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Unlabelled: In the genetic male, mutations of the androgen receptor (AR) gene cause phenotypes ranging from female to subfertile male. Binding assays on genital skin fibroblasts and DNA analysis alone provide incomplete information about receptor function. We used the sex hormone-binding globulin (SHBG) response to stanozolol as a measure of AR function and correlated the results with phenotypes which were classified according to the degree of defective masculinization. Of the 34 patients investigated, 9 had complete, and 14 had partial androgen insensitivity syndrome (AIS) with predominantly female, ambiguous, or predominantly male phenotype. Eleven subjects served as controls. Mutations were characterized using polymerase chain reaction-single strand conformation polymorphism analysis and direct DNA sequencing. DNA analysis revealed two major deletions, two minor defects leading to premature stop codons in exon 1, and 19 point mutations in the DNA- and hormone-binding domains of the AR gene. After stanozolol, SHBG remained unchanged in patients with complete AIS (102.0 +/- 3.8 [SE]%; range 92.4%-129% of the initial value). The SHBG decrease was diminished in partial AIS with predominantly female (83.8% +/- 1.7%; range 81.3%-87.0%), ambiguous (80.4% +/- 4.4%, range 68.4%-89.1%), and predominantly male (mean 65.9% +/- 4.9%, range 48.6%-80.8%) phenotypes, and normal in controls (51.4% +/- 2.1%, range 35.6%-62.1%). Differences between controls and each AIS group were statistically significant (P < 0.05 - < 0.0001). A close correlation was found between the degree of undermasculinization (AIS phenotype) and the SHBG response.Conclusions: The SHBG test provides functional information about the severity of the receptor defect in vivo and hence adds to the structural information provided by DNA analysis. It detects receptor defects due to mutations within the entire gene, including the DNA-binding domain, and is a rapid, simple, and cost effective procedure. It may provide useful information for the diagnosis and management of affected children. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Molecular causes of phenotypic diversity in androgen insensitivity syndrome, occurring even in the same family, have rarely been identified. We report on a family with four affected individuals, three brothers (B1-3) and their uncle, displaying strikingly different external genitalia: B1, ambiguous; B2, severe micropenis; B3, slight micropenis; and uncle, micropenis and penoscrotal hypospadias. All had been assigned a male gender. We detected the same L712F mutation of the androgen receptor (AR) gene in each subject. Methyltrienolone binding on cultured genital skin fibroblasts of B2 suggested moderate impairment of the ligand-binding domain [maximal binding capacity, 38.2 fmol/mg protein (normal); Kd, 0.21 nmol/L; normal range, 0.03-0.13 nmol/L]. In trans-activation assays, the mutant 712F-AR showed considerable deficiency at low concentrations of testosterone (0.01-0.1 nmol/L) or dihydrotestosterone (0.01 nmol/L). Remarkably, this could be fully neutralized by testosterone concentrations greater than 1.0 nmol/L. Hence, the 712F-AR could switch its function from subnormal to normal within the physiological concentration range of testosterone. This was reflected by an excellent response to testosterone therapy in B1, B2, and the uncle. Taking into account the well documented individual and time-dependent variation in testosterone concentration in early fetal development, our observations clearly illustrate the potential impact of varying ligand concentrations for distinct cases of phenotypic variability in androgen insensitivity syndrome.

Abnormal human spermatogenesis is caused by a variety of genetic and acquired conditions. Because spermatogenesis is dependent on androgens, some males may have a minimal form of androgen insensitivity that does not inhibit virilization but impairs fertility. This has lead us to investigate the possibility of abnormalities in the androgen receptor (AR) gene in a large cohort of males suffering from infertility of unknown cause. We studied 180 males with variable impairment of spermatogenesis. In all patients, serum levels of testosterone and gonadotropins were analyzed to define an androgen sensitivity index (ASI). Single-strand conformation analysis and direct DNA sequencing of PCR-amplified blood leukocyte DNA were used to identify mutations within the whole coding region of the AR-gene. Endocrine and molecular investigations were compared with 53 normal males with proven fertility. In three infertile males, mutations in the AR were identified. Two unrelated males had the same variation within the first exon encoding for the transactivation domain of the receptor (Pro390Ser), whereas, in the third, a mutation in the hormone-binding region was characterized (Gln798Glu). All identified mutation carriers had a significantly elevated ASI. A proportion of males with idiopathic infertility carry relevant variations within the AR-gene. These males may be distinguished on the basis of hormone levels, calculating the ASI, although this index lacks specificity.

