[Karyotyping and analysis of 5α -reductase-2 gene mutation in 25 patients with hypospadias].

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.

[Single nucleotide polymorphisms of the DGKK gene and hypospadias in Chinese children].

OBJECTIVE: To investigate the role of single nucleotide polymorphisms of the gene of diacylglycerol kinase κ (DGKK) in hypospadias in Chinese children. METHODS: We performed direct sequencing on 2 hypospadias-related candidate single nucleotide polymorphisms of the DGKK gene (rs1934179 and rs7063116, never previously reported in the Chinese population) from 300 children with sporadic hypospadias and 200 healthy controls, and compared the results between the two groups. RESULTS: The mutation frequencies of rs1934179 and rs7063116 were 5.0% (15/300) and 5.67% (17/300) respectively in the hypospadias patients, significantly higher than 1.5% (3/200) and 2.0% (4/200) in the normal controls (P <0.05). The mutation frequencies of rs1934179 and rs7063116 in the cases of distal and middle hypospadias were also remarkably higher (6.5%, [13/200] and 7.5% [15/200], P <0.05), but those in the proximal cases (both 2.0% [2/100]) showed no statistically significant difference from the control (P >0.05). CONCLUSION: The polymorphisms of the DGKK gene may be associated with hypospadias, particularly distal and middle hypospadias, in Chinese children.

c-Jun N-terminal kinase is upregulated in patients with hypospadias.

OBJECTIVE: To investigate the expression of c-Jun N-terminal kinase (JNK) 1/2 in human hypospadiac tissue compared with that in normal penile tissue. MATERIALS AND METHODS: Penile skin tissue specimens obtained at surgery during hypospadias repair or during elective circumcision were divided into 3 groups according to the position of the urethral meatus: children with mild hypospadias (n=16), severe hypospadias (n=16), and normal controls (n=16). The expression of total and phosphorylated JNK1 and JNK2 at the messenger ribonucleic acid and protein levels were assessed using real-time quantitative polymerase chain reaction, immunochemistry, and Western blot analysis. RESULTS: JNK1 messenger ribonucleic acid expression and JNK1 and JNK2 phosphorylated protein levels increased significantly in subjects with mild or severe hypospadias compared with the controls (P<.05). JNK2 phosphorylated protein levels increased significantly in those with severe hypospadias compared with those with mild hypospadias (P<.05). Those with hypospadias had increased phosphorylation protein expression of JNK1/2 in the mesenchymal cell layers of the preputial subcutaneous mesenchymal cell layer. CONCLUSION: Our findings suggest that JNK upregulation might contribute to the development of hypospadias and might associated with mesenchymal cell migration in the process of external male genitalia defect development.

Recent progress on type II diacylglycerol kinases: the physiological functions of diacylglycerol kinase δ, η and κ and their involvement in disease.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DAG) to produce phosphatidic acid (PA) and plays an important role in signal transduction by modulating the balance between these signalling lipids. To date, 10 mammalian DGK isozymes have been identified, and these isozymes are subdivided into five groups according to their structural features. The type II DGKs, consisting of δ1, δ2, η1, η2 and κ isoforms, possess a pleckstrin homology (PH) domain at their N-termini in addition to the separate catalytic region. Moreover, DGKs δ1, δ2 and η2 have a sterile α motif domain at their C-termini. Recent studies have revealed that type II DGKs play pivotal roles in a wide variety of mammalian signal transduction pathways for cell proliferation and differentiation and glucose metabolism and that the DGKs are involved in cancer, type II diabetes, seizures, hypospadias and bipolar disorder. This review summarizes the current knowledge on the properties and physiological functions of type II DGKs and their involvement in disease.

Aldosterone synthase deficiency type II with hypospadias.

Aldosterone synthase deficiency (ASD) type II was diagnosed in a 3 week old boy with severe dehydration. Elevated plasma renin activity, low-normal aldosterone, increased levels for 18-OH corticosterone (18-OHB) and 18-OH-deoxycorticosterone were measured. Sequencing revealed a homozygous mutation for c554C > T in exon 3 (p.T185I) (CYP11B2). Hypospadias has so far not been reported in ASD.

