Search

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.

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.

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.

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.

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.

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.

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.