Abstract
Diagnosis and management strategy of disorders of sex development (DSD) are difficult and various due to heterogeneous phenotype and genotype. Under widespread use of genomic sequencing technologies, multiple genes and mechanisms have been identified and proposed as genetic causes of 46,XY DSD. In this study, 178 46,XY DSD patients were enrolled and underwent gene sequencing (either whole-exome sequencing or targeted panel gene sequencing). Detailed clinical phenotype and genotype information were summarized which showed that the most common clinical manifestations were micropenis (56.74%, 101/178), cryptorchidism (34.27%, 61/178), and hypospadias (17.42%, 31/178). Androgen synthesis/action disorders and idiopathic hypogonadotropic hypogonadism were the most frequent clinical diagnoses, accounting, respectively, for 40.90 and 21.59%. From all next-generation sequencing results, 103 candidate variants distributed across 32 genes were identified in 88 patients. The overall molecular detection rate was 49.44% (88/178), including 35.96% (64/178) pathogenic/likely pathogenic variants and 13.48% (24/178) variants of uncertain significance. Of all, 19.42% (20/103) variants were first reported in 46,XY DSD patients. Mutation c.680G>A (p.R227Q) on SRD5A2 (steroid 5-alpha-reductase 2) (36.67%, 11/30) was a hotspot mutation in the Chinese population. Novel candidate genes related to DSD (GHR (growth hormone receptor) and PHIP (pleckstrin homology domain-interacting protein)) were identified. Overall, this was a large cohort of 46,XY DSD patients with a common clinical classification and phenotype spectrum of Chinese patients. Targeted gene panel sequencing covered most of the genes contributing to DSD, whereas whole-exome sequencing detected more candidate genes.
Introduction
Disorder of sex development (DSD) is a general term for diseases with inconsistencies between chromosomal karyotypes, external genitalia, and gonadal development (1). According to the DSD etiology classification defined in the 2006 Chicago Expert Consensus meeting by the European Society for Pediatric Endocrinology and the Lawson Wilkins Pediatric Endocrine Society, patients can be divided into three categories according to karyotype: (i) sex chromosome DSD, (ii) 46,XY DSD, and (iii) 46,XX DSD. The worldwide incidence of DSD is between 1/500 and 1/10,000 (3). However, there are no relevant DSD incidence statistics for the Chinese population (4, 5).
Compared with sex chromosome DSD and 46,XX DSD, 46,XY DSD patients present with more remarkable clinical heterogeneity among different classifications and ages (6). The phenotypes vary from mild to severe as the clinical classification varies. Gene sequencing technologies can help confirm the pathogenesis of different classifications of 46,XY DSD at an early stage. However, to date, only about 20% of 46,XY DSD patients have been identified with a specific genetic cause (7). It is important to determine the diagnosis protocol and genetic pathogenesis of 46,XY DSD patients, which not only helps to establish specialized medical care for affected individuals but also helps to provide future genetic counseling for the next generation (8).
In this study, we intended to expand the clinical and genetic characteristics of 178 46,XY DSD patients between 2016 and 2022 and compare the efficiency of two different next-generation sequencing (NGS) methods, targeted gene panel sequencing (TPS), and whole-exome sequencing (WES). Through analysis and discussion of sequencing results, we provide a better overview of DSD diagnosis, especially in the Chinese population.
Materials and methods
Study design and participants
This study was conducted at the Shanghai Children’s Medical Center, affiliated with the Shanghai Jiao Tong University School of Medicine. This single-center prospective study enrolled pediatric patients (aged ≤18 years) suspected of having DSD by experienced physicians from December 2016 to March 2022. The diagnosis of DSD was based on clinical manifestations, laboratory tests, and imaging examinations according to the 2006 Chicago consensus criteria (2). The inclusion criteria of suspected DSD were as follows: patients presenting clinical manifestations of DSD, such as ambiguous external genitalia, male-type external genitalia with bilateral cryptorchidism and/or micropenis and/or hypospadias, female-type external genitalia with clitoral enlargement and/or labia fusion and/or inguinal mass and/or inguinal hernia, gonadal dysplasia (GD), absent secondary sexual characteristics during puberty, gynecomastia, and gender inconsistency between gonads and chromosomes showing 46,XY. Complete clinical data and genetic analysis results were evaluated. Subjects with any one of the following conditions were excluded: (i) other chronic diseases or malnutrition (such as uremia and anorexia) and (ii) history of intracranial lesions or pituitary tumors. Patient characteristics were collected by clinicians, including age at diagnosis, sex of rearing, main concern, medical history, relative familial history, consanguinity, history of pregnancy and delivery, previous treatment, and surgical intervention.
Physical examination
Physical examination methods were based on articles previously published by our team (9, 10). The body weight and height of the patients were measured and recorded using the same type of apparatus and following the standard procedures recommended by Cameron (11), and then the body mass index was calculated. The secondary sexual characteristics of all patients were evaluated and recorded by qualified endocrinologists using the Tanner stage grade (12). The testicular volume was measured by endocrinologists using both the Prader measurement apparatus and ultrasonography. Clinical manifestations of DSD were evaluated and recorded by following terminology: ambiguous external genitalia, male-type external genitalia with bilateral cryptorchidism and/or micropenis and/or hypospadias, female-type external genitalia with clitoral enlargement and/or labia fusion and/or inguinal mass and/or inguinal hernia, GD, absent secondary sexual characteristics during puberty, gynecomastia, and gender inconsistency between gonad and chromosome.
Paraclinical examination
All suspected DSD patients underwent basic laboratory tests, including testosterone, estrogen, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and gonadotropin-releasing hormone (GnRH) stimulation tests, and imaging examinations including bone age (13), pelvic ultrasonography (14), and magnetic resonance imaging of the pituitary gland and olfactory bulb. All the patients underwent karyotyping and were recommended to undergo gene sequencing.
Chinese smell identification tests (CSITs) (15, 16, 17) were performed in patients with suspected hypogonadotropic hypogonadism and healthy control groups (1:1 matched). Inclusion criteria were as follows: (i) aged 6–18 years old; (ii) willing to participate in this study; (iii) no difficulty in language communication. Cases and controls were excluded if they had a history of respiratory allergies/upper respiratory infections/rhinitis within the past 14 days or had any chronic diseases. The control’s data were initially collected as described in Zhang et al. (17).
Gonadorelin acetate (Gonadorelin, BBCA, Anhui, China) was used for the GnRH stimulation test, which was conducted between 08:00 and 10:00 h in fasting subjects by intravenous injection of gonadorelin acetate at a dose of 2.5 mg/kg body weight (the maximum total dose 100 mg) (18). Venous blood samples were collected to determine the serum concentrations of testosterone, estrogen, LH, and FSH at 0, 30, 60, 90, and 120 min after the injection of gonadorelin acetate. Serum was separated from the blood samples less than 1 h after collection, and testosterone, estrogen, LH, and FSH levels were tested immediately. The concentration of testosterone, estrogen, LH, and FSH were detected by chemiluminescence immunoassay (Abbott Diagnostics) on an Architect i2000SR (Abbott Diagnostics), with detection limits of 0.02 nmol/L, 10 pg/mL, 0.09 IU/L, and 0.05 IU/L, respectively; intra-assay and inter-assay coefficients of variation were less than 6.4% and 8.4%, respectively.
Genetic analysis
Subjects from December 2016 to December 2018 underwent TPS and subjects from January 2019 to March 2022 underwent WES. Genetic sequencing was performed as previously described by Xu et al. (19). Peripheral blood (2 mL) was collected from the children and their parents to extract DNA. The experimental process included the digestion of sample DNA fragments, library hybridization, and capture library amplification and purification. Agilent Sure Select method was used for exon capture (using All Exome V6 kit and panel kit including 2742 hereditary disease-causing genes (cat No. 5190–7519; Agilent Technologies Inc.), see Supplementary Table 1 (see section on supplementary materials given at the end of this article)). Hiseq2500 sequencing platform (Illumina, Inc., San Diego, CA, USA) was used for high-throughput sequencing. For data quality control and removal of the adaptor sequence, Fastqc (Babraham Research Institute, Cambridge, UK) and Fastp (Visible Genetics, Inc., Toronto, Canada) were applied. Speedseq (Ira Hall Lab, St. Louis, MO, USA) was aligned to the reference genome, and bamdst and mosdepth were used to count the sequencing indexes of BAM files after alignment. The Genome Analysis Toolkit (Broad Institute, Cambridge, MA, USA) was used to detect the variations in the BAM file that passed the quality control, and a VCF format file was generated. Variations in VCF files were annotated by Ingenuity Variant Analysis (Ingenuity Systems, Redwood City, CA, USA). These candidate variants were verified by Sanger sequencing and manually classified according to the American College of Medical Genetics and Genomics (ACMG) variant classification criteria guideline. We analyzed the allele frequencies of the detected variants and checked whether the variants had been reported before by retrieving relevant information from the population database Genome Aggregation Database (gnomAD) (http://gnomad.broadinstitute.org/). Alamut functional software was used to predict the functions of the mutated proteins. The accessible DNA of the patients’ parents (94.32%, 83/88) was also isolated and subjected to Sanger sequencing to confirm the origin of the detected candidate variants.