SRD5A2 gene mutations--a population-based review.

Knowledge of steroid 5 alpha-reductase type 2 (SRD5A2) gene mutations is expanding, and its role has been implicated in various disease susceptibilities concerning reproductive health. Extensive research has revealed the tendency for specific SRD5A2 gene mutations to be passed along certain racial, ethnic and geographically isolated groups, which suggests population specificity of these mutations. The review provides evidence of variation in the mutational spectrum of the SRD5A2 gene leading to population-specific high prevalence of characteristic disease or phenotypic expression.

Mitochondrial encephalocardio-myopathy with early neonatal onset due to TMEM70 mutation.

OBJECTIVE: Mitochondrial disturbances of energygenerating systems in childhood are a heterogeneous group of disorders. The aim of this multi-site survey was to characterise the natural course of a novel mitochondrial disease with ATP synthase deficiency and mutation in the TMEM70 gene. METHODS: Retrospective clinical data and metabolic profiles were collected and evaluated in 25 patients (14 boys, 11 girls) from seven European countries with a c.317-2A-->G mutation in the TMEM70 gene. RESULTS: Severe muscular hypotonia (in 92% of newborns), apnoic spells (92%), hypertrophic cardiomyopathy (HCMP; 76%) and profound lactic acidosis (lactate 5-36 mmol/l; 92%) with hyperammonaemia (100-520 micromol/l; 86%) were present from birth. Ten patients died within the first 6 weeks of life. Most patients surviving the neonatal period had persisting muscular hypotonia and developed psychomotor delay. HCMP was non-progressive and even disappeared in some children. Hypospadia was present in 54% of the boys and cryptorchidism in 67%. Increased excretion of lactate and 3-methylglutaconic acid (3-MGC) was observed in all patients. In four surviving patients, life-threatening hyperammonaemia occurred during childhood, triggered by acute gastroenteritis and prolonged fasting. CONCLUSIONS: ATP synthase deficiency with mutation in TMEM70 should be considered in the diagnosis and management of critically ill neonates with early neonatal onset of muscular hypotonia, HCMP and hypospadias in boys accompanied by lactic acidosis, hyperammonaemia and 3-MGC-uria. However, phenotype severity may vary significantly. The disease occurs frequently in the Roma population and molecular-genetic analysis of the TMEM70 gene is sufficient for diagnosis without need of muscle biopsy in affected children.

Steroid 5alpha-reductase 1 polymorphisms and testosterone/dihydrotestosterone ratio in male patients with hypospadias.

BACKGROUND/AIM: Defects in the steroid 5alpha-reductase type 2 (SRD5A2) activity cause decreased formation of dihydrotestosterone (DHT) from testosterone (T), resulting in defective masculinization of external genitalia; the T/DHT ratio is increased. We investigated 10 patients with elevated T/DHT ratios in whom mutations in the SRD5A2 and AR genes had been excluded to find out whether structural alterations of the SRD5A1 gene could contribute to their genital malformations. METHODS: Single-strand conformation polymorphism analysis and direct sequencing were used to detect variations in the SRD5A1 gene of the patients and of 49 adult fertile men who served as controls. RESULTS: The sequence analysis of exon 3 of the SRD5A1 gene indicated an adenine-to-guanine change (ACA vs. ACG), both triplets encoding the amino acid residue threonine. The ACG sequence was detected in 57% of all subjects and was equally distributed in patients and controls. The T/DHT ratio was significantly higher in controls with the ACG variant as compared with those having the ACA variant. However, no particular sequence aberration was found in the SRD5A1 genes of either group. CONCLUSION: Mutant SRD5A1 isoenzyme does not seem to play a crucial role in the development of hypospadias.

5alpha-reductase type 2 mutations are present in some boys with isolated hypospadias.

