term outcomes through personalized surveillance and family counseling.

Limitations and future directions

While this study expands the genetic spectrum of hypospadias, validation in larger cohorts is required because of the limited sample size (n = 30). Future studies should combine multicenter recruitment with functional assays, such as transcriptomics or animal models, to confirm the pathogenicity of variants (e.g., NR5A1 c.1344dupC/c.244+1G>T and SOX3 c.1273G>C).

CONCLUSION

This study shows substantial genetic heterogeneity in southeastern Chinese patients with severe hypospadias. WES outperforms targeted panels by concurrently identifying known variants, such as ARX c.644C>G and 22q11.2 deletions, novel variants, including NR5A1 c.1344dupC/c.244+1G>T, SOX3 c.1273G>C, GLI3 c.4731delA, PTCHD1 c.667G>A, and EHMT1 c.3081C>T, and novel CNVs with a 18q11.2 deletion and MYRF exon 18 deletion. Dual detection of SNVs and CNVs by WES enables refinement of the genetic architecture of hypospadias and precise clinical management, demonstrating its indispensability in modern diagnostics.

AUTHOR CONTRIBUTIONS

WHH and HGW participated in sample collection, experimental design, and manuscript drafting. WZ and XC performed data acquisition and curation. QQT and YZ participated in sample testing and validation. EHW and WHN performed data analysis, visualization, and manuscript review. WHN and CMZ supervised the project, validated the findings, and are responsible for the overall content as corresponding authors. CMZ conceptualized the research proposal and provided critical revision of the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

Although the authors (QQT, YZ, EHW, and WHN) are affiliated with commercial entities Puluo (Wuhan) Medical Biotechnology Co., Ltd. and Wuhan Kindstar Clinical Diagnostic Institute Co., Ltd., these companies had no role in the design of the study; in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. All authors declare no competing interests.