Search

Objective: To explore the heterogeneity and correlation of clinical phenotypes and genotypes in children with disorders of sex development (DSD). Methods: A retrospective study of 1 235 patients with clinically proposed DSD in 36 pediatric medical institutions across the country from January 2017 to May 2021. After capturing 277 DSD-related candidate genes, second-generation sequencing was performed to analyzed the heterogeneity and correlation combined with clinical phenotypes. Results: Among 1 235 children with clinically proposed DSD, 980 were males and 255 were females of social gender at the time of initial diagnosis with the age ranged from 1 day of age to 17.92 years. A total of 443 children with pathogenic variants were detected through molecular genetic studies, with a positive detection rate of 35.9%. The most common clinical phenotypes were micropenis (455 cases), hypospadias (321 cases), and cryptorchidism (172 cases) and common mutations detected were in SRD5A2 gene (80 cases), AR gene (53 cases) and CYP21A2 gene (44 cases). Among them, the SRD5A2 mutation is the most common in children with simple micropenis and simple hypospadias, while the AMH mutation is the most common in children with simple cryptorchidism. Conclusions: The SRD5A2 mutation is the most common genetic variant in Chinese children with DSD, and micropenis, cryptorchidism, and hypospadias are the most common clinical phenotypes. Molecular diagnosis can provide clues about the biological basis of DSD, and can also guide clinicians to perform specific clinical examinations. Target sequence capture probes and next-generation sequencing technology can provide effective and economical genetic diagnosis for children with DSD.

Objective: To explore the clinical characteristics and genetic basis of a child with 5α-reductase type 2 deficiency., Methods: Clinical data of the child was retrospectively analyzed. Targeted capture-next generation sequencing and Sanger sequencing were carried out to detect potential variants., Results: The patient's main features included micropenis and hypospadia. He was found to harbor compound heterozygous c.680G>A (p.R227Q) and c.3G>T (p.M1I) variants of the SRD5A2 gene. Among these, c.680G>A (p.R227Q) was inherited from his father and was a known pathogenic mutation, while c.3G>T (p.M1I) was inherited from his mother and was unreported previously., Conclusion: The compound heterozygous variants of the SRD5A2 gene probably underlay the disease in this child, who was eventually diagnosed with 5α-reductase 2 deficiency.

This article reported the clinical characteristics and SRD5A2 gene mutation pattern of a child with steroid 5-α reductase type 2 deficiency. The 2-month-old boy showed hypospadias and short penis shortly after birth. DNA was extracted from the peripheral blood of the child and his parents. The endocrine disease-related genes were captured and sequenced by high-throughput sequencing technology, and the family DNA samples were verified by Sanger sequencing. The results showed that c.680G>A(p.R227Q) and c.608G>A(p.G203D) compound heterozygous mutations existed in the SRD5A2 gene of the child. The c.680G>A mutation inherited from his father, which was a known pathogenic mutation. The c.608G>A mutation originated from his mother, which was a novel mutation discovered in this study. These results provide molecular evidence for the etiological diagnosis of the child and genetic counseling for the family, as well as extend the mutation spectrum of SRD5A2 gene.

