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The human prostate is a complex organ composed of four glandular zones that differ in their histology and biology. In addition to this anatomical organization, the prostate is mainly composed of epithelial and mesenchymal tissues. Two pathologies affect the growth of the prostate: cancer and benign prostatic hyperplasia. Anatomical and pathological studies have shown that stromal enlargement is the first step in the process of BPH and that both transition and periurethral regions are the exclusive sites of origin. Because the first step of androgen action is the reduction of testosterone into dihydrotestosterone by the 5 alpha-reductase, it have been postulated that modification of this enzymatic activity may play an important role in BPH development. However DHT production alone cannot explain the hyperplastic development of the gland. Thus it has also been postulated that androgen-growth factor interaction may be an important feature of this growth: these growth factors include the IGF axis. An interaction between androgen pathway and sympathethic system, via alpha-adrenoreceptor may also be suspected.

Finasteride is a potent 5 alpha-reductase inhibitor which has proven useful in the clinical management of benign prostatic hyperplasia. To determine a potential mode of action for finasteride in prostatic cell proliferation, we have studied the incorporation of [3H]-thymidine into the DNA of cultured epithelial and stromal cells from normal and hyperplastic human prostates. The effects of short treatment with 10(-9) M and 10(-6) M finasteride (48 hrs.) on the incorporation of labelled thymidine were studied. A significant effect of finasteride on prostatic cell proliferation was observed at 10(-6) M for both epithelium and stroma from normal prostate: the rate of thymidine incorporation decreased to 80 +/- 3% (p < 0.001) and 55 +/- 10% (p < 0.01), respectively, compared to the control cells. As observed for normal prostates, this rate of thymidine incorporation was less for hyperplastic epithelium (70 +/- 4%, p < 0.001) than that observed for the hyperplastic stroma (74 +/- 4%, p < 0.01). These data clearly demonstrate that the reduction of the prostate volume observed in BPH treatment by finasteride is partly due to an inhibition of cell proliferation. However, the absence of complete inhibition of cell proliferation at 10(-6) M, a concentration known to strongly inhibit the 5 alpha-reductase activity, supports the hypothesis that factors other than DHT are necessary to induce prostatic cell proliferation.

A wide spectrum of androgen receptor (AR) gene mutations has been reported in complete androgen insensitivity syndromes. The molecular basis of androgen resistance was investigated in a female newborn with complete testicular feminization. Sequencing identified a point mutation in exon 4 responsible for a leucine (CTG) to arginine (CGG) replacement at codon 707. This novel mutation is located in the amino-terminal part of the ligand-binding domain of the AR. To determine the functional properties of the mutated AR and to establish the correlation with the clinical phenotype of androgen resistance, the mutation was reproduced in AR wild-type complementary DNA, and the plasmid was transfected into AR-free mammalian cells. In vitro studies showed that the mutant AR was functionally defective as an androgen-binding molecule. Electrophoretic mobility shift assay revealed that the binding of mutated AR to DNA was reduced. Finally, the mutant was unable to induce the transcriptional activation of androgen-responsive reporter gene. This amino acid defect in the primary sequence probably involves the rupture of hydropathicity in a region that is conserved among members of the steroid receptor subfamily. Our data substantiate the major contribution of leucine 707 to normal AR function and demonstrate that its substitution by an arginine caused the complete androgen insensitivity in this patient. Our findings also contribute to the elaboration of the structure-function map of the AR based on naturally occurring mutations.

In two families with complete androgen insensitivity, we have identified naturally occurring point mutations in the human androgen receptor gene that encode amino acid substitutions within the DNA-binding domain. The two amino acid substitutions, a valine to phenylalanine and a leucine to proline, occur at positions 581 and 616, respectively, of the androgen receptor. The mutations were recreated by site-directed mutagenesis. Expression of the mutants androgen receptors in COS 7 and CV 1 cells revealed a normal size 110-kDa receptor protein on Western blots, normal androgen binding capacities and affinities, but absence of binding to target DNA on electrophoretic mobility shift assays. In cotransfection assays, the mutant ARs failed to activate transcription of an androgen-responsive reporter gene. To study the possible structural impact of these mutations, three-dimensional models of the normal androgen receptor and of each mutant were built by homology with the glucocorticoid receptor. Analysis of the models predicts that mutation Val581Phe would affect interaction between the protein and DNA, whereas the Leu616Pro mutation would more likely be involved in destabilizing the protein structure or protein dimerization. Taken together, the experimental investigations and the molecular modeling studies of the mutant androgen receptors observed in families with complete androgen insensitivity syndrome, highlight the role of Val-581 and Leu-616 in receptor structure and function.