We recently found that postzygotic de novo mutations occur at the expected high rate of an X-linked recessive mutation in androgen insensitivity syndrome. The resulting somatic mosaicism can be an important molecular determinant of in vivo androgen action caused by expression of the wild-type androgen receptor (AR). However, the clinical relevance of this previously underestimated genetic condition in androgen insensitivity syndrome has not been investigated in detail as yet. Here, we present the clinical and molecular spectrum of somatic mosaicism considering all five patients with mosaic androgen insensitivity syndrome, whom we have identified since 1993: Patient 1 (predominantly female, clitoromegaly), 172 TTA(Leu)/TGA(Stop); patient 2 (ambiguous), 596 GCC(Ala)/ACC(Thr); patient 3 (ambiguous), 733 CAG(Gln)/ CAT(His); patient 4 (completely female), 774 CGC(Arg)/TGC (Cys); and patient 5 (ambiguous), 866 GTG(Val)/ATG(Met). Serum sex hormone binding globulin response to stanozolol, usually correlating well with in vivo AR function, was inconclusive for assessment of the phenotypes in all tested mosaic individuals. An unexpectedly strong virilization occurred in patients 1, 3, and 5 compared with phenotypes as published with corresponding inherited mutations and compared with the markedly impaired transactivation caused by the mutant ARs in cotransfection experiments. Only the prepubertal virilization of patients 2 and 4 matched appropriately with transactivation studies (patient 4) or the literature (patients 2 and 4). However, partial pubertal virilization in patient 4 caused by increasing serum androgens and subsequent activation of the wild-type AR could not be excluded. We conclude that somatic mosaicism is of particular clinical relevance in androgen insensitivity syndrome. The possibility of functionally relevant expression of the wild-type AR needs to be considered in all mosaic individuals, and treatment should be adjusted accordingly.

Objective: The objective of this study was to assess somatic and inherited androgen receptor gene mutations in families with only one affected individual., Methods: Molecular genetic analysis of the androgen receptor gene in DNA derived from blood leukocytes from 30 families with single-strand conformation analysis, direct sequencing, and restriction fragment analysis was performed., Results: In 22 families the mothers and all investigated grandmothers were heterozygous carriers. However, within the sisters and aunts, both heterozygous carriers and noncarriers were present. In eight families a de novo mutation was characterized. In three of these patients indication for somatic mosaicism was found., Conclusions: De novo mutations occur at a high rate within the androgen receptor gene (8 of 30 = 26.7%); a high proportion (3 of 8) arise after the zygote stage. Thus only direct analysis of the underlying mutation of the androgen receptor gene in the proband and his or her family can provide the basis for genetic counseling.

Deficiency of 5alpha-reductase type 2 activity causes deficient masculinization of 46,XY individuals caused by a lack of dihydrotestosterone. At puberty, virilization is often observed. A precise diagnosis with correct gender assignment at an early age is very important. Recently, the molecular basis of the enzyme defect was discovered; however, only a few cases of 5alpha-reductase deficiency with a complete molecular genetic analysis have been published. We report on a Turkish patient clinically classified with steroid 5alpha-reductase deficiency (SRD) type 3b (karyotype 46,XY) who was raised as a girl and presented to us at the age of 14 years because the male phenotype had become predominant at puberty. Endocrinological investigations revealed an elevated serum testosterone/dihydrotestosterone ratio (17.3, normal: <16). PCR-SSCP analyses detected a deletion of methionine on exon 3 of the 5alpha-reductase type 2 gene.