Objective: To investigate the novel genetic cause associated with hypospadias and the strategy for preventing offspring genetic defects in these patients. Methods: In March 2019, a patient with gonadal dysplasia (hypospadias associated with cryptorchidism) was referred to Shanghai General Hospital. His secondary sex characters, level of sex hormones and the development of male reproductive system was assessed through physical examination, sex hormone examination, male reproductive system B-ultrasound and computed tomography (CT). Whole-exome sequencing (WES) was preformed to investigate the pathogenic genetic variations associated with hypospadias and cryptorchidism. Also, Sanger sequencing was conducted to verify the WES results in the pedigree. Semen analysis was used to assess the fertility of the proband and the SRD5A2 gene analysis of his spouse was performed to assess the risk of genetic defects in the offspring. Results: The patient suffered from gonadal dysplasia (hypospadias associated with cryptorchidism). Physical examination showed an inverted triangular distribution of pubic hair, small penis and the volume of the testis was 8 ml. Sex hormone examination revealed the level of FSH, LH, Pituitary prolactin (PRL), estrogen (E(2)), testosterone (T), and sex hormone-binding globulin (SHBG) was 25.81 U/L, 10.84 U/L, 21.09 μg/L, 153 pmol/L, 16.95 nmol/L, and 36.15 nmol/L respectively. B-ultrasound and computed tomography (CT) showed left inguinal testis. Also, semen analysis illustrated that the volume was 0.05 ml and sperm concentratio n <2×10(6)/ml, suggesting oligospermia in this case. WES sequencing and Sanger sequencing showed compound heterozygous LoF mutations in SRD5A2 [NM&#95;000348.3:C.679C>T(p.Arg227Ter) and NM&#95;000348.3:C.16C>T(p.Gln6Ter)] in this patient. And there were no pathogenic genetic variations of SRD5A2 in the spouse. Conclusion: Novel compound heterozygous LoF mutations in SRD5A2[NM&#95;000348.3:C.679C>T(p.Arg227Ter) and NM&#95;000348.3:C.16C>T(p.Gln6Ter)] may be the primary cause of disorders of sex development.

Objective: Molecular genetics and clinical phenotypic characteristics of 5 alpha reductase deficiency were analyzed. Methods: The genetic results and clinical features classied as Prader grade of external genitalia of 86 children with SRD5A2 mutation seen from 2007 to 2017 at Department of Endocrinology of Beijing Children's Hospital were analyzed, and the mutation differences in different were compared regions according to the literatures. Results: Among the 86 children, 15 had were homozygous mutations, accounting for 17%, and 71 cases of compound heterozygous mutations accounted for 83%. Totally 172 alleles mutations in this series. The mutation was mainly located on exon 1 and exon 4, in which the mutation frequency of exon 1 was 23.8% (41/172), and the frequency of exon 4 mutation was 55.8% (96/172). A total of 19 mutation types of the SRD5A2 gene in this group were detected, of which 5 were new mutations (p.A228F, p.E57D, p.V124D, p.A117D, p.E197K); 65 patients had p.R227Q mutation, accounting for 76%, while 31 had p.Q6* mutation, accounting for 36%. Other rare types such as p.R246W, p.R103* and so on were also seen in the present study, there was no significant difference between north China and south China ( P> 0.05). The clinical phenotypes of p.R227Q variation varied, mainly in Prader 3-4, accounting for 82%, while (Prader 0-1) were less, accounting only 2%. The variation of p.Q6* was mainly manifested in Prader 3, accounting for 50%. p.R246Q mainly presented Prader 3. The variation of p.G203S appeared to have Prader 2 and Prader 4-5, accounting for 20% and 73% respectively. There was no significant difference in clinical phenotype corresponding to each protein type ( P >0.05) . Conclusion: Among the 86 children have identified 19 SRD5A2 mutation types, p.R227Q is a hotspot mutation in Chinese. Variations at different types may have different clinical phenotypes, while the same variations may have different clinical features. There was no significance different in the variation types between the north and the south.

Objective: To analyze the karyotypes and SRD5A2 gene mutations in 25 patients with sporadic or familial hypospadias., Methods: The patients included 10 adults and 15 children, whose chromosomes were analyzed by G-banded karyotyping, and the SRD5A2 genes were sequenced., Results: Two patients were found to have an abnormal karyotype, while eight have carried compound heterozygous mutations of the SRD5A2 gene, which included 5 genotypes formed by 6 types of mutations, i.e., p.G203S/p.R227Q, p.R227Q/p.R246Q, p.Q6X/p.Q71X, p.L20P/p.G203S, and p.Q71X/p.R227Q. Mutations of the SRD5A2 gene were present in 32% (8/25) of all patients, 35% (8/23) in those with a normal karyotype, and 44.4% (8/18) in those with proximal type hypospadia. Bioinformatic analysis, literature review and pedigree analysis confirmed that all such mutations are pathogenic., Conclusion: Chromosomal anomalies and mutations of the SRD5A2 gene are the main cause of hypospadias. Sequencing of the SRD5A2 gene may explain the etiology of nearly half of the patients with proximal type of hypospadas but a normal karyotype, which can facilitate genetic consulting.