Statistical analysis
Data were analyzed using GraphPad Prism 9.4.0 (Macintosh Version by Software MacKiev GraphPad Software, LLC) and RStudio (2021.09.1+372, RStudio, PBC). Descriptive statistics were presented as mean ± standard deviation for continuous variables, and percentages were used for categorical variables. The Mann–Whitney U-test or the Kruskal–Wallis test was used for non-normal distribution, and the chi-square test was used to compare the proportion of manifestations between groups.
Results
A total of 178 patients with 46,XY DSD (163 boys and 15 girls) who underwent NGS were enrolled in this study (Fig. 1). Of these, 79 pathogenic/likely pathogenic (P/LP) variants were found in 64 subjects (35.96%, 64/178) under NGS (NGS-positive group). Twenty-four variants of uncertain significance (VUS) were detected in 24 subjects (13.48%, 24/178). The overall candidate variant detection rate was 49.44% (88/178).
Flowchart of current study. In this study, 383 children suspected of DSD between 2016 and 2022 were initially included. Among these, 189 were SRY positive with A chromosome karyotype of 46,XY. A total of 107 subjects were excluded due to other chronic diseases, and 11 were excluded due to refusal of gene sequencing and incomplete examination results for personal reasons. Thus, a total of 178 subjects were finally included in this study.
Citation: Endocrine Connections 12, 10; 10.1530/EC-23-0029
Based on NGS results, all subjects were divided into the NGS-positive group (subjects with P/LP variants, n = 64) and the NGS-negative group (subjects with no variants detected by current methods, n = 90). Subjects with VUS or undefined variants (n = 24) were not included in the NGS-positive groups during statistical analysis, since the pathogenicity of these variants was not clarified yet. Here, we mainly focus on the phenotype and genotype in the subjects who were diagnosed both clinically and genetically (NGS-positive group, n = 64). Furthermore, subjects were divided into the primary gonadal/genital disorders group (n = 48) and central hypogonadism group (n = 16) according to their phenotypes and paraclinical results.
Clinical characteristics
Age
Among all the 46,XY subjects, the median age at diagnosis was 8.63 (2.61, 12.00) years (n = 178), ranging widely from 1 month to 17 years. Most of these patients were diagnosed before puberty, and the youngest diagnosed patient was of 1 month.
A significant difference was observed (P < 0.05) in age at diagnosis between NGS-positive (median age 4.00 years old, n = 64) and NGS-negative patients (median age 10.04 years old, n = 90). In the NGS-positive group, the median age of primary gonadal/genital disorders subjects (n = 48) was 2.92 (0.67, 7.33) years old, and that of central cause subjects (n = 16) was 7.88 (2.69, 12.07) years old. A significant difference was observed in two groups (P < 0.05).
Clinical diagnosis
In primary gonadal/genital disorders group (n = 48), subjects were diagnosed as 5α reductase deficiency (5αRD, 35.42%, 17/48), androgen insensitivity syndrome (AIS, 27.08%, 13/48), GD (12.50%, 6/48), congenital adrenal hyperplasia (CAH, 4.17%, 2/48), persistent Mullerian duct syndrome (PMDS, 4.17%, 2/48), and syndromic hypogonadism (10.42%, 5/48).
In the central cause group (n = 16), idiopathic hypogonadotropic hypogonadism (IHH) subjects accounted for 68.75% (11/16), hypopituitarism accounted for 25.00% (4/16), and another Ulnar-mammary syndrome accounted for 6.25% (1/16), respectively.
Clinical manifestations
46,XY DSD patients manifested various clinical manifestations, including bifid scrotum, penoscrotal transposition, penile curvature, ambiguous external genitalia, gynecomastia, and olfactory dysfunction. Overall, in all 178 subjects, the most frequent clinical manifestations were small testis (66.29%, 118/178), micropenis (56.74%, 101/178), unilateral or bilateral cryptorchidism (34.27%, 61/178), and hypospadias (30.34%, 54/178). Of all, 15 subjects were raised as girls from birth because of female external genitalia; the most common concern of these female patients was the predominance of female external genitalia (80.00%, 12/15), unilateral or bilateral cryptorchidism (73.33%, 11/15), clitoromegaly (53.33%, 8/15), ambiguous external genitalia (20.00%, 3/15), delayed mammary development in pubertal subjects (100.00%, 5/5), and primary amenorrhea in subjects >14 years old (100.00%, 3/3).
Among NGS-positive subjects (n = 64), the most common clinical manifestations were micropenis (59.38%, 38/64), unilateral or bilateral cryptorchidism (50.00%, 32/64), and hypospadias (21.88%, 14/64). Most of them showed undeveloped testis volume and short stretched penile length (see Table 1). In comparison, in the NGS-negative group (n = 90), a minor proportion of unilateral or bilateral cryptorchidism (21.11%, 19/90) and hypospadias (13.33%, 12/90) phenotypes was observed.
Clinical phenotype and biomarkers of NGS-positive subjects (n = 64).
| Clinical characteristics | NGS-positive subjects (n = 64) | Primary group (n = 48) | Central group (n = 16) | P-valuea |
|---|---|---|---|---|
| Age (years) | 4.00 (0.87, 10.00) | 2.92 (0.67, 7.33) | 7.88 (2.69, 12.07) | 0.033 |
| Micropenis, % | 59.38% | 54.17% | 75.00% | 0.142 |
| Cryptorchidism, % (unilateral or bilateral) | 50.00% | 52.08% | 43.76% | 0.774 |
| Hypospadias, n/N (%) | 21.88% | 27.08% | 6.25% | 0.159 |
| Olfactory disorder, % (n/N) | NA | NA | 62.50% | / |
| Other phenotypes | Prepenile scrotum, bifid scrotum, ambiguous external genitalia, penile curvature, blind perineal pouch resembling vagina, female external genitalia, transverse testicular ectopic, persistent Müllerian duct syndrome, gonadal dysplasia, enlarged clitoris, gynecomastia. | Prepenile scrotum, bifid scrotum, ambiguous external genitalia, penile curvature, blind perineal pouch resembling vagina, female external genitalia, transverse testicular ectopic, persistent Müllerian duct syndrome, gonadal dysplasia, enlarged clitoris, gynecomastia. | Intellectual disability, delayed motor development, skeletal deformity, bilateral pinky joint malformation, brain structure abnormalities, and hearing disorders | / |
| Average testis volume (mL) | 1.00 (1.00, 2.00) (n = 47) | 1.00 (1.00, 2.00) (n = 33) | 1.00 (1.00, 3.00) (n = 16) | 0.384 |
| Stretched penis length (cm) | 3.00 (1.00, 3.45) (n = 48) | 3.00 (1.50, 3.50) (n = 34) | 2.75 (2.00, 3.00) (n = 16) | 0.643 |
| Testosterone (ng/ml) | 0.11 (0.09, 0.24) (n = 51) | 0.12 (0.09, 0.66) (n = 35) | 0.11 (0.09, 0.12) (n = 16) | 0.652 |
| DHT (pg/mL) | 70.18 (40.85, 133.91) (n = 51) | 54.85 (34.98, 110.32) (n = 35) | 132.87 (66.65, 145.52) (n = 14) | 0.001 |
| FSH (mIU/mL) | 2.77 (1.57, 4.88) (n = 51) | 3.31 (1.78, 6.79) (n = 35) | 0.91 (0.43, 1.71) (n = 16) | 0.089 |
| LH (mIU/mL) | 0.80 (0.29, 2.07) (n = =51) | 0.88 (0.30, 2.63) (n = 35) | 0.19 (0.08, 0.31) (n = 16) | 0.075 |
Sixty-fout subjects with pathogenic/likely pathogenic variants in this cohort were classified as NGS positive and were further divided into primary cause group (n = 48) and central cause group (n = 16) according to clinical diagnosis. The phenotypes (cryptorchidism, micropenis, and hypospadias), physical examination (testicular volume and penis length), concentrations of testosterone, estrogen, FSH, and LH in serum or plasma, and other clinical or laboratory findings are listed separately.
aThe Mann–Whitney U-test and chi-square test were used between primary and central groups. P < .05 was considered statistically significant.