PURPOSE: We determined whether 5a-reductase type 2 mutations are present in boys with isolated hypospadias. MATERIALS AND METHODS: Penile skin tissues obtained at surgery during hypospadias repair were examined for 5alpha-reductase type 2 mutations by single strand conformational polymorphism and deoxyribonucleic acid sequence analysis. Clinical data, including family history of hypospadias and preoperative position of the urethral meatus, were correlated with the genetic findings. RESULTS: Of the 81 specimens examined 7 (8.6%) involved a mutation in at least 1, 5alpha-reductase type 2 gene, while 2 patients had mutations in both alleles. The mutations identified were A49T, L113V and H231R. The A49T mutation in 5 patients was the most common (71%) and it was generally present in less severe forms of hypospadias. To our knowledge neither the A49T nor the L113V mutation has been previously reported in association with 5alpha-reductase type 2 deficiency and to date they have only been identified in cases of isolated hypospadias. Family history was negative in the 7 patients with 5a-reductase type 2 mutations but positive in 5 without mutations. CONCLUSIONS: Some boys with isolated hypospadias have a mutation in at least 1 gene for 5alpha-reductase type 2. This finding suggests that a partial deficiency of 5alpha-reductase activity and inadequate levels of dihydrotestosterone in the fetal urethra may be sufficient to cause the phenotype of hypospadias without other clinical features of 5alpha-reductase deficiency. Family history may not be reliable for determining which boys with hypospadias are likely to have such mutations.

Defects of the testosterone biosynthetic pathway in boys with hypospadias.

PURPOSE: We determined the incidence of defects in 3 enzymes, namely 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase and 17,20-lyase, on the testosterone biosynthetic pathway in boys with hypospadias. MATERIALS AND METHODS: We evaluated 30 boys with a 46,XY karyotype, fully descended testes and penoscrotal or proximal shaft hypospadias. Serum concentrations of the metabolites mediated by these enzymes were measured, from which the precursor-to-product ratios were calculated. Seven patients underwent adrenocorticotropic hormone stimulation. Findings were compared to previously published data on age matched normal boys. RESULTS: A total of 11 boys had evidence of impaired function of 3beta-hydroxysteroid dehydrogenase alone or in combination with impaired 17,20-lyase or 17alpha-hydroxylase activity. An additional 4 boys had evidence of isolated 17,20-lyase deficiency. Thus, of the 30 boys studied 15 (50%) had evidence of a testosterone biosynthetic defect. The effect of adrenocorticotropic hormone stimulation varied with widening of the precursor-to-product ratios in some boys and narrowing in others. CONCLUSIONS: A high incidence of 3beta-hydroxysteroid dehydrogenase and 17,20-lyase deficiency was found in boys with proximal hypospadias. The response to adrenocorticotropic hormone stimulation suggests that enzymes in the adrenal glands and testes may be affected independently. Our findings support the hypothesis that hypospadias is the result of fetal endocrinopathy.

Molecular genetic analysis and human chorionic gonadotropin stimulation tests in the diagnosis of prepubertal patients with partial 5 alpha-reductase deficiency.

UNLABELLED: Reduced conversion of testosterone (T) to dihydrotestosterone (DHT) results in defective virilization in karyotypic males. Different mutations in the 5 alpha-reductase type 2 gene cause the phenotypic variability of the disease. In this report we describe four prepubertal patients with a predominantly male phenotype who carry homozygous point mutations in the 5 alpha-reductase type 2 gene and address the specific T and DHT response to different human chorionic gonadotropin (hCG) stimulation tests. For molecular genetic analysis, DNA from peripheral blood leucocytes was studied. The coding region of the 5 alpha-reductase type 2 gene was characterized by exon-specific polymerase chain reaction amplification, non-radioactive single strand polymorphism analysis, and direct sequencing. Three different homozygous point mutations (Gly196-Ser, Arg227-Gln and Ala228-Thr) were identified in the patients. In contrast, in the DNA from 100 phenotypically normal males only two heterozygous abnormalities (Ile196-Ile, delta Met157) were characterized. For hormonal studies, T and DHT were measured in serum before and after hCG stimulation employing different protocols. HCG stimulation with 5000 IU/m2 once and prolonged stimulation with seven injections of 1500 IU hCG per single dose every other day were used. CONCLUSION: While abnormal T/DHT ratios were identified with both hCG protocols in the patients, prolonged stimulation lead to higher T values and to higher T/DHT rations, and hence to a better discrimination of pathologic results.