Benign prostatic hyperplasia (BPH) is the most common benign proliferative disorder of unknown etiology found in men. Dysregulation of testosterone conversion to dihydrotestosterone (DHT) by 5 alpha-reductase has been described as a key step in the development of BPH. We investigated the 5 alpha-reductase activity in primary cultures of epithelial and stromal cells from the four different regions (periurethral, transition, central and peripheral zones) of normal and hyperplastic human prostates. No significant difference in DHT production was observed among the four epithelial zones and the hyperplastic epithelial cells, or among the four normal zones and the hyperplastic stromal cells. To investigate whether the two known 5 alpha-reductase isoenzymes were expressed in prostate, we performed specific enzymatic amplification after reverse transcription which showed the presence of 274 and 383 base-pair fragments, respectively, specific for 5 alpha-reductase type 1 and 5 alpha-reductase type 2, demonstrating the expression of mRNA for both isoenzymes in BPH tissue. To pharmacologically characterize the isoenzyme present in the two cell components of hyperplastic prostate, we used finasteride, a well known inhibitor specific to 5 alpha-reductase type 2, which only slightly inhibits 5 alpha-reductase type 1. In BPH, IC50 was estimated at 62 nM and 45 nM, for stromal and epithelial cells respectively, suggesting predominately 5 alpha-reductase type 2 activity. Our results provide further evidence that 5 alpha-reductase type 2 activity is mainly responsible for DHT production by human prostatic cells.

Unlabelled: The molecular basis for male pseudohermaphroditism produced by the 5 alpha-reductase deficiency is becoming increasingly understood., Objective: We have performed biochemical and molecular analyses of the 5 alpha-reductase type 2 gene in a Turkish family with a 5 alpha-reductase deficiency., Patient: A 46,XY prepubertal Turkish patient with female phenotype showing clitoral hypertrophy, high plasma testosterone and dihydrotestosterone, and normally differentiated and developed testosterone-dependent internal genitalia., Measurements: 5 alpha-Reductase activity, measured by the conversion of 3H-T into 5 alpha-reduced compounds, was determined from cultured genital skin fibroblasts by both intact monolayer assay and cell-free extracts at various pH values. The five exons of the 5 alpha-reductase type 2 gene were sequenced after enzymatic amplification (PCR) of the patient's genomic DNA. Labelled PCR of the consanguineous parents' DNA was submitted to electrophoresis on a sequencing gel., Results: A marked decrease in the transformation of T into 5 alpha-reduced compounds by intact cells and a diminished 5 alpha-reductase activity at acidic pH by sonicated cell extracts strongly suggested a 5 alpha-reductase type 2 deficiency. Molecular analysis of the 5 alpha-reductase type-2 gene showed a trinucleotide deletion straddling codons 156 and 157, responsible for a methionine residue deletion at position 157 of the protein. The parents' DNA contained both normal and deleted alleles., Conclusions: This is the third deletion described in the 5 alpha-reductase type 2 gene. The deleted methionine 157 is conserved in both types 1 and 2 of human and rat 5 alpha-reductase, which suggests its crucial role in the functioning of the enzyme. This gene rearrangement was thus clearly responsible for the reduced 5 alpha-reductase activity and abnormal genital development in this patient.