Specifically, in primary gonadal/genital disorders subjects, the main phenotype was micropenis (54.17%, 26/48), bilateral or unilateral cryptorchidism (52.08%, 25/48), and hypospadias (27.08%, 13/48) (see Table 1). Those subjects who presented hypospadias (n = 13) could also manifest as cryptorchidism and/or micropenis. Details of different subgroups are shown in Table 2.
Clinical features of primary gonadal/genital disorders.
| Clinical characteristics | 5αRD (n = 17) | AIS (n = 13) | Gonadal dysplasia (n = 6) |
|---|---|---|---|
| Age (years) | 2.00 (0.58, 4.46) | 10.00 (0.92, 14.17) | 2.75 (0.17, 6.00) |
| Female external genitalia, % | 5.88% (1/17) | 53.85% (7/13) | 16.67% (1/6) |
| Micropenis, % | 76.47% (13/17) | 53.85% (7/13) | 16.67% (1/6) |
| Cryptorchidism, % | 29.41% (5/17) | 53.85% (7/13) | 66.67% (4/6) |
| Hypospadias, n/N (%) | 35.29% (6/17) | 23.08% (3/13) | 50.00% (3/6) |
| Other phenotypes | Bifid scrotum, ambiguous external genitalia, penile curvature, penile curvature. | Penoscrotal transposition, bifid scrotum, enlarged clitoris, gynecomastia. | Penoscrotal transposition, bifid scrotum, ambiguous external genitalia, penile dysplasia |
| Average testis volume (mL) | 1.00 (1.00, 2.00) (n = 13) | 1.00 (0.75, 3.00) (n = 6) | 1.00 (0.88, 1.25) (n = 4) |
| Stretched penis length (cm) | 2.80 (1.25, 3.25) (n = 14) | 2.50 (1.00, 3.50) (n = 6) | 3.00 (2.23, 3.85) (n = 5) |
| Testosterone (ng/mL) | 0.09 (0.09, 0.49) (n = 14) | 0.19 (0.09, 2.32) (n = 13) | 0.13 (0.09, 0.86) (n = 5) |
| DHT (pg/mL) | 47.36 (38.89, 60.63) (n = 14) | 100.11 (77.86, 141.51) (n = 13) | 88.48 (33.18, 131.63) (n = 5) |
| FSH (mIU/mL) | 2.07 (0.68, 2.97) (n = 14) | 13.44 (2.94, 47.98) (n = 13) | 6.40 (3.76, 9.54) (n = 5) |
| LH (mIU/mL) | 0.78 (0.09, 1.58) (n = 14) | 9.31 (2.75, 20.04) (n = 13) | 1.05 (0.84, 1.59) (n = 5) |
The top three classifications of primary cause DSD were 5α-reductase deficiency (n = 17), androgen insensitive syndrome (n = 13), and gonadal dysplasia (n = 6). The median age of 5α-reductase deficiency was lower than that of AIS subjects. The patients’ testosterone and DHT levels in these diseases remained similar, but the gonadotropin levels in 5α-reductase-deficient subgroup were much lower than those in the AIS subgroup.
Among central DSD subjects, micropenis was the most common phenotype (75.00%, 12/16); unilateral or bilateral cryptorchidism was found in 43.75% (7/16) patients; and few subjects presented hypospadias (6.25%, 1/16) (see Table 1). Different from the primary cause group, subjects in the central cause group, mostly those IHH subjects, could be presented with other systemic manifestations, such as olfactory dysfunction (62.50%, 10/16), intellectual disability, delayed motor development, skeletal deformity, brain structure abnormalities, and hearing disorders. CSIT score, a toolkit specifically designed to assess olfactory function in the Chinese population, was analyzed (Supplementary Fig. 1).
Genetic characteristics
Overall distribution
The overall genetic diagnosis rate was 49.44% (88/178). A total of 103 candidate variants distributed across 32 genes were identified in 88 patients. Of all, 79 P/LP variants (79/103) were detected in 64 subjects in this cohort (see Fig. 2, Tables 3 and4). The genetic diagnostic rate (including patients with P/LP variants only) of this cohort was 35.96% (64/178). According to specific phenotype, the genetic diagnostic rate of micropenis phenotype, cryptorchidism phenotype, and hypospadias phenotype was 67.09% (53/79), 79.25% (42/53), and 79.17% (19/24), respectively. According to the ACMG guidelines, all 103 variants were classified as pathogenic (41.75%, 43/103), likely pathogenic (34.95%, 36/103), and variants of uncertain significance (VUS, 24/103, 23.30%) (details of each variant in Tables 2, 3, and 4).
Mutations detected in SRD5A2 gene in our cohort. All the variants and the hot spot (c.680G>A, p. Arg227Gln) detected in SRD5A2 in 5α reductase deficiency patients of the current cohort.
Citation: Endocrine Connections 12, 10; 10.1530/EC-23-0029
Phenotype and genotype of primary gonadal/genital disorders subjects with P/LP variants.
| Patient ID | Age | Phenotype | Transcript ID | Genetic variants | Zygosity | Family inheritance | Inheritance mode | Pathogenicity | gnomAD frequency | Popmax |
|---|---|---|---|---|---|---|---|---|---|---|
| 1880 | 2 months | Penoscrotal transposition, bifid scrotum | NM_003140.2 | SRY c.397C>T, p.Arg133Trp | Hemi | NA | YL | P | / | / |
| 1886 | 6 months | Ambiguous genitalia, blind perineal pouch resembling vagina, cryptorchidism | NM_000348.3 | SRD5A2 c.419_421delinsATTC, p.Trp140Tyrfs*8 | Het | F | AR | P | / | / |
| SRD5A2 c.680G>A, p.Arg227Gln | Het | M | AR | P | 0.0002235 | 0.004817 (East Asian) | ||||
| 2367 | 11 years | Predominantly female external genitalia | NM_000102.3 | CYP17A1 c.985_987delinsAA, p.Tyr329Kfs*90 | Homo | F + M | AR | P | 0.00004599 | 0.0006317 (East Asian) |
| 2420 | 7 months | Micropenis, cryptorchidism | NM_000348.3 | SRD5A2 c.680G>A, p.Arg227Gln | Homo | F + M | AR | P | 0.0002235 | 0.004817 (East Asian) |
| 2439 | 2 months | Ambiguous external genitalia | NM_004959.4 | NR5A1 c.721C>T, p.Arg241Trp | Het | M | AD | LP | / | / |
| 2540 | 17 years | Predominantly female external genitalia, cryptorchidism | NM_000044.3 | AR c.1787G>A, p.Cys596Tyr | Hemi | De novo | XR | P | / | / |
| 2752 | 1 year | Micropenis, hypogonadism | NM_000044.3 | AR c.2528T>C, p.Ile843Thr | Hemi | M | XR | LP | / | |
| 2782 | 2 years 9 months | Perineal hypospadias, micropenis | NM_000348.3 | SRD5A2 c.680G>A, p.Arg227Gln | Het | F | AR | P | 0.0002235 | 0.004817 (East Asian) |
| SRD5A2 c.281G>A, p.Arg94Lys | Het | M | AR | LP | / | / | ||||
| 2892 | 7 months | Ambiguous genitalia, micropenis, severe hypospadias, cryptorchidism | NM_000348.3 | SRD5A2 c.607G>A, p.Gly203Ser | Het | NA | AR | P | 0.00002629 | 0.0001567 (East Asian) |
| SRD5A2 c.737G>A, p.Arg246Gln | Het | NA | AR | LP | 0.000126 | 0.0004087 (South Asian) | ||||
| 3167 | 6 years | Predominantly female external genitalia, cryptorchidism | NM_000233.3 | LHCGR c.681-1Gly>Ala | Het | F | AR | P | / | / |
| Predominantly female external genitalia, cryptorchidism | LHCGR c.1582_1585delATAT, p.Ile528* | Het | M | AR | P | / | / | |||
| 4004 | 11 years | Micropenis, hypogonadism | NM_000044.3 | AR c.1175C>G, p.Pro392Arg | Hemi | M | XR | LP | 0.00001776 | 0 |
