Phenotypic diversity and testosterone-induced normalization of mutant L712F androgen receptor function in a kindred with androgen insensitivity.

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.

Nonisotopic single strand conformation analysis of the 5 alpha-reductase type 2 gene for the diagnosis of 5 alpha-reductase deficiency.

5 alpha-Reductase deficiency is a rare autosomal recessive disorder of defective virilization in karyotypic males due to reduced conversion of testosterone to dihydrotestosterone. The gene encoding the affected 5 alpha-reductase type 2 enzyme has recently been cloned, and mutations within the coding region have been discovered as the cause of this disease. We address the possibility of a rapid nonradioactive molecular genetic screening technique for initial diagnosis and report different point mutations in this gene in eight unrelated patients with clinical features of 5 alpha-reductase deficiency. For molecular genetic analysis, DNA from peripheral blood leukocytes was studied. The coding region of the 5 alpha-reductase type 2 gene was characterized by exon-specific PCR amplification, nonradioactive single strand conformation analysis, and direct sequencing. In seven patients, homozygous point mutations were identified (Leu55-Gln, delta Met157, Gly196-Ser, Arg227-Gln, Ala228-Thr, and His231-Arg). One individual was a compound heterozygote carrier of two mutations (Ile112-Asn and Gln126-Arg). We conclude that molecular genetic characterization of point mutations in the 5 alpha-reductase type 2 gene may be used as an additional valuable procedure for the diagnosis of this disorder.

Significance of mutations in the androgen receptor gene in males with idiopathic infertility.

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.

Single strand conformation polymorphism analysis of androgen receptor gene mutations in patients with androgen insensitivity syndromes: application for diagnosis, genetic counseling, and therapy.

Recent studies indicate that mutations in the androgen receptor gene are associated with androgen insensitivity syndromes, a heterogeneous group of related disorders involving defective sexual differentiation in karyotypic males. In this report, we address the possibility of rapid mutational analysis of the androgen receptor gene for initial diagnosis, genetic counseling, and molecular subclassification of affected patients and their families. DNA from peripheral blood leukocytes of six patients from five families with various degrees of androgen insensitivity was studied. Exons 2 to 8 of the androgen receptor gene were analyzed using a combination of single strand conformation polymorphism analysis and direct DNA sequencing. Female family members were also studied to identify heterozygote carriers. Point mutations in the AR gene were identified in all six patients, and all mutations caused amino acid substitutions. One patient with incomplete androgen insensitivity was a mosaic for the mutation. Four of the five mothers, as well as a young sister of one patient, were carriers of the mutation present in the affected child. Our data show that new mutations may occur in the androgen receptor gene leading to sporadic androgen insensitivity syndrome. Molecular genetic characterization of the variant allele can serve as a primary tool for diagnosis and subsequent therapy, and can provide a basis for distinguishing heterozygous carriers in familial androgen resistance. The identification of carriers is of substantial clinical importance for genetic counseling.

The role of androgen receptor gene mutations in male breast carcinoma.

Certain germline mutations (607Arg-Gln, 608Arg-Lys) in the androgen receptor gene have been associated with the occurrence of breast cancer in males suffering from partial androgen insensitivity. To assess whether somatic mutations in this gene could be detected in breast carcinoma, archival tumor tissue of males without clinical evidence of androgen insensitivity was screened for point mutations in the androgen receptor gene. DNA was retrieved by chloroform-phenol extraction from formalin-fixed, paraffin-embedded tissues. Exons 2-8 of the androgen receptor gene, encoding the DNA- and hormone-binding regions of the receptor, were amplified by polymerase chain reaction and subjected to nonisotopic single strand conformation assay (SSCA) to screen for point mutations. In the tumor DNA, no variations suggestive of mutations were encountered on SSCA. However, in a control patient with partial androgen insensitivity and predominantly female phenotype, the germline mutation 607Arg-Gln was identified in blood leukocyte DNA. Our results indicate that somatic mutations of the androgen receptor are not required for the development of male breast cancer. This, however, does not exclude an increased risk of breast carcinoma in patients with androgen insensitivity.

Mosaicism due to a somatic mutation of the androgen receptor gene determines phenotype in androgen insensitivity syndrome.

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.

The clinical and molecular spectrum of androgen insensitivity syndromes.