The molecular basis of 5 alpha-reductase (5 alpha R) deficiency was investigated in four patients from three European families. In the French family, the first patient was raised as a female, and gonadectomy was performed before puberty. The second sibling, also raised as female, differed in that gonadal removal was performed after the onset of pubertal masculinization. The other two patients, both from Polish families, developed masculinization of external genitalia during puberty. All patients developed a female sexual identity. In all cases, no known consanguinity or family history of 5 alpha R deficiency was reported. The genomic DNAs of the patients were sequenced after polymerase chain reaction amplification of the five exons of the 5 alpha R type 2 gene. We found two homozygous mutations responsible for glutamine to arginine and histidine to arginine substitution in families 1 and 3, respectively. In family 2, we found a heterozygous mutation responsible for an asparagine to serine substitution at position 193. The glutamine/arginine 126 mutation in the French family was previously reported in a Creole ethnic group, and the Polish histidine/arginine 231 mutation was previously reported in a patient from Chicago. Moreover, all of the mutations created new restriction sites, which were used to determine the kindred carrier status in the three families. Because 5 alpha R deficiency is known to be a heterogenous disease in terms of clinical and biochemical expression, our data suggest that molecular biology analysis of the type 2 gene could be an essential step in diagnosing 5 alpha R deficiency.

We studied the androgen receptor gene in a large kindred with complete androgen insensitivity syndrome and negative receptor-binding activity, single-strand conformation polymorphism (SSCP) analysis and sequencing identified a 13 base pair deletion within exon 4. This was responsible for a predictive frameshift in the open reading frame and introduction of a premature stop codon at position 783 instead of 919. The deletion was reproduced in androgen receptor wildtype cDNA and transfected into mammalian cells. Western blot showed a smaller androgen receptor of 94 kDa for the transfected mutated cDNA instead of 110 kDa. Androgen-binding assay of the mutated transfected cells assessed the lack of androgen-binding. Gel retardation assay demonstrated the ability of the mutant to bind target DNA; however, the mutant was unable to transactivate a reporter gene. Although the role of the partial deletion in the lack of androgen action was expected, in vitro analyses highlight the role of the abnormal C-terminal portion in the inhibition of the receptor transregulatory activity of the protein causing androgen resistance in this family.

The various processes involved in sexual differentiation have been considerably clarified over the last few years through advances in biochemistry and molecular genetics. The cloning of the gene responsible for testicular determination SRY, of the anti-Müllerian hormone and anti-Müllerian hormone receptor genes, of the several steroidogenic enzymes genes, of the 5 alpha-reductase type 2 gene and of the androgen receptor gene has permitted to elucidate the molecular defects causing abnormal sexual differentiation. These data have brought a substantial impact on the understanding of human male sexual differentiation and its main disorders.

The androgen receptor belongs to the family of nuclear receptors and contains three functional domains: a carboxy-terminal hormone binding region, a central cystein rich DNA binding region and an amino-terminal region involved in the expression of androgen regulated genes. Cloning of the complementary DNA encoding the androgen receptor enabled the characterization of the molecular defects associated with androgen insensitivity syndromes, X-linked disorders resulting from androgen action defects in target cells. Moreover, androgen receptor gene alterations have been recently described in two unrelated diseases: male breast cancer and spinal and bulbar muscular atrophy. Our group have identified 16 androgen receptor gene alterations in patients with androgen insensitivity syndrome, an amino acid substitution in a patient with a partial androgen insensitivity syndrome and a breast cancer. In 2 families, the molecular diagnosis of spinal and bulbar muscular atrophy has been performed.

In a large family with Reifenstein syndrome, we previously performed molecular analysis of the androgen receptor gene. Direct sequencing showed a G-A point mutation at position 2818 of exon 7, which was responsible for an arginine-histidine substitution at position 840 of the androgen receptor. In this family, the proband's mother became pregnant and wished to know whether she was carrying an unaffected fetus. Polymerase chain reactions of the sex-determining region of the Y chromosome (the SRY gene) on trophoblastic DNA at week 14 revealed a 46,XY genotype. Sequencing analysis showed the canonical sequence (CGT, encoding an Arg residue), suggesting that the fetus was not affected. The expectation of normal male sexual development was confirmed by detection of normal male external genitalia through ultrasonography at week 24. These data confirm that sequence analysis of the androgen receptor gene on trophoblastic DNA is the most reliable method for prenatally diagnosing or excluding androgen insensitivity syndrome in high-risk families.