Urinary type IV collagenase: elevated levels are associated with bladder transitional cell carcinoma.

Accumulating experimental evidence has linked the overproduction of extracellular matrix-degrading metalloproteinases with tumor cell invasion. In the present study one member of the metalloproteinase family, type IV collagenase (M(r) 72,000 gelatinase), is shown to be elevated in the urine of patients with transitional cell carcinoma of the bladder. The form of the enzyme in the urine was studied by three independent methods: enzyme-linked immunosorbent assay, Western immunoblotting; and gelatin zymography. Immunoblotting revealed that the enzyme was present as a series of fragments, each retaining the amino terminus of the mature proenzyme. A prominent M(r) 43,000 fragment was associated with the transitional cell carcinoma cases. Zymography demonstrated that multiple enzyme species with gelatinase activity were present in urine and that high-molecular-weight bands of substrate lysis corresponded to complexes between type IV collagenase and tissue inhibitor of metalloproteinases 2. The total amount of type IV collagenase antigen was significantly elevated in the urine of 37 transitional cell carcinoma patients (range, 0-1081 ng/ml; mean, 318.4 +/- 147.3) compared to 19 normal controls (P < or = 0.004) and 17 inflammatory disease controls (P < or = 0.011). Immunohistochemical staining of bladder tumor biopsies verified that the transitional cell carcinoma cells were producing the M(r) 72,000 enzyme. Thus, M(r) 72,000 type IV collagenase, which is present in the urine in many forms including fragments and complexes with inhibitors, may be a useful marker for bladder cancer diagnosis or prognosis.

[A case of pseudo-vaginal, perineoscrotal hypospadia with 5-alpha reductase deficiency]. / Ein Fall von pseudovaginaler, perineoskrotaler Hypospadie mit 5 alpha-Reduktase-Defizienz.

A case of pseudovaginal perineoscrotal hypospadia (PPSH) is presented. This autosomal recessive disorder, also termed incomplete male pseudohermaphroditism type 2, is mostly caused by a deficiency of 5 alpha-reductase, which controls the conversion of testosterone to 5 alpha-dihydrotestosterone. In genital skin fibroblasts, the activity of the 5 alpha-reductase was strongly reduced, compared with a normal male. The 5 alpha-reductase activity in nongenital skin fibroblasts, however, was in the range of the normal male control. For complete diagnostic evaluation of PPSH 5 alpha-reductase activity it should be determined simultaneously in genital and non genital skin fibroblasts. The conversion of T to DHT in genital skin fibroblasts of a patient with testicular feminisation (Tfm) was found to be of the same order of magnitude as in PPSH. This suggests, that the expression of 5 alpha-reductase is androgen-dependent.

Variable expression of 5 alpha-reductase deficiency: presentation with male phenotype in a child of Greek origin.

A male infant with perineal hypospadias and a small phallus bound in chordee is described. Biochemical investigation at age 9 months after hCG stimulation revealed a testosterone to dihydrotestosterone (DHT) ratio of 40, a markedly elevated value suggestive of deficient steroid 5 alpha-reductase activity. The diagnosis of 5 alpha-reductase deficiency was confirmed by elevated urinary 5 beta/5 alpha-steroid metabolite ratios and demonstration of defective 5 alpha-reductase activity in cultured fibroblasts from the patient's scrotum and foreskin. Application of DHT cream to the patient's abdomen raised circulating levels of DHT to the adult male range. Two courses of DHT given nightly for 3 and 4 months resulted in phallic enlargement. Surgical release of the chordee and hypospadias repair have resulted in normal male appearance of the genitalia. This case illustrates the heterogeneity of the 5 alpha-reductase deficiency phenotype.

Male pseudohermaphroditism due to 3 beta-hydroxysteroid dehydrogenase-isomerase deficiency associated with atrial septal defect.