Premature stop codons of the human androgen receptor (AR) gene are usually associated with a complete androgen insensitivity syndrome. We, however, identified an adult patient with a 46,XY karyotype carrying a premature stop codon in exon 1 of the AR gene presenting with signs of partial virilization: pubic hair Tanner stage 4 and clitoral enlargement. No other family members were affected. A point mutation at codon position 172 of the AR gene was detected that replaced the original TTA (Leu) with a premature stop codon TGA (opal). Careful examination of the sequencing gel, however, also identified a wild-type allele, indicating a mosaicism. In addition, elimination of the unique AflII recognition site induced by the mutation was incomplete, thus confirming the coexistence of mutant and wild-type AR alleles in the patient. Normal R1881 binding and a normal 110/112-kDa AR doublet in Western immunoblots consolidated the molecular genetic data by demonstrating the expression of the wild-type AR in the patient's genital skin fibroblasts. Transfection analysis revealed that only relatively high plasmid concentrations carrying the mutated AR complementary DNA lead to expression of a shortened AR due to downstream reinitiation at methionine 189. Thus, reinitiation does not play a role in the presentation of the phenotype; rather, the partial virilization is caused by the expression of the wild-type AR due to a somatic mosaic. We conclude that somatic mosaicism of the AR gene can represent a substantial factor for the individual phenotype by shifting it to a higher degree of virilization than expected from the genotype of the mutant allele alone.

Objective: Classification of severe hypospadias employing a broad array of diagnostic tools. Standardization of a diagnostic approach to children with hypospadias. Indentification of patients at risk of having malignancies and endocrine problems., Design: Retrospective analysis of patients in a single-center study., Subjects: Thirty-three patients with severe (scrotal or penoscrotal) hypospadias, aged 1 to 18 years., Methods: Clinical assessment, ultrasonography, karyotyping, endocrine evaluation including adrenal steroid concentrations, sex hormone-binding globulin test for androgen sensitivity, human chorionic gonadotropin stimulation with determination of testosterone and dihydrotestosterone concentrations to exclude 5 alpha-reductase deficiency, and molecular genetic analysis of the androgen receptor gene and the 5 alpha-reductase gene., Results: In 12 patients the cause was clarified. Diagnoses included Drash syndrome with Wilms tumor in infancy (3 patients), partial androgen insensitivity resulting from androgen receptor mutations (2), true hermaphroditism (2), chromosomal aberration (1), deficiency of antimüllerian hormone (1), gonadal dysgenesis (1), partial 5 alpha-reductase deficiency caused by a novel point mutation (1), and XX-male syndrome (1). Twelve patients had associated findings such as cardiac malformations (3 patients), rectal atresia (1), dilation of urinary tract (2), cystinuria (1), and others., Conclusions: Patients with severe hypospadias should be submitted to a standardized set of diagnostic procedures in infancy. A stepwise diagnostic study avoids unnecessary, invasive, and expensive testing. A high proportion of classified causes can be expected. Patients at risk of having malignancies or hormonal disorders must remain under close surveillance.

Non-enzymic interactions of carbohydrates and proteins are a major feature of cumulative modification in basement membranes in the course of diabetic microvascular complications. To evaluate the significance of both glycation and glycoxidation reactions for subsequent alterations of biochemical properties, we examined the effects of in vitro glycation on distinct collagen IV domains under different experimental conditions. The 7 S domain and the major triple-helical domain from human placental collagen IV were incubated for various time intervals up to 14 days at 37 degrees C in the presence of different concentrations of either glucose or ribose under oxidative and antioxidative conditions. Carbohydrate-induced non-enzymic modification in two collagen IV domains was revealed by increased cross-linking and fluorescence. In addition, these non-enzymic modifications apparently have a major impact on molecular conformation and thermal stability of collagen IV, which in turn might influence both cell-matrix interactions and matrix assembly.

Androgen insensitivity syndromes (AIS) are due to end-organ resistance to androgenic steroids in males leading to defective virilization of the external genitalia. The phenotype encompasses a wide array of genital ambiguity and may range from completely female to undervirilized but unequivocally male with infertility. This disorder is caused by mutations of the androgen receptor and is an X-linked recessive trait. We have studied 47 patients with AIS and have characterized the underlying molecular abnormality in the androgen receptor gene. Twenty patients had complete AIS and twenty-seven had partial AIS. Of the latter, 11 were of predominantly female phenotypic appearance and gender was assigned accordingly, while 16 were raised as males. Within the group of complete AIS, two patients had gross deletions within the gene, one had a small deletion, and one had an insertion. In the other patients with complete AIS, as well as all individuals with partial AIS, single nucleotide substitutions within the coding region were detected, each leading to an amino acid alteration. Seven codons were involved in more than one mutation in different cases. In addition, in one patient with spinal and bulbar muscular atrophy, an elongation of a glutamine-repeat was characterized. We conclude that mutations in the androgen receptor gene may be present throughout the whole coding region. However, our study provides evidence that several mutational hot spots exist.