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.

Differential display RT PCR of total RNA from human foreskin fibroblasts for investigation of androgen-dependent gene expression.

Male sexual differentiation is a process that involves androgen action via the androgen receptor. Defects in the androgen receptor, many resulting from point mutations in the androgen receptor gene, lead to varying degrees of impaired masculinization in chromosomally male individuals. To date no specific androgen regulated morphogens involved in this process have been identified and no marker genes are known that would help to predict further virilization in infants with partial androgen insensitivity. In the present study we first show data on androgen regulated gene expression investigated by differential display reverse transcription PCR (dd RT PCR) on total RNA from human neonatal genital skin fibroblasts cultured in the presence or absence of 100 nM testosterone. Using three different primer combinations, 54 cDNAs appeared to be regulated by androgens. Most of these sequences show the characteristics of expressed mRNAs but showed no homology to sequences in the database. However 15 clones with significant homology to previously cloned sequences were identified. Seven cDNAs appear to be induced by androgen withdrawal. Of these, five are similar to ETS (expression tagged sequences) from unknown genes; the other two show significant homology to the cDNAs of ubiquitin and human guanylate binding protein 2 (GBP-2). In addition, we have identified 8 cDNA clones which show homologies to other sequences in the database and appear to be upregulated in the presence of testosterone. Four of these clones again are similar to ETS from unknown genes. Three differential expressed sequences that appear to be upregulated in the presence of testosterone show significant homology to the cDNAs of L-plastin and one to the cDNA of testican. This latter gene codes for a proteoglycan involved in cell social behavior and therefore of special interest in this context. The results of this study are of interest in further investigation of normal and disturbed androgen-dependent gene expression.

Phenotypic classification of male pseudohermaphroditism due to steroid 5 alpha-reductase 2 deficiency.

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.

Applicability of the SHBG androgen sensitivity test in the differential diagnosis of 46,XY gonadal dysgenesis, true hermaphroditism, and androgen insensitivity syndrome.

The sex hormone-binding globulin (SHBG) androgen sensitivity test has been used as a simple method to assess androgen receptor function in vivo. After a short term oral administration of the anabolic-androgenic steroid stanozolol the mean nadir serum concentration of SHBG is used as a measure of androgen response. We performed this test in order to evaluate its applicability in 16 patients with intersexual genital status: eleven with 46,XY gonadal dysgenesis and three with true hermaphroditism (group I), and in two patients with androgen insensitivity syndrome (AIS, group II). Ten healthy adult volunteers served as controls. In the two patients with AIS (group II) we found a diminished decrease of serum SHBG to 80.1 % and 80.7 %, respectively, indicating slight residual androgen responsiveness. In eleven patients of group I who were not on hormone replacement therapy, a mean nadir level of 51.7 +/- 8.7 % was found. In the controls the mean nadir serum SHBG level was significantly higher (62.7 +/- 5.2 %), probably due to interference of endogenous androgens and contraceptive medication with the stanozolol-induced SHBG decrease. In three gonadectomised patients who were on hormone replacement therapy the initial SHBG concentration was increased (513.5 +/- 239.1 nmol/l); the mean nadir SHBG concentration of 45.6 +/- 9.8 % of the initial level indicates an increased sensitivity of the test in patients in whom the counteracting ovarian androgens are absent. Our findings confirm that under standard test conditions the SHBG androgen sensitivity test is a simple diagnostic tool for the detection of androgen receptor malfunction.

Diagnosis of 5alpha-reductase deficiency in a teenage Turkish girl.

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.

[Non-radioactive SSCP for the detection of androgen receptor gene mutation--a diagnostic tool for androgen resistance]. / Nicht-radioaktive SSCP zum Nachweis von Androgenrezeptor Genmutationen--Ein Diagnostikum bei Androgenresistenz.

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.

Detection of point mutations in the androgen receptor gene using non-isotopic single strand conformation polymorphism analysis. German Collaborative Intersex Study Group.

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.

Functional assessment and clinical classification of androgen sensitivity in patients with mutations of the androgen receptor gene. German Collaborative Intersex Study Group.

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.

Intracellular sex hormone-binding globulin (SHBG) in normal and neoplastic breast tissue--an additional marker for hormone dependency?