Objective: Partial androgen insensitivity syndromes are the cause of genital ambiguity that is at times quite severe; there is, therefore, a high demand for prenatal diagnosis in families already afflicted with this syndrome. When the mutation has not been identified, the diagnosis can be made by the study of the polymorphisms of the androgen receptor gene. To perform molecular prenatal diagnosis in a family with partial androgen insensitivity syndrome, we studied the Hind III polymorphism of the androgen receptor gene on the trophoblastic DNA. The use of this restriction fragment length polymorphism tracked maternal X chromosome segregation and established prenatal diagnosis although the mutation had not yet been identified in this family. FAMILY: The mother had been previously described as heterozygous for the Hind III polymorphism and chromosomal segregation analysis showed that the affected allele was associated with the 6.7-kb Hind III fragment., Measurements: Hind III RFLP with an androgen receptor gene cDNA probe was realized on the trophoblastic DNA, along with measurement of androgen binding activity on the trophoblastic cells., Results: We detected the presence of the 6.7-kb fragment in the DNA of the trophoblastic cells suggesting the fetus was affected. Partial androgen insensitivity syndrome was confirmed by a considerable decrease in androgen binding activity on the trophoblastic cells and by sonography of the fetus. After a therapeutic abortion requested by the parents, the diagnosis was confirmed by clinical examination of the fetus, biochemical analyses of the fetal androgen receptor, and molecular studies of the fetal DNA., Conclusions: When the mutation of the androgen receptor gene has not been identified, Hind III polymorphism of the trophoblastic DNA is useful in the prenatal diagnosis of androgen insensitivity syndrome in high-risk families.

Turner syndrome is a complex human phenotype most commonly seen in association with a 45,X karyotype and it has been proposed that the phenotype is the result of monosomy for genes common to the X and Y chromosomes. Detection of unrecognized Y derived material is now possible by PCR of the SRY gene. Its presence is correlated with the presence of testicular tissue, known to increase the risk of developing gonadal neoplasia. Study of Y chromosome allowed the localisation of a candidate gene for the development of gonadoblastoma, GBY. Moreover, some groups described genes on the Y chromosome whose defects seem to be involved in the development of Turner stigmata: ZFY and RPS4Y. In conclusion; molecular genetics of the Y chromosome develops new pathophysiological and fundamental perspectives of the molecular genetics of the Turner syndrome.

We screened thirteen male breast cancers for the presence of germline mutations in exons 2 and 3 encoding the DNA-binding domain of the androgen receptor. These two exons were amplified from genomic DNA extracted from patients' white blood cells. In one of these thirteen patients, single strand conformation polymorphism and direct sequencing detected a guanine-adenine point mutation at nucleotide 2185 that changes Arg608 into Lys in a highly conserved region of the second zinc finger of the androgen receptor. This mutation occurred in a 38 year old man with partial androgen insensitivity syndrome and normal androgen-binding capacity in cultured genital skin fibroblasts. To our knowledge, only one germline Arg to Gln androgen receptor gene mutation has been previously reported at position 607 in male breast cancer. This androgen receptor mutation along with the Arg608 into Lys mutation we describe, suggests that this genetic abnormality is not fortuitous: a decrease in androgen action within the breast cells could account for the development of male breast cancer by the loss of a protective effect of androgens on these cells. Activation of estrogen regulated genes by the change of DNA-binding characteristics of the mutant androgen receptor cannot, however, be ruled out.

Objective: To study the androgen receptor gene in a large kindred with complete androgen insensitivity syndrome and positive receptor-binding activity., Design: Enzymatic amplification coupled with single strand conformation polymorphism and DNA sequencing were used., Results: In all the affected members, single strand conformation polymorphism showed a mobility shift in exon 2, suggesting a sequence alteration. Sequencing identified a valine into phenylalanine substitution at position 581 in the first zinc finger of the DNA-binding domain., Conclusions: This valine 581 is conserved in all nuclear receptors and has an important role for the recognition of the androgen response elements by the activated androgen receptor. This amino acid substitution has not been previously described in the androgen receptor in patients with androgen insensitivity syndrome. Moreover, single strand conformation polymorphism allowed determination of the generation in which the mutation appeared and performance of carrier diagnosis in this family.