Conversion of testosterone (T) to dihydrotestosterone (DHT) in genital tissue is catalysed by the enzyme 5 alpha-reductase 2, which is encoded by the SRD5A2 gene. The potent androgen DHT is required for full masculinization of the external genitalia. Mutations of the SRD5A2 gene inhibit enzyme activity, diminish DHT formation, and hence cause masculinization defects of varying degree. The classical syndrome, formerly described as pseudovaginal perineoscrotal hypospadias, is characterized by a predominantly female phenotype at birth and significant virilization without gynecomastia at puberty. We investigated nine patients with steroid 5 alpha-reductase 2 deficiency (SRD). Phenotypes, which were classified according to the severity of the masculinization defect, varied between completely female (SRD type 5), predominantly female (SRD type 4), ambiguous (SRD type 3), predominantly male with micropenis and hypospadias (SRD type 2), and completely male without overt signs of undermasculinization (SRD type 1). T/DHT-ratios were highly increased ( > 50) in the classical syndrome (SRD type 5), but variable in the less severe affected patients (SRD types 1-4) (14-35). Mutations in the SRD5A2 gene had been characterized using PCR-SSCP analysis and direct DNA sequencing. A small deletion was encountered in two patients, while all other patients had single base mutations which result in amino acid substitutions. We conclude that phenotypes may vary widely in patients with SRD5A2 gene mutations spanning the whole range from completely female to normal male without distinctive clinical signs of the disease. Hence, steroid 5 alpha-reductase deficiency should be considered not only in sex reversed patients with female or ambiguous phenotypes, but also in those with mild symptoms of undermasculinization as encountered in patients with hypospadias and/or micropenis. A classification based on the severity of the masculinization defect may be used for correlation of phenotypes with enzyme activities and genotypes, and for comparisons of phenotypes between different patients as the basis for clinical decisions to be made in patients with pseudohermaphroditism due to steroid 5 alpha-reductase 2 deficiency.

Point mutations in the androgen receptor gene cause androgen insensitivity syndromes, clinically characterized by masculinization defects in karyotypic males due to endorgan resistance to androgenic steroids. Characterization of these mutations with single strand conformation polymorphism analysis utilizing radioactive PCR can serve as a diagnostic tool for molecular subclassification of these syndromes. It is the basis for genetic counseling and for therapeutic decisions. Here we report an improved non-radioactive single strand polymorphism analysis for rapid detection of androgen receptor gene mutations in affected individuals. In addition to previously reported mutations, 10 patients with clinical features of androgen resistance were studied. DNA was isolated from peripheral blood leucocytes and exons 1 to 8 of the coding region of the androgen receptor gene amplified by PCR. Amplification products were denatured and run on non-denaturing gels. These were subjected to fixation and silver staining. Variations were directly sequenced. In all patients a different point mutation in one of the exons was detected. While one insertion mutation was found in a patient with complete androgen insensitivity, all other mutations cause amino acid substitutions. These data suggest that the described non-radioactive single strand polymorphism analysis is a useful tool for the characterization of androgen receptor gene mutations. The omission of radioisotopes is advantageous in a clinical setting. The mutations described emphasize the clinical and molecular heterogeneity of this disease.

Point mutations in the androgen receptor gene cause androgen insensitivity syndromes, clinically characterized by masculinization defects in karyotypic males due to endorgan resistance to androgenic steroids. Characterization of these mutations with single strand conformation polymorphism analysis utilizing radioactive PCR can serve as a diagnostic tool for molecular subclassification of these syndromes. It is the basis for genetic counseling and for therapeutic decisions. Here we report an improved non-radioactive single strand polymorphism analysis for rapid detection of androgen receptor gene mutations in affected individuals. In addition to previously reported mutations, 9 patients with clinical features of androgen resistance were studied. While one insertion mutation was detected, in all other patients different point mutations initiating amino acid substitutions were characterized.