| 4119 | 5 years | Micropenis, bilateral cryptorchidism, gonadal dysplasia | NM_003309.3 | TSPYL1 c.67C>T, p.Gln23* | Het | De novo | AR | LP | 0.0001441 | 0.001463 (East Asian) |
| 4208 | 10 months | Female external genitalia, clitoromegaly | NM_000044.3 | AR c.2256G>A, p.Trp752* | Hemi | M | XR | P | / | / |
| 4622 | 1 year 7 months | Hypospadias, penile curvature, cryptorchidism | NM_000348.3 | SRD5A2 c.269A>C, p.His90Pro | Het | F | AR | LP | / | / |
| SRD5A2 c.680G>A, p.Arg227Gln | Het | M | AR | P | 0.0002235 | 0.004817 (East Asian) | ||||
| 4986 | 6 months | Ambiguous genitalia, hypospadias, cryptorchidism | NM_004959.4 | NR5A1 c.251G>A, p.Arg84His | Het | M | AD | P | / | / |
| 5015 | 8 months | Cryptorchidism, inguinal fallopian tubes | NM_000479.3 | AMH c.585C>A, p.Tyr195* | Het | F | AR | LP | / | / |
| AMH c.1165G>T, p.Glu389* | Het | M | AR | LP | 0.0000198 | / | ||||
| 6791 | 2 years 9 months | Micropenis | NM_000348.3 | SRD5A2 c.680G>A, p.Arg227Gln | Homo | F+M | AR | P | 0.0002235 | 0.004817 (East Asian) |
| 7847 | 9 years 9 months | Hypospadias, micropenis, penile curvature | NM_000348.3 | SRD5A2 c.656delT, p.Phe219Serfs*60 | Het | F | AR | P | 0.00000657 | 0 |
| 7929 | 6 years | Penoscrotal hypospadias, cryptorchidism | NM_004959.4 | NR5A1 c.848G>T, p.Cys283Phe | Het | M | AD | LP | / | / |
| 8065 | 10 months | Female external genitalia, cryptorchidism | NM_000044.3 | AR del Exon5-8 | Hemi | NA | XR | P | / | |
| 8803 | 10 years | Micropenis, hypospadias | NM_000044.4 | AR c.2270A>G, p.Asn757Ser | Hemi | M | XR | LP | 0.000008892 | 0 |
| 8945 | 1 month | Hyperpigmentation, cryptorchidism, electrolyte disturbance | NM_000475.4 | NR0B1 c.1169-2Ala>Gly | Hemi | De novo | XL | P | / | / |
| 9052 | 1 year | Female external genitalia, cryptorchidism | NM_000044.4 | AR c.2495G>A, p.Arg832Gln | Hemi | M | XR | LP | 0.000008987 | 0 |
| 9242 | 1 year 3 months | Perineal hypospadias, bifid scrotum | NM_000348.3 | SRD5A2 c.650C>A, p.Ala217Glu | Het | F | AR | LP | / | / |
| SRD5A2 c.680G>A, p.Arg227Gln | Het | M | AR | P | 0.0002235 | 0.004817 (East Asian) | ||||
| 9431 | 3 years 1 month | Ambiguous genitalia, cryptorchidism | NM_000348.3 | SRD5A2 c.607G>A, p.Gly203Ser | Het | F | AR | P | 0.00002629 | 0.0001567 (East Asian) |
| SRD5A2 c.680G>A, p.Arg227Gln | Het | M | AR | P | 0.0002235 | 0.004817 (East Asian) | ||||
| 9471 | 12 years | Predominantly female external genitalia, clitoromegaly, cryptorchidism | NM_000044.4 | AR c.4G>A, p.Glu2Lys | Hemi | M | XR | LP | / | |
| 9482 | 5 years | Micropenis, cryptorchidism | NM_000348.3 | SRD5A2 c.680G>A, p.Arg227Gln | Homo | F + M | AR | P | 0.0002235 | 0.004817 (East Asian) |
| 10066 | 5 months | Micropenis, cryptorchidism | NM_000044.4 | AR c.899G>C, p.Gly300Ala | Hemi | M | XR | LP | 0.00002657 | 0.00001494 (European) |
| 10239 | 2 years | Bilateral cryptorchidism, motor development retardation, ptosis, short stature, hydrocephalus | / | 7q33-q35Del | Het | M | - | P | / | / |
| 10482 | 7 years 6 months | Bilateral cryptorchidism, short stature, funnel chest | NM_002934.4 | PTPN11 c.1472C>A, p.Pro491His | Het | De novo | AD | P | / | / |
| 10800 | 2 months 12 days | Hypospadias, penoscrotal transposition, micropenis, bifid scrotum | / | 2q33.1Del | Het | De novo | - | LP | / | / |
| 10892 | 3 years 5 months | Unilateral cryptorchidism, short stature | NM_002934.4 | PTPN11 c.922A>G, p.Asn308Asp | Het | De novo | AD | P | 0.00001193 | 0 |
| 10908 | 14 years 6 months | Female external genitalia, cryptorchidism, gynecomastia | NM_000044.4 | AR c.1737delC, p.Cys580Alafs*46 | Hemi | M | XR | P | / | / |
| 11072 | 15 years 6 months | Female external genitalia, cryptorchidism, clitoromegaly | NM_000044.4 | AR c.2113C>T, p.Leu705Phe | Hemi | M | XR | LP | / | |
| 11757 | 8 months | Hypospadias, micropenis | NM_000348.3 | SRD5A2 c.16C>T, p.Gln6* | Het | M | AR | P | / | / |
| SRD5A2 c.680G>A, p.Arg227Gln | Het | F | AR | P | 0.0002235 | 0.004817 (East Asian) | ||||
| 11867 | 6 months | Micropenis | NM_000348.3 | SRD5A2 c.680G>A, p.Arg227Gln | Homo | F + M | AR | P | 0.0002235 | 0.004817 (East Asian) |
| 12480 | 5 years | Bilateral cryptorchidism, mental retardation, intelligence disability, ptosis, strabismus | / | 18p11.31-p11.21del | Het | De novo | - | P | / | / |
| 12888 | 13 years | Bilateral cryptorchidism, micropenis, intelligence disability | / | 16p11.2del | Het | De novo | - | P | / | / |
| 13048 | 5 years | Micropenis, mental retardation | / | 17p11.2del | Het | De novo | - | P | / | / |
| 13421 | 4 years | Micropenis, hypospadias, penoscrotal transposition | NM_000044.4 | AR c.2020T>C, p.Phe674Leu | Hemi | M | XR | LP | / | / |
| 14250 | 4 months 3 days | Micropenis, hypospadias, bifid scrotum | NM_000348.3 | SRD5A2 c.607G>A, p.Gly203Ser | Het | F | AR | P | 0.00002629 | 0.0001567 (East Asian) |
| SRD5A2 c.680G>A, p.Arg227Gln | Het | M | AR | P | 0.0002235 | 0.004817 (East Asian) | ||||
| 15362 | 1 year 9 months | PMDS, transverse testicular ectopia | NM_000479.3 | AMH c.259-270del, p.Val187-Ala90del | Het | M | AR | LP | / | / |
| AMH c.1432C>T, p.Leu478Phe | Het | F | AR | LP | / | / | ||||
| 16213 | 3 years 11 months | Micropenis | NM_000348.3 | SRD5A2 c.680G>A, p.Arg227Gln | Homo | F + M | AR | P | 0.0002235 | 0.004817 (East Asian) |
| 16746 | 5 years 4 months | Micropenis | NM_000348.3 | SRD5A2 c.680G>A, p.Arg227Gln | Homo | F + M | AR | P | 0.0002235 | 0.004817 (East Asian) |
| 18423 | 6 years 10 months | Bilateral cryptorchidism, dysmorphic facial features (ocular hypertelorism, low nose bridge, flapping ear, ptosis), chicken breast, lumbar lateral curvature, brachydactylia, pulmonic stenosis, atrial septal defect, patent foramen ovale, lymphangioma of right lower limb | NM_006912.6 | RIT1 c.170C>G, p.Ala57Gly | Het | F | AD | P | / | / |
| 18553 | 11 years 9 months | Micropenis | NM_000348.3 | SRD5A2 c.680G>A, p.Arg227Gln | Homo | F + M | AR | P | 0.0002235 | 0.004817 (East Asian) |
| 19697 | 2 years 8 months | Micropenis | NM_000348.3 | SRD5A2 c.467G>A, p.Gly156Glu | Het | M | AR | LP | 0.000006677 | 0 |
| 20472 | 13 years 10 months | Hypospadias, penoscrotal transposition | NM_000044.6 | AR c.2521C>T, p.Arg841Cys | Hemi | M | XR | LP | / | / |
| AR c.1175C>G, p.Pro392Arg | Hemi | M | XR | LP | 0.00001776 | 0 |
AD, autosomal dominant; AR, autosomal recessive; F, father; Hemi, hemizygote; Het, heterozygote; Homo, homozygote; LP, likely pathogenic; M, mother; P, pathogenic; PMDS, persistent Müllerian duct syndrome; XL, X chromosome linked; YL, Y chromosome linked.