Objective: To investigate the association of V89L polymorphism of the SRD5A2 gene with the prognostic factors of prostate cancer (PCa)., Methods: We identified the V89L polymorphic sites of the SRD5A2 gene after Rsa-1 restriction enzyme digestion, observed the distribution of V89L (VV, VL and LL) polymorphism in 112 PCa and 89 benign prostate hyperplasia (BPH) patients, and determined the association of V89L polymorphism with the age, free PSA (fPSA), total PSA (tPSA), fPSA/tPSA ratio, tumor stage and Gleason score of the PCa patients., Results: No statistically significant differences were found in the V89L polymorphism-induced genetic risk frequencies between the PCa and BPH groups (chi2 = 3. 606, df = 2, P = 0. 165), nor any significant correlation between the genotypes of VV and VL + LL and the differences in the fPSA, tPSA, fPSA/tPSA ratio, tumor stage, Gleason score and age of the PCa patients. VV and VL + LL showed no obvious association with the prognostic factors of PCa., Conclusion: V89L polymorphism is not related with the prognosis of PCa, but may be indirectly associated with its risk.

Objective: To explore possible molecular mechanism of hypospadias and relationship of the mutation of SRD5A2 gene to hypospadias., Methods: 96 blood samples from the patients with hypospadias were obtained and DNA was extracted from blood leukocytes. Polymerase chain reaction and direct sequencing were performed to analyze the coding regions of SRD5A2 gene., Results: 8 mutations were detected from 14 cases, including 5 missense mutations, 1 synonymous mutation, 1 nonsense mutation and 1 frameshift mutation. The mutations are in 1st, 4th and 5th exon. Gln6stop, His232His, Phe234Leu and frameshift mutations are novel., Conclusions: Exon 4 is a hot spot region of mutation within SRD5A2 gene, and about 10 percent of hypospadiac patients complicated with SRD5A2 dysfunction or deficiency.

Objective: To investigate the association between A49T polymorphism of SRD5A2 gene and risk of prostate cancer., Methods: PCR was used to examine the A49T polymorphisms of SRD5A2 gene in the tissues of prostate cancer resected from 112 patients (CaP group) and the specimens of benign prostate hyperplasia (BPH group) resected from 89 patients. The association of A49T polymorphism with age of onset, FPSA, TPSA, F/T, T stage, and Gleason score were analyzed., Results: There was no significant difference in A49T polymorphism between the CaP and BPH groups (P > 0.05). The average age of CaP patients was significantly higher than that of the BPH patients (P < 0.05). In the CaP patients, the Gleason score was significantly higher, and the age of onset was significantly lower in the AT + TT genotype than in the AA genotype (both P < 0.05) 2. The age of onset of the AA + AT group was significantly lower than that of the AA group (P < 0.05)., Conclusion: AA + AT genotype may be of worse prognosis, however, without significant difference. Rank scoring may reflect the relation between Gleason score and A49T genotype and estimate the prognosis better than two-level discrete evaluation.

Objective: The correlation were studied between testosterone 5-alpha-reductase II (SRD5A2) gene polymorphisms and prognosis factors., Methods: V89L and A49T variants was identified with Mwo1 and Rsa1. The differences of V89L and A49T between cancer of prostate (CaP) and benign prostatic hyperplasia (BPH) were studied. In addition, we also researched the association of polymorphisms with age of onset, free prostate specific antigen (FPSA), total PSA (TPSA), FPSA/TPSA (F/T), Gleason score, and T stage in cancer group., Results: We found no differences of V89L and A49T polymorphisms between CaP and BPH. In CaP group the A49T variant was associated with lower age of onset (P = 0.03) and higher Gleason score (P = 0.015). There were no differences between VV and VL+LL polymorphisms with any of the characteristics studied. When the characteristics above were regarded as two-level discrete variable, there were no differences by A49T and V89Lvariants., Conclusion: In CaP group, the AT+TT genotype was perhaps associated with poor prognosis. VL+LL genotype has no relation with prognosis.