The androgen receptor belongs to the family of steroid-thyroid hormone-retinoid nuclear receptors. It contains 3 major domains: a hormone-binding region, a DNA-binding region and an amino-terminal region. Cloning of the cDNA encoding the androgen receptor and elucidation of the androgen receptor gene structure enabled the characterization of the molecular defects associated with androgen insensitivity. Mutations of the androgen receptor in 46,XY individuals cause a spectrum of androgen insensitivity syndromes, ranging from female phenotype (testicular feminization) to minor degrees of undervirilization or infertility. Reports on androgen receptor gene structure in patients with complete or partial forms of androgen insensitivity demonstrate that gene deletions are very rare. Several categories of mutations have been reported and are reviewed in this paper. Nucleotide substitutions in the androgen-binding domain or the N-terminal region that cause insertion of premature termination codons result in failure to form a functional protein. Missense mutations within the androgen-binding domain are responsible for a decrease or absence of receptor-binding activity. Mutations within the DNA-binding domain are associated with a positive receptor-binding form of androgen insensitivity. Analysis of described mutations indicates that they are spread throughout the gene, either associated with partial or complete androgen insensitivity. Furthermore, the same point mutation was reported to be associated with variable phenotypic expression of androgen insensitivity syndrome. It is thus difficult to define a genotype/phenotype relationship. However, mutations causing androgen insensitivity will certainly yield important new insights into the molecular basis of androgen action.

In a French child with complete androgen insensitivity syndrome and negative receptor-binding, no gross deletion has been found. Using single-strand conformation polymorphism assay, a useful screening method for rapid detection of DNA sequence alterations, and direct DNA sequencing, a G-T nucleotide substitution in exon 5 of the androgen receptor gene at nucleotide 2590 was found. This changed codon 743, glycine to valine, in the hormone-binding domain and created a new recognition sequence for the restriction endonuclease Asp HI. Amplification of exon 5 by polymerase chain reaction followed by digestion with Asp HI enabled easy recognition of the described mutation. Since the mother's exon 5 was undigested, we suspected the de novo nature of this nucleotide substitution. This was confirmed by direct sequencing of the mother's DNA which only showed the canonical sequence. To our knowledge, there has been no previous report of a de novo mutation described within the androgen receptor gene in patients with androgen insensitivity syndrome.

Objective: We explored the possibility of a genetic anomaly in the sex determining region of the Y chromosome, SRY gene, known to be equated to the testis determining region., Patients: Four patients with bilateral congenital anorchia, absence of testicular tissue, elevated FSH and a lack of testosterone response to human chorionic gonadotrophin stimulation tests were studied., Measurements: Amplification by polymerase chain reaction of the SRY gene and direct double stranded DNA sequencing were performed with the same primers., Results: The expected 648 basepairs band of SRY was detected in the four DNA samples from patients with bilateral congenital anorchia. Direct sequencing did not show any difference with the previous published sequence., Conclusions: These data suggest that, in the four patients, bilateral congenital anorchia is not related to an anomaly of the opening reading frame sequence of the SRY gene.

In a large kindred with Reifenstein syndrome, we performed the molecular analysis of the androgen receptor gene. Since the biochemical characteristics of the androgen receptor, determined on the cultured genital skin fibroblasts, showed a drastic decrease of the androgen binding capacity, we assumed that a point mutation was located in exons 4-8 encoding the carboxy-terminal domain of the receptor. Enzymatic amplifications of these exons did not point out any deletions. Direct sequencing showed a G-A point mutation at position 2818 of exon 7 responsible for an arginine-histidine substitution at position 840 of the androgen receptor. The presence of the same mutation has been reported by other groups in four unrelated patients. Its association with different phenotypes of androgen insensitivity and different biochemical characteristics of the androgen receptor pointed out the complexity of the genotype-phenotype relationship in androgen insensitivity. Moreover the identification of the point mutation gave us the opportunity to perform a prenatal exclusion diagnosis of Reifenstein syndrome in this high-risk family.