Phenotype and genotype of central hypogonadotropic DSD subjects with P/LP variants.
| Patient ID | Age | Phenotype | Transcript ID | Genetic variants | Zygosity | Family inheritance | Inheritance mode | Pathogenicity | gnomAD frequency | Popmax |
|---|---|---|---|---|---|---|---|---|---|---|
| 2061 | 15 years | Hypogonadotropic hypogonadism, hyposmia | NM_000216.2 | ANOS1 del Exon 4-5 | Hemi | M | XR | LP | / | / |
| 2356 | 7 years | micropenis, small testes | NM_000406.2 | GNRHR c.806C>T, p.Thr269Met | Homo | F + M | AR | LP | 0.00001194 | 0.00000293 (European non-Finnish) |
| 2955 | 6 years | Hypogonadotropic hypogonadism, bilateral cryptorchidism, small testes | NM_000216.2 | ANOS1 c.1712G>A, p.Trp571* | Hemi | De novo | XR | P | / | / |
| 3879 | 15 years | Hypogonadotropic hypogonadism, hyposmia, absence of olfactory bulb | NM_000216.2 | ANOS1 c.984C>G, p.Tyr328* | Hemi | M | XR | LP | / | / |
| 3973 | 11 years | Gonadal dysplasia, cryptorchidism, short stature | NM_005634.2 | SOX3 c.424C>A, p.Pro142Thr | Hemi | M | XL | LP | / | / |
| 4136 | 11 years | Gonadal dysplasia, hypogonadotropic hypogonadism | NM_001059.2 | TACR3 c.196dupG, p.Ala66fs*84 | Het | F + M | AR | LP | / | / |
| 5175 | 1 year | Micropenis, short stature | NM_005270.4 | GLI2 c.3463_3464delGA, p.Asp1155Arg fs*39 | Het | De novo | AD | P | / | / |
| 5589 | 2 years 3 months | Micropenis, cryptorchidism | NM_005270.4 | GLI2 c.3137delG, p.Gly1046Ala fs*84 | Het | M | AD | LP | / | / |
| 6606 | 3 years | Micropenis, small testes | NM_005270.4 | GLI2 c.3640C>T, p.Gln1214* | Het | M | AD | LP | / | / |
| 12343 | 10 years | Micropenis, gonadal dysgenesis, short stature | NM_017780.3 | CHD7 c.5211-2A>G | Het | De novo | AD | P | / | / |
| 14317 | 12 years 5 months | Micropenis, hypogonadotropic hypogonadism, mild intelligence disability, absence of olfactory bulb (normal olfactory function) | NM_003106.3 | SOX2 c.259A>G, p.Lys87Glu | Het | De novo | AD | LP | / | / |
| 14376 | 8 years 9 months | Hypogonadotropic hypogonadis, micropenis, bilateral cryptorchidism, hyposmia | NM_000216.2 | ANOS1 c.515G>A, p.Cys172Tyr | Hemi | M | XR | LP | / | / |
| 15071 | 4 years 8 months | Micropenis, motor development retardation | NM_006941.3 | SOX10 c.506C>T, p.Pro169Leu | Het | M | AD | LP | / | / |
| 15128 | 5 months | Bilateral cryptorchidism, bilateral pinky joint malformation | NM_005996.3 | TBX3 c.932dupA, p.Glu312Glyfs*6 | Het | De novo | AD | P | / | / |
| 15562 | 1 year 1 month | Micropenis, bilateral cryptorchidism, hyposmia | NM_006941.3 | SOX10 c.506C>T, p.Pro169Leu | Het | De novo | AD | LP | / | / |
| 16255 | 13 years 3 months | Bilateral cryptorchidism, micropenis, absence of olfactory bulb, intelligence disability, dysmorphic facial features (long philtrum, congenital cup ear, ocular hypertelorism), brachydactyly (finger), syndactyly (toe), abnormal brain structure | NM_023110.2 | FGFR1 c.2173T>C, p.Cys725Arg | Het | De novo | AD | LP | / | / |
| 2061 | 15 years | Hypogonadotropic hypogonadism, hyposmia | NM_000216.2 | ANOS1 del Exon 4-5 | Hemi | M | XR | LP | / | / |
| 2356 | 7 years | Micropenis, small testes | NM_000406.2 | GNRHR c.806C>T, p.Thr269Met | Homo | F + M | AR | LP | 0.00001194 | 0.00000293 (European non-Finnish) |
| 2955 | 6 years | Hypogonadotropic hypogonadism, bilateral cryptorchidism, small testes | NM_000216.2 | ANOS1 c.1712G>A, p.Trp571* | Hemi | De novo | XR | P | / | / |
| 3879 | 15 years | Hypogonadotropic hypogonadism, hyposmia, absence of olfactory bulb | NM_000216.2 | ANOS1 c.984C>G, p.Tyr328* | Hemi | M | XR | LP | / | / |
| 3973 | 11 years | Gonadal dysplasia, cryptorchidism, short stature | NM_005634.2 | SOX3 c.424C>A, p.Pro142Thr | Hemi | M | XL | LP | / | / |
| 4136 | 11 years | Gonadal dysplasia, hypogonadotropic hypogonadism | NM_001059.2 | TACR3 c.196dupG, p.Ala66fs*84 | Het | F + M | AR | LP | / | / |
| 5175 | 1 year | Micropenis, short stature | NM_005270.4 | GLI2 c.3463_3464delGA, p.Asp1155Arg fs*39 | Het | De novo | AD | P | / | / |
| 5589 | 2 years 3 months | Micropenis, cryptorchidism | NM_005270.4 | GLI2 c.3137delG, p.Gly1046Ala fs*84 | Het | M | AD | LP | / | / |
| 6606 | 3 years | Micropenis, small testes | NM_005270.4 | GLI2 c.3640C>T, p.Gln1214* | Het | M | AD | LP | / | / |
| 12343 | 10 years | Micropenis, gonadal dysgenesis, short stature | NM_017780.3 | CHD7 c.5211-2A>G | Het | De novo | AD | P | / | / |
| 14317 | 12 years 5 months | Micropenis, hypogonadotropic hypogonadism, mild intelligence disability, absence of olfactory bulb (normal olfactory function) | NM_003106.3 | SOX2 c.259A>G, p.Lys87Glu | Het | De novo | AD | LP | / | / |
| 14376 | 8 years 9 months | Hypogonadotropic hypogonadis, micropenis, bilateral cryptorchidism, hyposmia | NM_000216.2 | ANOS1 c.515G>A, p.Cys172Tyr | Hemi | M | XR | LP | / | / |
| 15071 | 4 years 8 months | micropenis, motor development retardation | NM_006941.3 | SOX10 c.506C>T, p.Pro169Leu | het | M | AD | LP | / | / |
| 15128 | 5 months | Bilateral cryptorchidism, bilateral pinky joint malformation | NM_005996.3 | TBX3 c.932dupA, p.Glu312Glyfs*6 | Het | De novo | AD | P | / | / |
| 15562 | 1 year 1 month | Micropenis, bilateral cryptorchidism, hyposmia | NM_006941.3 | SOX10 c.506C>T, p.Pro169Leu | Het | De novo | AD | LP | / | / |
| 16255 | 13 years 3 months | Bilateral cryptorchidism, micropenis, absence of olfactory bulb, intelligence disability, dysmorphic facial features (long philtrum, congenital cup ear, ocular hypertelorism), brachydactyly (finger), syndactyly (toe), abnormal brain structure | NM_023110.2 | FGFR1 c.2173T>C, p.Cys725Arg | Het | De novo | AD | LP | / | / |
| 2061 | 15 years | Hypogonadotropic hypogonadism, hyposmia | NM_000216.2 | ANOS1 del Exon 4-5 | Hemi | M | XR | LP | / | / |
| 2356 | 7 years | Micropenis, small testes | NM_000406.2 | GNRHR c.806C>T, p.Thr269Met | Homo | F + M | AR | LP | 0.00001194 | 0.00000293 (European non-Finnish) |
| 2955 | 6 years | Hypogonadotropic hypogonadism, bilateral cryptorchidism, small testes | NM_000216.2 | ANOS1 c.1712G>A, p.Trp571* | Hemi | De novo | XR | P | / | / |
| 3879 | 15 years | Hypogonadotropic hypogonadism, hyposmia, absence of olfactory bulb | NM_000216.2 | ANOS1 c.984C>G, p.Tyr328* | Hemi | M | XR | LP | / | / |
| 3973 | 11 years | Gonadal dysplasia, cryptorchidism, short stature | NM_005634.2 | SOX3 c.424C>A, p.Pro142Thr | Hemi | M | XL | LP | / | / |
| 4136 | 11 years | Gonadal dysplasia, hypogonadotropic hypogonadism | NM_001059.2 | TACR3 c.196dupG, p.Ala66fs*84 | Het | F + M | AR | LP | / | / |
| 5175 | 1 year | Micropenis, short stature | NM_005270.4 | GLI2 c.3463_3464delGA, p.Asp1155Arg fs*39 | Het | De novo | AD | P | / | / |
| 5589 | 2 years 3 months | Micropenis, cryptorchidism | NM_005270.4 | GLI2 c.3137delG, p.Gly1046Ala fs*84 | Het | M | AD | LP | / | / |