Objective: To investigate the genetic polymorphisms of the three major susceptibility genes, AR, VDR, and SRD5A2 genes, in the Han nationality population in Northern China with a low risk of prostate cancer (Pca) and the relationship of these polymorphisms to the susceptibility to Pca, and to discuss the possible reason causing racial difference of prevalence of PCa., Methods: Restriction fragment length polymorphism (RFLP) technique and denaturing high-performance liquid chromatography (DHPLC) were used to detect the polymorphic sites in the DNAs from the peripheral blood samples of 116 patients with PCa and 190 normal male controls, all of Han nationality in Northern China. The distribution frequencies of different polymorphic sites were compared with those of the populations with high risk of prostate cancer., Results: The frequencies of SRD5A2 and VDR genotypes were not significantly different between the Pca patients and the controls (P > 0.05). The frequency of CAG repeat < 22 was significantly higher in the Pca group than in the control group (P < 0.05). Statistical analyses of the AR genotype prevalence showed significant differences between the PCa patients and the controls (P < 0.05). The frequencies of AR, VDR, and SRD5A2 genotypes showed significant difference between the Han population in Northern China and the high-PCa risk populations in Western countries. The frequency of CAG > or = 22 was 80.4% in the Han people in Northern China, significantly higher than that in the US Caucasians (52.1%) and Afro-Americans (25%, both P < 0.001)., Conclusion: There is a significant association between the AR gene CAG polymorphisms and PCa in the Han nationality population in Northern China. The distributions of the genotypes of AR, SRD5A2 and VDR gene are different among different ethnic population, which may be one of the reasons causing the racial difference in prostate cancer risk.

Objective: To study the expression levels and cell-specific expression patterns of steroid 5alpha-reductase type 1 and type 2 isoenzymes in human benign prostatic hyperplasia (BPH) tissues., Methods: Immunohistochemistry and RT-PCR were used to investigate qualitatively and semi-quantitatively the expression of 5alpha-reductases type 1 and type 2 in 15 specimens of normal prostate tissues from dead kidney donor or patients undergoing cystectomy and 25 BPH tissues obtained from suprapubie enucleation., Results: Both steroid 5alpha-reductase type 1 mRNA and protein and 5alpha-reductase type 2 mRNA and protein were present in the normal and BPH tissues, especially in the glandular epithelial cells and mainly in the cytoplasm. In the BPH group the expression of type 2 reductase in the periurethral tissues was significant higher than that in the subcapsular tissues (1.50 +/- 0.42 vs. 1.07 +/- 0.35, P < 0.01), however, there was no significant difference in the expression of type 1 reductase between these 2 zones (P > 0.05). The expression level of 5alpha-reductase type 2 mRNA in BPH tissues was significantly higher than that in the normal specimens (e.g. in periurethral zone 1.50 +/- 0.42 vs. 0.98 +/- 0.32, P < 0.01), but there was no statistically significant difference in the expression of 5alpha-reductase type 1 mRNA between these 2 groups (e.g. in periurethral zone 0.62 +/- 0.31 vs 0.51 +/- 0.24, P > 0.05). In BPH tissues, the expression of type 2 mRNA was significantly higher in the larger prostate tissues (>or= 40 g) than in the smaller ones (e.g. in periurethral zone 1.58 +/- 0.47 vs. 1.14 +/- 0.46, P < 0.05), however, there was no significant difference in the expression of 5alpha-reductase type 1 mRNA between the BPH tissues with different volumes., Conclusion: Type 2 5alpha-reductase plays an important role in the pathogenesis and maintenance of BPH. The role of type 1 expression in the pathogenesis of BPH needs further research.