Exon 1 polymorphism of the androgen receptor (AR) gene is characterized by a (CAG)n(CAA) repeat at position 172 following the translation start codon. The aim of this study was to determine whether AR gene exon 1 polymorphism could be used to perform prenatal diagnosis in high risk families with complete or partial androgen insensitivity syndrome. After enzymatic amplification of a 1 kilobase exon 1 fragment, each DNA was simultaneously digested by MspI and PstI restriction enzymes. After electrophoresis on a 15% electrophoresis on a 15% acrylamide gel or a 6% Nusieve gel, we measured the size of the obtained fragments and determined the number of CAG repeats since a 282 basepair fragment corresponds to 21 CAG. We previously showed that the number of CAG repeats within the AR gene exon 1 in 23 families with complete or partial androgen insensitivity syndrome was 19 +/- 4. By this method, we detected heterozygosity in 50% of the mothers. We present here 2 exclusion prenatal diagnoses using exon 1 polymorphism of the AR gene. Family A presented a boy with a severe form of partial androgen insensitivity syndrome. The mother had 2 uncles with ambiguous genitalia. In family B, the affected child had a complete androgen insensitivity syndrome. In both families, analysis of the AR gene exon 1 polymorphism of the trophoblastic DNA showed the presence of the normal maternal X chromosome. The parents decided to carry on the gestation. In family A, the newborn had normal male external genitalia. In family B, sonography confirmed the presence of normal male external genitalia. These data suggest that exon 1 polymorphism of the AR gene could be prenatally used to predict androgen insensitivity syndrome.

In Turner patients, the presence of a Y chromosome or derivative Y is correlated with the risk of gonadoblastoma induction. "Marker" chromosomes originating from Y, may not show characteristic fluorescence and then be very difficult to identify by conventional cytogenetic techniques, although they still predispose the patients to gonadal tumors. Using polymerase chain reaction of the gene from the sex-determining region of the Y chromosome, we screened 40 Turner patients (thirty seven 45X and three 45X,46XX) for the presence of Y chromosomal DNA. We were able to identify karyotypically unrecognized Y chromosome material in 1 patient out of the 40 studied. In this patient mild clinical and biological hyperandrogenism was observed. Reliability of our technique was ascertained by the detection of the expected 648 base pairs amplified DNA fragment in all normal male controls as well as in 3 Turner patients with confirmed 45X,46XY mosaicism. Despite the low frequency of unrecognized Y chromosome material (1 case over 40 in our experience), our data suggest that polymerase chain reaction of the gene from the sex-determining region of the Y chromosome is worthy of being performed in Turner patients considering the potential risk of the presence of a Y chromosome.

In the Frasier syndrome there is an association between XY gonadal dysgenesis and chronic renal failure. Owing to an observed sex reversal, the Y chromosomes of two girls with this syndrome have been analyzed. Using molecular-biology techniques, no major alterations of the known sex-determining area of the Y chromosome were found. Furthermore, the sequence did not reveal impairment of the recently described testis-determining factor SRY. These data suggest that in the Frasier syndrome, XY sex reversal and renal failure could be the result of either faulty gene(s) located downstream in the sex differentiation pathway during embryogenesis, or impaired SRY regulation. Preliminary results on the Wilms' tumor suppressor gene WT1, a candidate for acting downstream to SRY, are also provided.

Analysis of the sexual determination and differentiation factors has progressed by the important findings made in biochemistry (hormonal assays, determination of the receptor concentration), in cellular and molecular endocrinology (mechanism of the hormonal action) and in genetics (identification, isolation and cloning of the genes implied in the sexual determination and differentiation pathways). We first describe the basis of sexual determination and differentiation, and then we analyze the different roles of SRY gene, encoding for the testis determining factor, androgen receptor gene, 5 alpha-reductase gene and anti-Müllerian hormone gene.

Sexual ambiguity can be a difficult and sometimes confusing diagnostic problem in children. Recent developments in molecular biology have provided the opportunity to analyze the gene responsible for testicular determination, SRY, the androgen receptor gene and the gene encoding the cP450 enzyme specific for 21-hydroxylation, CYP21B, whose defects are responsible for congenital adrenal hyperplasia. Southern-blotting studies and PCR analyses of SRY, androgen receptor and CYP21B genes can be routinely used for the direct diagnosis of gonadal dysgenesis, androgen insensitivity syndromes and congenital adrenal hyperplasia, respectively. In sex-reversed XY females, several de novo mutations or deletions in the SRY gene have been reported. Defects in the human androgen receptor cause a spectrum of defects in male phenotypic sexual development associated with abnormalities in the receptor protein. Analyses of the androgen receptor gene structure have identified the causative mutation in some families: mutations that result in large-scale alterations of the structure of the androgen receptor, mRNA or gene mutations that alter the primary structure of the androgen receptor protein and mutations that alter the level of mRNA. The diversity of clinical phenotypes, apparent in 21-hydroxylase deficiency, is paralleled by a considerable degree of mutational heterogeneity in the CYP21 gene locus. Various changes causing severe 21-hydroxylase deficiency have been reported: point mutations, gene conversions and gene deletions. In conclusion, substantial progress has been made elucidating genetic defects causing sex reversal in XY females, the androgen insensitivity syndrome and congenital adrenal hyperplasia. Molecular genetics can also be applied for carrier identification and prenatal diagnosis.