Recent studies indicate that in addition to free diffusion, uptake of sex hormones into target cells is mediated by sex hormone-binding globulin (SHBG). The purpose of this study was to investigate localization and distribution of SHBG in normal and neoplastic breast tissue. We examined 31 normal, 21 non-invasive, 52 invasive breast cancer tissues and 33 cases of recurrences and metastases of breast cancer immunohistochemically for SHBG by the ABC-peroxidase method, using a polyclonal, monospecific antiserum derived from rabbit. The proportion of stained cells was evaluated semiquantitatively. In 81 malignant cases the oestrogen receptor (ER) content was evaluated by the ER-ICA method. Positive staining for SHBG was found exclusively in epithelial cell cytoplasm. Benign tissue was focally SHBG-positive and showed more stained cells in proliferating epithelium. Staining of neoplastic tissue was more heterogeneous. Half of the non-invasive carcinomas were SHBG-positive; particularly the highly differentiated. Independent of subtype and differentiation, invasive tumours were SHBG-negative in 32.5% of cases, while 19.3% were SHBG-positive in most cells. In 13 cases of invasive carcinomas, associated intraductal parts showed more staining for SHBG than the invasive tissue. Recurrences and metastases of breast cancer were SHBG-negative in 45.5% of cases, while only 3% were positive in most cells. SHBG-staining was unrelated to ER content. These results suggest that the demonstration of cytoplasmic SHBG represents a physiological feature of breast epithelium and its presence is compatible with a mechanism for cellular uptake of SHBG-bound sex hormones preceding their interaction with nuclear receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Inherited and de novo androgen receptor gene mutations: investigation of single-case families.

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.

Etiologic classification of severe hypospadias: implications for prognosis and management.

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.

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.

Biochemical and biophysical alterations of the 7S and NC1 domain of collagen IV from human diabetic kidneys.

Glycation of basement membrane collagen IV has been implicated as a major pathogenetic process leading to diabetic microvascular complications. To evaluate the relevance of carbohydrate-induced modifications on collagen IV in diabetic nephropathy, we isolated the cross-linking domains 7S and NC1 from the glomerular basement membrane (GBM) of patients with diabetes mellitus. Modifications characteristic for glycated proteins were identified when the domains from diabetic kidney were compared with the same domains from human placenta as an unmodified control. In both domains a marked formation of inter-and intramolecular cross links could be demonstrated by SDS-PAGE. Furthermore circular dichroism studies showed a decrease in helicity of the 7S domain from human diabetic kidneys of 13%, indicating denaturation already at room temperature. Thermal transition profiles, showing a shift of the denaturation temperature towards a lower temperature, with loss of a distinct second melting point, confirmed this observation. Our data provide further evidence for a possible role of protein-modification by glycoxidative reactions in the onset of diabetic nephropathy in vivo.

Expression of two functionally different androgen receptors in a patient with androgen insensitivity.

Recently, we demonstrated a previously unknown high rate of de novo mutations of the androgen receptor (AR) gene in androgen insensitivity syndrome (AIS) with some resulting in somatic mosaicism of mutant and wild type AR alleles. However, data on the genotype-phenotype relationship in the latter patients are sparse. We present here a 46,XY newborn with ambiguous genitalia carrying a mosaic of an 866 GTG (Val) --> ATG (Met) mutation with the wild type AR gene. This mutation has usually been associated with complete AIS. Accordingly, we found markedly impaired transactivation due to the mutant Met866 AR. Essential information arose from Scatchard analysis of methyltrienolone binding on cultured genital skin fibroblasts. We demonstrated for the first time the expression of two functionally different ARs (Kd1: 5.58 nM = mutant, Kd2: 0.06 nM = wild type) in one AIS individual. This finding not only represents an important confirmation for the presence of the somatic mosaicism in the patient, it also indicates the most likely molecular mechanism responsible for the unexpectedly strong virilization of the patient: Androgen action through the wild type AR expressed by part of the somatic cells. The present case clearly demonstrates the molecular mechanism by which somatic mosaicism of the androgen receptor gene can modulate in vivo androgen action. It underlines the importance of particular notice on somatic mosaicism in all androgen insensitivity syndrome patients carrying de novo mutations of the androgen receptor gene.