Male pseudohermaphroditism in a 6 month old boy, due to congenital 3 beta-hydroxysteroid dehydrogenase deficiency, associated with atrial septal defect, is reported. At 2 weeks he required therapy for severe dehydration and projectile vomiting. The parents were first cousins and one female sibling had died suddenly at 2 months. The patient presented with melanoderma, perineal hypospadias with testicles in a bifid scrotum and atrial septal defect (ostium secundum). Complete cytogenetic studies showed a 46,XY karyotype. Serum sodium ranged from 129 to 140 mEq/l and serum potassium from 5.1 to 4.6 mEq/l. Basal plasma hormonal studies showed normal androstenedione (delta 4A), decreased cortisol (F), slightly elevated ACTH, 17-hydroxy-progesterone (17-OH-P) and testosterone (T), and highly increased dehydroepiandrosterone-sulphate (DHEA-S) levels. ACTH stimulation increased and DXM suppression decreased the plasma levels of DHEA-S, 17-OH-P and T but not that of F; hCG stimulation during cortisone therapy did not change the levels of DHEA-S and T. Corticosteroid therapy normalized electrolyte levels and reduced melanoderma and hormonal hypersecretion. Moderately elevated plasma levels of 17-OH-P and T suggest a partial testicular 3 beta-HSD deficiency. The multifactorial inheritance and the relatively high prevalence of atrial septal defect vs the rarity of adrenal enzymatic defect suggest a causal association even if a common genetic factor cannot be excluded.

An improved method for evaluating testosterone biosynthetic defects.

A double-label, double-substrate incubation technique has been developed and used to study the conversion of progesterone to testosterone in testes extracts from incompletely virilized males. The procedure involves separation of the microsomes from a testicular homogenate, incubating the microsomes with 1 microM [7-3H] progesterone, 1 microM 17-hydroxy[4-14C]progesterone, and 0.25 mM NADPH in pH 7.4 phosphate buffer at 37 degrees C. Steroid precursors and products are separated by column chromatography on Sephadex LH-20 with a solvent system of isoctane:ethyl acetate:methanol (4:1:1 by volume). These procedures can be completed in 2 days, and thus the method represents an improvement in time, reproducibility, and simplicity when compared to techniques based on thin layer or paper chromatography. The method has been used to distinguish the biochemical abnormality in three cases with XY sex chromatin, posterior labial fusion, clitoromegaly, and hypospadias. The abnormalities identified were: Case 1, no defect in testosterone synthesis (probable androgen insensitivity); Case 2, 17-ketosteroid reductase deficiency; and Case 3, steroid-17, 20-lyase deficiency.

Pseudovaginal perineoscrotal hypospadias: genetic heterogeneity.

Defects in either the production or the action of androgenic steroids have been demonstrated to cause pseudovaginal perineoscrotal hypospadias, a syndrome of male hermaphoditism. The ability to make a rapid, specific biochemical diagnosis in an infant with this syndrome, and thus to predict the infant's response to androgens, could be a valuable aid in the assignment of sex to an infant with ambiguous external genitalia.

Reduction of androstenedione by skin in vitro and serum levels of gonadotrophins and androgens in men with hypospadias.

The reduction of 4-[1,2-3H]androstene-3,17-dione (androstenedione) in vitro by scrotal skin was measured in samples from nine men (16--34 years old) with hypospadias and from ten male control subjects. The reduction of androstenedione was also studied in axillary and upper arm skin of seven control subjects. Androstenedione was reduced to material with chromatographic characteristics of 5 alpha-androstane-3,17-dione and to 3 alpha- and 3 beta-hydroxy-5 alpha-androstan-17-one. No difference in 5 alpha-reductase activity (defined as the sum of these three metabolites formed) was found in scrotal skin from hypospadic and control men. The mean concentration of 5 alpha-dihydrotestosterone in serum from men with hypospadias was lower than that in serum from control subjects (P less than 0.01). The mean ratio of the serum concentrations of testosterone and 5 alpha-dihydrotestosterone was higher in hypospadic men than in control subjects (P less than 0.05). No differences between the two groups were found in the mean serum concentrations of LH, FSH, prolactin, dehydroepiandrosterone, androstenedione, testosterone or testosterone-binding globulin.