| 6606 | 3 years | Micropenis, small testes | NM_005270.4 | GLI2 c.3640C>T, p.Gln1214* | Het | M | AD | LP | / | / |
| 12343 | 10 years | Micropenis, gonadal dysgenesis, short stature | NM_017780.3 | CHD7 c.5211-2A>G | Het | De novo | AD | P | / | / |
| 14317 | 12 years 5 months | Micropenis, hypogonadotropic hypogonadism, mild intelligence disability, absence of olfactory bulb (normal olfactory function) | NM_003106.3 | SOX2 c.259A>G, p.Lys87Glu | Het | De novo | AD | LP | / | / |
| 14376 | 8 years 9 months | Hypogonadotropic hypogonadis, micropenis, bilateral cryptorchidism, hyposmia | NM_000216.2 | ANOS1 c.515G>A, p.Cys172Tyr | Hemi | M | XR | LP | / | / |
| 15071 | 4 years 8 months | Micropenis, motor development retardation | NM_006941.3 | SOX10 c.506C>T, p.Pro169Leu | Het | M | AD | LP | / | / |
| 15128 | 5 months | Bilateral cryptorchidism, bilateral pinky joint malformation | NM_005996.3 | TBX3 c.932dupA, p.Glu312Glyfs*6 | Het | De novo | AD | P | / | / |
AD, autosomal dominant; AR, autosomal recessive; F, father; Hemi, hemizygote; Het, heterozygote; Homo, homozygote; LP, likely pathogenic; M, mother; P, pathogenic; XL, X chromosome linked.
Of all the P/LP variants (n = 78), 68 were heterozygote (87.18%, 68/78) and 10 were homozygote (12.82%, 10/78). The variant types included missense (64.10%, 50/78), deletion (11.54%, 9/78), nonsense (11.54%, 9/78), frameshift (8.97%, 7/78), and splicing (3.85%, 3/78) mutations. The accessible DNA of these subjects’ parents (95.31%, 61/64) was then isolated and subjected to Sanger sequencing to confirm the origin of the detected candidate variants. The verification test results showed that 21.79% variants (17/78) were found to be de novo mutations.
In primary gonadal/genital disorders subjects (n = 48), SRD5A2 (steroid 5-alpha-reductase 2) and AR (androgen receptor) were the most common candidate genes. SRD5A2 mutations were detected in 35.42% (17/48) of the patients, and AR mutations were detected in 27.08% (13/48) of the patients, accounting for 42.19% (27/64) and 21.88% (14/64) of all the variants detected in this group, respectively (Figure 2 and 3). Of the variants in SRD5A2 (n = 27), most were located on exon 4. The same mutation site (c.680G>A, p.R227Q) was detected in 16 patients, which is considered a hotspot mutation in Chinese patients with 5αRD. Of the variants in the AR gene (n = 14), most were located on exon 1. To date, no clear hotspot mutations in AR have been found in a Chinese cohort.
Mutations detected in AR gene in our cohort. Most of the variants detected in AR located on Exon1 in our androgen insensitivity syndrome patients.
Citation: Endocrine Connections 12, 10; 10.1530/EC-23-0029
In central DSD subjects (n = 16), variants in genes related to Kallmann syndrome were detected frequently in our cohort, including ANOS1 (anosmin 1), CHD7 (chromodomain helicase DNA-binding protein 7), FGFR1 (fibroblast growth factor receptor 1), GNRHR (gonadotropin-releasing hormone receptor), SOX2 (SRY-box transcription factor 2), SOX10 (SRY-box transcription factor 10), and TACR3 (tachykinin receptor 3) (see Table 3). Mutations in ANOS1 accounted for 25.00% (4/16) in this section. Besides, GLI2 (GLI family zinc finger 2) variants (18.75%, 3/16) were reported in three subjects diagnosed as hypopituitarism (Fig. 4).
Genetic expansion of primary gonadal/genital disorders and central hypogonadism in this cohort. 79 variants accross 21 genes were detected in 65 subjects of the current study. AMH, anti-Mullerian hormone; ANOS1,anosmin 1; AR, androgen receptor; CHD7, chromodomain helicase DNA binding protein 7; CYP17A1, cytochrome P450 family 17 subfamily A member 1; FGFR1, fibroblast growth cactor receptor 1; GLI2, GLI family zinc finger 2; GNRHR, gonadotropin releasing hormone receptor; LHCGR, luteinizing hormone/choriogonadotropin receptor; NR0B1, nuclear receptor subfamily 0 group B member 1; RIT1, Ras like without CAAX 1; SOX10, SRY-box transcription factor 10; SOX2, SRY-box transcription factor 2; SOX3, SRY-box transcription factor 3; SRD5A2=Steroid 5 Alpha-reductase 2; SRY, sex determining region Y; TACR3,tachykinin receptor 3; TBX3, T-box transcription factor 3; TSPYL1, TSPYlike 1.
Citation: Endocrine Connections 12, 10; 10.1530/EC-23-0029
Rare variants
To be mentioned, we have reported a rare variant in TBX3 (T-box transcription factor 3). TBX3 is a critical developmental regulator of heart, mammary glands, limbs, and lungs. It is considered a pathogenic gene for Ulnar-mammary syndrome according to limited literature from the HGMD database. In Ulnar-mammary syndrome, DSD phenotypes such as hypospadias, micropenis, or cryptorchidism could be observed (20). Here we report a 5-month-old boy presented with bilateral cryptorchidism and bilateral fifth finger joint malformation. Abnormal mammary development was not found up to now. After prediction of the variant’s (c.932dupA, p. Glu312Glyfs*6) function by Alamut software, this subject was thus diagnosed as Ulnar-mammary syndrome with a de novo pathogenic TBX3 mutation c.932dupA, p. Glu312Glyfs*6.
Another likely pathogenic variant (c.600+1G>C) in pleckstrin homology domain-interacting protein (PHIP) was found in a 12.5-year-old boy, presented with intellectual disability, unilateral renal agenesis, obesity, and bilateral cryptorchidism. PHIP is known as an implicated gene for Chung–Jansen syndrome, characterized by developmental delay, intellectual disability, obesity, and dysmorphism (21). According to the previous case, it is reported that genital disorders can be observed in Chung–Jansen syndrome (21), differed from our case.
Among VUS variants, we also noticed a homo variant (c.136+1G>A) in growth hormone receptor (GHR) which has not been verified to be related to 46,XY DSD. The 5-year-old patient presented with dominant female external genitalia, suspicious uterine-like structure between bladder and rectum, absent gonad structure and short stature. The paraclinical results showed low level of testosterone (<0.10 ng/mL), DHT (26.63 pg/mL), and estrogen (<20.00 pg/mL). The gonadotropin was augmented as LH 0.26 mIU/mL and FSH 10.73 mIU/mL. The evaluation of the adrenal axis showed normal function. The patient was clinically diagnosed as growth hormone insensitivity and 46,XY DSD. According to the literature review, other than growth hormone insensitivity, GHR could also cause Laron syndrome characterized by a typical phenotype including dwarfism, obesity, and hypogenitalism (22). However, no cases of dominant female external genitalia were reported, and functional experiments are still lacking. Whether GHR variant could cause sex differential or sex development disorder remains a question.