Objective: To investigate methodology of cloning full-length cDNA from tissues of normal adrenal gland and pheochromocytoma and predict their function., Methods: 104 samples from normal adrenal gland and 22 samples from pheochromocytoma were examined by expressed sequence tags (EST) sequencing, bioinformatics analysis, in silico cloning, rapid amplification of cDNA ends and RT-PCR., Results: Among the 126 samples of novel full-length cDNA cloning 104 were from normal adrenal gland, and 22 from pheochromocytoma (PC). Among the novel genes 50 were cloned by direct sequencing of clones, 74 by in silico cloning, and 2 by rapid amplification of cDNA ends, and the localization of chromosomes was made with UniGene searching and radiation hybrid (RH) procedure. Comparison of homology and analysis of motif or domain showed that some genes may be important functional genes, such as those coding signaling proteins, proteins associated with ion channel, enzymes involved in hormone production, important transcription factors, translation initiation factors, etc. 7 of the novel full-length cDNAs are new isoforms produced by alternative splicing., Conclusion: 126 novel full-length cDNAs have been cloned from adrenal tissues by large-scale EST sequencing and bioinformatic tools in a rather short time, some of which may play an important role in regulating adrenal functions and tumorigenesis of adrenal gland.

Objective: To study the relative tissue distribution and expression pattern of type I and type type II 5alpha-reductase isozymes in prostate cancer tissues., Methods: Immunohistochemistry and RT-PCR method were used to investigate qualitatively and semi-quantitatively the expression of type I and type II 5alpha-reductases in prostate tissues from 15 normal persons and 15 patients with prostate cancer., Results: Two isozymes were detected in both normal and cancerous prostate tissues with the two methods. Both 5alpha-reductases were mainly localized in the cytoplasm, and higher degree of staining was observed in epithelial cells than in stroma. Significantly higher level of type I 5alpha-reduetase expression was observed in cancerous tissue than in normal tissue. The expression level of type I 5alpha-reduetase was positively correlated with the tumor stage and grade, and serum PSA concentration. The expression of type II 5alpha-reductase was very weak in prostate cancer tissue., Conclusion: The type I 5alpha-reductase, not type II, is involved in the pathogenesis of protate cancer. Selective type I 5alpha-reductase inhibitors or dual inhibitors of both type human 5alpha-reductase isoforms can be used in the treatment of prostate cancer.

Objective: To set up a simple method for identifying the mutation of R227Q of SRD5alpha2 gene which is one of the most common mutations in congenital hypospadias in China., Methods: The amplification refractory mutation system(ARMS) was employed in detecting the SRD5alpha2 gene R227Q mutation in congenital hypospadias confirmed by PCR-SSCP and DNA sequencing., Results: The ARMS was successfully applied to the detection of the R227Q mutation of SRD5alpha2 gene. Three out of 23 congenital hypospadias were positive for R227Q mutation. The mutations determined by ARMS were in full agreement with those obtained by the DNA sequencing., Conclusion: This is a simple, rapid and accurate method. It can be used for detecting the SRD5alpha2 gene R227Q mutation in congenital hypospadias and male pseudohermaphroditism.

Objective: To identify possible molecular mechanism underlined hypospadias and any relationship of the mutations of SRD5A2 gene and SRY gene to hypospadias., Methods: Twenty-three blood samples from the patients with hypospadias were obtained from Aug.1996 to Jan. 1998. DNA was extracted from blood leukocytes. Exons 1 to 5 of the SRD5A2 gene and exon of the SRY gene were amplified by PCR. Mutation detection was performed using PCR-SSCP/silver staining and direct DNA sequencing., Results: In 3 cases (named 5R2-China-1, 5R2-China-2, and 5R2-China-3), DNA sequencing revealed that a homozygous change from nucleotide G to A occurred in 5R2-China-1 and 2, leading to a substitution of glutamine to arginine in the codon 227(Arg 227 to Gln). In the third patient (5R2-China-3), DNA sequencing revealed two different heterozygous mutations(Arg 227 to Gln, Phe 186 to Leu) in exon 4 of the SRD5A2 gene. No mutation of SRY gene was found in all patients., Conclusion: The mutation of SRD5A2 gene affects the differentiation of the external genitalia and may play a role in the etiology of hypospadias. Since no mutation of SRY gene was found, this study might suggest that the mutation of SRY gene is not an important event in hypospadias. Codon 227 is a hotspot site of mutation within the gene. The mutation of codon 186(Phe 186Leu) represents a new form of SRD5A2 mutation.