In patients with androgen insensitivity syndrome (AIS), RFLP study of the androgen receptor gene made it possible to analyze whether deletions or mutations could be responsible for abnormalities in androgen responsiveness. We studied RFLPs of DNA from 25 46,XY patients with partial AIS (PAIS), defined as a concentration of androgen receptor in genital-skin fibroblasts less than 340 fmol/mg DNA, and DNA from 27 46,XY patients with complete AIS (CAIS) with no detectable androgen receptor site. DNA samples were digested with BamHI, EcoRI, HindIII and TaqI restriction enzymes and hybridized with three cDNA probes covering the three domains of the androgen receptor. When we had the maternal and an unaffected brother's DNA, we analyzed the two androgen receptor gene polymorphisms described, the HindIII and the exon 1 CAG repeat polymorphisms, in order to distinguish the two maternal X chromosomes, and to detect carriers of AIS. We did not find any large deletion among the 52 patients. We observed a heterozygous mother in 3 of 14 families studied with the HindIII polymorphism, and in 12 of 25 families using the exon 1 CAG repeat polymorphism. This study suggests that in AIS, abnormalities in androgen receptor response could be related to point mutations or microdeletions rather than to gross structural alterations of the androgen receptor gene. Furthermore, unless the point mutation has been described, exon 1 and HindIII polymorphism studies would enable the identification of carriers in 50% of families, and the prenatal diagnosis of AIS.

Partial androgen insensitivity syndrome (PAIS) is an X-linked disorder resulting from defects in the intracellular androgen receptor (AR). The cloning of the AR cDNA has provided the molecular tools to identify gene abnormalities. Gene deletions being the exception in PAIS, prenatal diagnosis of PAIS resulting from a single base mutation in high risk families is not practical unless the mutation is already known. Brown et al. (1989) reported that 10% of normal X chromosomes present a Hind III 6.7/3.5 kb polymorphism. In this study, we report the association of the Hind II polymorphism in a woman whose son has a PAIS associated with a very low androgen receptor concentration: we differentiated the two maternal X chromosomes and characterized the affected allele. These data demonstrate that the presence of Hind III polymorphic fragments could be used in prenatal diagnosis of androgen insensitivity syndrome in high risk families.

Molecular genetic study of androgen insensitivity syndrome is now easier by the development of powerful molecular tools. Complementary DNA of the androgen receptor gene has been recently cloned and sequenced. The development of cDNA probes gave the opportunity to study DNA restriction fragment length polymorphisms of patients with complete or partial androgen insensitivity syndrome. These studies demonstrated that deletions are rarely observed. Using PCR and sequencing of exons, several groups described point mutations within the androgen receptor gene. Enzymatic amplification along with denaturing gradient gel electrophoresis and single strand conformational polymorphism study allowed the description of new mutations. These powerful tools together with mRNA study, expression of muted gene, anti-receptor study had also permitted to analyze correlations between structure-function activity of the androgen receptor gene in patients with androgen insensitivity syndrome.

Androgen insensitivity syndromes are X-linked disorders. Restriction fragment length polymorphism analysis of the androgen receptor gene showed that deletions were infrequent. Some mutations have been described. In these conditions, in high-risk family, carrier diagnosis is impossible unless identification of mutations is made. It is thus necessary to detect androgen receptor gene polymorphism in order to differentiate the two maternal X chromosomes. Two androgen receptor gene polymorphisms have been reported (Hind III and exon 1). In this study we analyzed these two gene polymorphisms to detect carriers in at-risk families. The combined results of the two analyses allowed us to detect carriers in 45% of the studied families. In two families the prenatal diagnosis of androgen insensitivity syndrome was performed.