A VUS variant (c.2129A>G, p. Tyr710Cys) in NOTCH3 (Notch Receptor 3) was found in a 2-month-old boy who presented with short stature, penile curvation, hypospadias, bilateral cryptorchidism, micropenis, penile scrotal translocation, scrotal division, special features (high hairline, forehead protrusion), premature delivery, and small for gestational age. The patient showed a remarkable DSD phenotype. However, at the moment, we only found a VUS variant in NOTCH3 by WES. Cases related to NOTCH3, for example, CADASIL patients, do not include sexual disorders (23). Thus, the pathogenicity of NOTCH3 in sex development is still under-researched. Further genetical evaluation and functional verification are required to determine the pathogenesis mechanism.
Comparison of TPS and WES
After all, we intended to compare the efficacy and results of two different sequencing technologies in the current cohort. As mentioned earlier, subjects between 2016 and 2018 (n = 92) underwent TPS (group TPS), while those during 2019 and 2022 (n = 86) underwent WES (group WES). We compared the characteristics of these two groups to different degrees (see Table 5).
Comparison of targeted panel sequencing group (group TPS) and whole-exome sequencing group (group WES) in our cohort.
| Features | TPS (n = 92) | WES (n = 86) | P-value |
|---|---|---|---|
| Clinical manifestation | |||
| Age (years) | 8.00 (1.58, 12.00) | 8.67 (3.23, 11.79) | 0.808 |
| Micropenis, % | 50.00% | 63.95% | 0.060 |
| Cryptorchidism, % | 32.61% | 36.06% | 0.629 |
| Hypospadia, % | 19.57% | 15.12% | 0.434 |
| Genetic characteristics | |||
| Diagnosis rate, % (n/N) | 57.61% (53/92) | 40.70% (35/86) | 0.024a |
| Pathogenic mutation, % (n/N) | 39.68% (25/63) | 45.00% (18/40) | 0.594 |
| Likely pathogenic mutation, % (n/N) | 33.33% (21/63) | 37.50% (15/40) | 0.666 |
| VUS mutation, % (n/N) | 25.87% (18/63) | 15.00% (6/40) | 0.267 |
| De novo mutation, % (n/N) | 6.34% (4/63) | 32.50% (13/40) | 0.001a |
| Missense mutation, % (n/N) | 71.43% (45/63) | 72.50% (29/40) | 0.906 |
| Nonsense mutation, % (n/N) | 11.11% (7/63) | 5.00% (2/40) | 0.284 |
| Deletion mutation, % (n/N) | 15.87% (10/63) | 20.00% (8/40) | 0.591 |
Subjects between 2016 and 2018 (n = 92) underwent targeted gene panel sequencing (group TPS), those between 2019 and 2022 (n = 86) underwent whole-exome sequencing (group WES). We compared the characteristics of these two groups to both clinical phenotype and genetic degrees. *aSignificant difference between group TPS and group WES, P value by the Pearson Chi-square, P < 0.05 was considered statistically significant.
aSignificant difference between group TPS and group WES, P-value by the Pearson chi-square, P < .05 was considered statistically significant.
No significant differences were observed in the average age of the subjects at diagnosis. Second, the proportion of the DSD phenotype among the 46,XY DSD patients slightly increased. The proportion of patients with micropenis increased from 50.00 to 63.95% and that of patients with cryptorchidism increased from 32.61 to 36.05%. However, the proportion of patients with hypospadias decreased from 19.57 to 15.12%. This might have contributed to the early surgical correction in these patients, which led to a decrease in the number of patients in the endocrinology department. In addition, the proportion of patients with single phenotype (micropenis, cryptorchidism, hypospadias, GD, hypogonadism, undeveloped testis) has increased since 2019 (46.2 vs 58.8%, P = 0.288). When the composition and clinical diagnosis between the two groups were compared, the percentage of AIS and 5αRD remained similar (22.64 vs 22.85% for AIS; 18.90 vs 17.14% for 5α RD), while the percentage of IHH slightly increased from 20.75 to 22.86%. Among all the variants detected, pathogenic and likely pathogenic variants accounted for 39.68 and 33.33% in group 2016−2018, respectively, while the percentage of pathogenic variants in group 2019−2022 has increased to 45.00% and that of likely pathogenic variants increased to 37.50%. Since 2019, under the application of WES technology, we have diagnosed much more complex syndromic DSD due to the accurate interpretation of variants at our center.
Discussion
Patients with 46,XY DSD present with various clinical manifestations and complex pathogenesis, which increases the difficulty of diagnosis (24, 25). To understand the diagnosis and pathogenesis and to improve the infertility consequences for patients, research is needed to expand the phenotype and genotype (26).
Overall speaking, the most common symptoms in 46,XY DSD patients are cryptorchidism, hypospadias, and micropenis. In our study, the main symptoms of patients in different age groups were different. Mini-pubertal and prepubertal children mostly presented with cryptorchidism and micropenis. Adolescent patients usually present with less cryptorchidism or hypospadias but are more likely to undergo micropenis and delayed puberty (characterized by undeveloped testicular volume and lack of secondary sex characteristics at the age of 14 in boys). A family history of delayed pubertal development is an important factor in distinguishing between DSD and a constitutional delay of growth and puberty (27). In other studies (28, 29), some children with DSD were reported to have spontaneous pubertal development. For patients with mild manifestations, it becomes more difficult to detect the symptoms accurately early, making the initial intervention difficult (30).
Currently, the age of diagnosis of 46,XY DSD has advanced as early as the neonatal period due to prenatal genetic testing and typical genital differences or associated symptoms observed soon after birth (31, 32). In our group, the overall average diagnosis of Chinese 46,XY patients was 8.63 (2.61,12.00) years, ranging from 1 month to 17 years. The median age of primary gonadal/genital disorders subjects was 2.92 (0.67, 7.33) years old. It is known that the detection of 46,XX CAH patients can be performed as early as 0.25 months old based on specific salt-losing features, electrolyte disturbances, and abnormal hormonal profiles. One of the early diagnosed patients (0.08 years) was a 46,XY atypical CAH boy (Patient ID 8945) whose symptoms manifested as hyperpigmentation, cryptorchidism, and electrolyte disturbance, indicating that early detection of CAH in 46,XY males is required and can be identified using NGS.
5αRD and AIS accounted for an important part in primary DSD (33). AIS was the most frequent cause of 46,XY DSD in the previous large Asian cohort, and 5αRD was ranked as the second. These two subgroups accounted for approximately 50.5% of all cases (33). AIS and 5αRD patients both presented abnormal androgen function. We have found that the median age of 5αRD was 2.00 (0.58, 4.46) years old and that of AIS subjects was 10.00 (0.92, 14.17) years old (P = 0.06) (see Table 2). The patients’ testosterone and DHT levels in these diseases remained similar, but the gonadotropin levels in 5α-reductase-deficient patients were much lower than those in AIS patients, thus indicating that gonadotropin may be a marker for distinguishing between these two diseases.
On the other side, among central hypogonadism subjects, the median age at diagnosis was 7.88 (2.69, 12.07) years old. The specific phenotypes of these pre-pubertal and adolescent subjects were micropenis and small tests (average testis volume). And the most common clinical diagnosis of central hypogonadism is IHH and isolated hypopituitarism (hypogonadotropin). In IHH patients, multi-system manifestation evaluations are important for disease detection and diagnosis, such as olfactory dysfunction, intellectual disability, delayed motor development, skeletal deformities, brain structure abnormalities, and hearing disorders. These non-endocrinological manifestations in elder children probably refer to IHH.
To further explore the distribution of 46,XY DSD, we then concluded the clinical characteristics in those who had a P/LP genetic variant (n = 64) and those whose genetic results were negative (n = 90). First, we found a significant age difference at diagnosis between the NGS-positive group (4.00 years old) and the NGS-negative group (8.75 years old) (P < 0.05). Second, the proportion of subjects presenting with unilateral or bilateral cryptorchidism in the NGS-negative group (21.11%) was smaller than that in the NGS-positive group (50.00%) (P < 0.05); the proportion of subjects presenting with isolated micropenis (28.89%, 26/90) was larger than that of NGS-positive group (9.38%, 6/64). Thus, we think that the value of gene sequencing in those young-age and cryptorchidism patients is especially important. And on the other hand, the mechanism of micropenis should be further explored in the future, since quite a part of subjects with isolated micropenis have not found a genetic explanation.