Hyperandrogenism in adolescent girls can be a troubling problem because of the difficulty in establishing a diagnosis and in prescribing appropriate therapy. Androgen excess in adolescent patients encompasses a spectrum of clinical presentations, including acne, hirsutism, oligomenorrhea, amenorrhea, virilism, and ovarian cysts. Androgen excess is a clinical and chemical feature of idiopathic hirsutism, late-onset forms of congenital adrenal hyperplasia, and polycystic ovarian disease; in some cases, functional hyperandrogenism is discussed. We recommend screening for hyperandrogenism by measuring blood levels of testosterone, dehydroepiandrosterone sulfate, and delta 4-androstenedione, while others propose a first dexamethasone suppression test for evaluation of free testosterone, dehydroepiandrosterone sulfate, and cortisol. Treatment will be chosen according to particular symptoms such as acne, hirsutism, obesity, or oligomenorrhea.

Defects in the enzyme, steroid 21-hydroxylase, result in congenital adrenal hyperplasia (CAH), a common autosomal recessive disorder of cortisol biosynthesis. The gene encoding this protein (CYP21B) and a closely linked pseudogene (CYP21A) have been mapped in the HLA complex on chromosome 6p, adjacent to the complement genes C4B and C4A, about 80 kb from the factor B gene. Molecular analyses of patients with CAH have shown that the cause of the defect may be either a deletion, a point mutation or a conversion of the active gene. Linkage of the disease to HLA has previously been studied by several groups. We have analyzed DNAs from patients with classical and non-classical CAH and from their family members, by probing with CYP21, C4 and BF cDNAs. In 70% of the CAH haplotypes studied, the defective CYP21B gene was indistinguishable from its structurally intact corresponding gene in Southern blot analysis, and presumably bore point mutations. In the remaining chromosomes, evidence for gene conversions, deletions and various deleterious mutations of the CYP21B gene is given. Moreover, our linkage studies show that a polymorphic TaqI cleavage site in the factor B gene, recently described by us, may be a new and useful genetic marker, because we found this TaqI restriction site only in unaffected haplotypes carrying functional CYP21B genes and, therefore, in negative association with the defective CYP21B gene.

Familial growth hormone deficiency has been often associated to homozygous gene deletions. In this work we have looked for the possible absence of this gene in patients with isolated GH deficiency. The patient genomic DNAs have been digested with two restriction enzymes and hybridized with a 32P labelled growth hormone cDNA. The presence of the growth hormone gene has been proved in the patients. This situation, in which the gene is present but not expressed, might be due to changes in gene regulation or to punctual gene deletions or mutations.

We studied a family in which one out of two children presented a non-salt wasting form of CAH. Genomic DNA of the patient, his brother, his parents and a normal control were digested by the Taq I and Bgl II restriction enzymes. The fragments were electrophoresed, transferred onto a nitrocellulose membrane and hybridized with two specific probes: pC21a for the CYP21 genes and pAT-A for the C4 genes. We performed simultaneous RFLP analyses of the CYP21 and C4 genes and determined the relative hybridization intensity of the genes using scanning densitometry of the X-ray films. The affected child had a CYP21B gene conversion in the CYP21A pseudogene on one chromosome inherited from his mother and a mutated CYP21B gene on the second chromosome inherited from his father. The second maternal chromosome, inherited by the unaffected brother, presented an unusual CYP21A gene deletion without a C4A or C4B gene deletion. Although CYP21A is a pseudogene, this type of complete CYP21A gene deletion associated with a CYP21B gene conversion has never been previously described.

21-hydroxylase gene analysis was performed on the genomic DNA from patients with congenital adrenal hyperplasia (CAH), their siblings, their parents as well as from a healthy individual serving as control. After digestion by the Taq I and Bgl II restriction enzymes, DNA was hybridized with specific nucleotidic probes: pC21a for the 21-hydroxylase genes, pAT-A for the C4 component Complement genes, closely linked to the 21-hydroxylase genes on the 6 chromosome. Likewise the pFB3B probe was used for the B factor gene located 80 kilobases upstream the 21-hydroxylase gene. From this molecular analysis on 11 families, we report here 4 investigations showing the most frequent genetic abnormalities we have encountered: gene deletions, gene conversions and point mutations. These data show that the molecular approach is a powerful tool for studying this endocrine disease at the clinical, genetic and fundamental point of view.