In the genetic aspect, with the development and application of NGS, the molecular detection rate of P/LP variants in 46,XY DSD has become easier and more efficient (34). To date, there are more than 300 candidate genes associated with DSD phenotypes (35), and the efficiency of molecular diagnosis of DSD patients has improved from 33% to 74.3% (36, 37, 38). The total molecular detection rate of the current cohort was 49.44% and the genetic diagnostic rate (including patients with P/LP variants only) was 35.96% (64/178).
In our study, among the genes associated with 46,XY DSD, the most common pathogenic variants in primary gonadal/genital disorders were variants in AR and SRD5A2 (Figures 2 and 3). Previous study indicated that variants in SRD5A2 are mostly distributed on exons 1, 4, and 5 (40), and the variant c.680G>A (p.R227Q) is the hotspot site in Chinese 5αRD, which is consistent with our results. The most common genetic change of central hypogonadism is in IHH-related genes, especially ANOS1, similar to the results in previous literature (39). The diagnosis of IHH is considered complicated also due to its genetic heterogeneity. Thus, the interpretation of ACMG guidelines for variants is extremely important. However, in China, due to regional differences and department settings, the interpretation of variants is usually performed by clinicians, instead of geneticists. Therefore, the prediction of variants’ function may differ among medical centers. Accurate sequencing protocol and proper interpretation methods are required to unify the standard method of gene sequencing.
In addition to common variants, we have also identified novel candidate variants (GHR, PHIP) that might be related to 46,XY DSD, As described in the result part, these three subjects presented with specific sexual disorders manifestation and abnormal sexual hormone levels. However, their genetic results showed undefined variants. Variant c.600+1G>C in PHIP was classified as likely pathogenic, and variant c.136+1G>A in GHR was classified as pathogenic. These two genes are known as causes of different diseases, of which, the common phenotypes do not involve DSD. Differing from our cases, previous cases of GHR and PHIP variants were reported to have DSD phenotypes such as genital disorders and hypogenitalism (21, 22). Combined with the functional prediction by Alamut software, the splicing variants in PHIP and GHR are predicted to have impacts on mRNA processing or expression. Thus, although according to current limited information, the pathogenicity of these variants cannot be clarified yet, we still consider it possible that DSD manifestations might be the new phenotypes of these genes. Functional verification in vivo and in vitro experiments are required to confirm the role of these variants.
After all, we explored the phenotype–genotype correlation in this study. Of variants in SRD5A2, most are missense mutations. Mutation sites c.680G>A, p. Arg227Gln and c.607G>A, p. Gly203Ser were frequently detected in Asian patients, especially in the Chinese population (40). Consistent with previous reports (41), in our study, patients carrying homozygous c.680G>A, p. Arg227Gln presented as milder phenotype (isolated micropenis), while those carrying c.607G>A, p. Gly203Ser (including in compound heterozygosity with p. R227Q) presented as partial androgen insensitivity syndrome (PAIS) with more severe phenotypes (ambiguous genitalia and hypospadias). Earlier work, however, suggested that p.R227Q might moderate the functional severity caused by other variants (42). Further relation between genotype and phenotype depends on the impact of genotype caused enzymatic damage.
Of the variants in the AR gene, most current reported mutations were located on exons 5–8, coding for the ligand-binding domain (43). Mutation on exon 1 could cause complete androgen insensitivity syndrome (CAIS), PAIS, or mild androgen insensitivity syndrome phenotypes. And it is reported in the earlier literature that most mutations causing CAIS were on exon 1 (44). However, in our cohort, mutations on exons 2–3, coding for the DNA-binding domain, are more likely to cause CAIS. This inconsistency may be due to the limited sample amount in our cohort.
On the other hand, there is still few information on the phenotype–genotype relation of central hypogonadism DSD. Clinical manifestations vary among patients carrying the same variant. Both in our cohort and previous reports, GLI2 demonstrates an autosomal dominant inheritance pattern with incomplete penetrance and variable expressivity in hypopituitary patients (45, 46). In IHH cases, two subjects in our cohort carried the same mutation site in SOX10, one manifested as isolated hypogonadotropic hypogonadism, while the other presented hypogonadism with obvious intelligence disability. However, certain multi-system phenotypes may help in confirming the pathogenic gene. Congenital unilateral renal agenesis and/or mirror movements of the upper limbs (synkinesis) in IHH patients refer to ANOS1 mutation (47); Hearing loss refers to CHD7 mutation (48); Bone anomalies (including syndactyly and dental agenesis) refers to FGF8/FGFR1 mutations (48).
Finally, when comparing the genetic findings of the two groups using different sequencing methods, we found that the molecular detection rate of group TPS (2016−2018) was 57.61%, while that of group WES (2019−2022) was 40.70%. Before 2018, our patient source was limited to the local population, where prenatal, neonatal, and pubertal screening of reproduction problems received high attention both in hospitals and in the community; After 2018, our center has widened the clinical patient resource and thus complicated the genetic diagnosis. Second, since male infertility has received much more attention in adolescent health, an increasing number of patients with milder manifestations such as isolated micropenis or isolated undeveloped testis seek endocrinology consultation. The pathogenic mechanisms and new candidate genetic variants that might contribute to mild manifestation are still under investigation. The difference between the two technologies can be concluded that TPS covers most of the genes contributing to DSD, which leads to a high efficiency of genetic diagnosis (19). However, WES can filter those new candidate genes or variants that have not been included in the panel. WES technology can also screen small copy number variations for certain diseases (49). In clinical practice, clinicians should select analysis methods according to the patients’ clinical performance and economic reasons.
Among the remaining large part of pathogenically undefined subjects, discussions on its etiology revealed that this might be related to oligo-/poly genetic reasons or other single nucleotide variations that could not be detected by currently applied methods (50). DSD disease is considered a monogenic disorder (51); however, some studies have shown that it may be a polygenic or oligogenic disease (52, 53). In a large international DSD cohort, oligogenic inheritance involving two testis development genes, MAP3K1 (mitogen-activated protein kinase kinase kinase 1) and ZFPM2 (zinc finger protein, FOG family member 2), was observed in three patients (34). There is an undefined molecular or environmental mechanism that may lead to various phenotypes and genetic heterogeneity. Moreover, recent research has focused on non-coding variants in regulatory elements that alter gene expression and cause disorders in gonadal development (54).
Limitations
First, this was a single-center study with limited patient resources. Second, some VUS and newly discovered DSD pathogenic genes were found, but it still lacks functional experiments to verify the pathogenicity of these variants and their potential molecular mechanisms. Also, additional testing could be performed on WES-negative patients using microarray for CNV, WGS technology, or RNA sequencing methods to identify additional pathogenic mechanisms among 46,XY DSD patients.
Conclusions
Here, we summarized the clinical and genetic characteristics of 178 Chinese children with 46,XY DSD. The total genetic detection rate was 49.44%. Most children manifested as cryptorchidism, micropenis, and hypogonadal hypogonadism. The top classifications of 46,XY DSD detected were 5αRD, IHH, and AIS. The hotspot mutation in the Chinese cohort was p.R227Q on SRD5A2. New candidate variants (GHR, PHIP) were detected in this cohort.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/EC-23-0029.
Declaration of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.
Funding
This work was supported by the National Nature Science Foundation of China (82170910), Shanghai Clinical Medical Research Center for Children’s Rare Diseases (20MC1920400), Joint Program of Shanghai Municipal Health Commission (PW2021D-13), and Single Disease Research Center of Shanghai Jiao Tong University School of Medicine (ELYZX202107).
Ethics statement
The studies involving human participants were reviewed and approved by the Research Ethics Committee of Shanghai Children's Medical Center, Affiliated to School of Medicine, Shanghai Jiao Tong University (No. SCMCIRB-K2016013). Written informed consent to participate in this study was provided by the participants’ legal guardian or next of kin. Written informed consent was obtained from the individual(s) and minor(s) legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.
Data availability statement
The data supporting the findings of this study are available from the corresponding authors upon reasonable request.
Author contribution statement
Conceptualization: Tang Yijun, Chen Yao, Li Xin, Wang Xiumin. Data curation: Tang Yijun, Wang Jiayi, Wang Yirou, Xu Yufei, Wang Jian. Acquisition: Wang Xiumin, Chen Yao. Methodology: Chen Yao, Wang Jian, Wang Xiumin. Investigation: Tang Yijun, Wang Jiayi, Zhang Qianwen. Supervision: Chen Yao, Wang Jian, Wang Xiumin. Writing—original draft: Tang Yijun. Writing—review and editing: Chen Yao, Wang Jiayi, Wang Xiumin.
Acknowledgements
We acknowledge the collaboration between patients and their guardians.
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