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Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease of steroid biosynthesis in humans. More than 90% of all CAH cases are caused by mutations of the 21-hydroxylase gene (CYP21A2), and approximately 75% of the defective CYP21A2 genes are generated through an intergenic recombination with the neighboring CYP21A1P pseudogene. In this study, the CYP21A2 gene was genotyped in 50 patients in Tunisia with the clinical diagnosis of 21-hydroxylase deficiency. CYP21A2 mutations were identified in 87% of the alleles. The most common point mutation in our population was the pseudogene specific variant p.Q318X (26%). Three novel single nucleotide polymorphism (SNP) loci were identified in the CYP21A2 gene which seems to be specific for the Tunisian population. The overall concordance between genotype and phenotype was 98%. With this study the molecular basis of CAH has been characterized, providing useful results for clinicians in terms of prediction of disease severity, genetic and prenatal counseling., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Congenital adrenal hyperplasia is caused by insufficient adrenal steroid biosynthesis due to impaired steroidogenic enzymes. The majority of patients suffer from deficiency of 21-hydroxylase (CYP21) coded by the CYP21A2 gene., Objective: Our objective was to study the functional and structural consequences of the novel CYP21A2 missense mutation c.364A > C (K121Q) detected in a female patient with nonclassical 21-hydroxylase deficiency. The patient was compound heterozygous for the novel K121Q mutation and the mild P453S mutation., Results: In vitro expression analysis of the mutant K121Q enzyme in transiently transfected COS-7 cells revealed reduced CYP21 activity of 14.0 +/- 5% for the conversion of 17-hydroxyprogesterone and 19.5 +/- 4% for the conversion of progesterone. K121 is located on helix C in the CYP21 protein, which is part of the heme coordinating system. In addition, helix C is involved in the interaction with the electron-providing enzyme P450 oxidoreductase. Protein modeling revealed that the substitution of glutamine for the basic amino acid lysine introduces an electrostatic change on the surface of CYP21 and may additionally change heme coordination. We hypothesize that the electron flux between P450 oxidoreductase and CYP21 is impaired and, moreover, that substrate affinity is altered due to heme dislocation with K121Q., Conclusion: Both the interaction of P450 oxidoreductase and CYP21 as well as heme coordination are likely to be disturbed due to the K121Q mutation. Our data exemplify how the combination of in vitro expression and structural protein analysis provide novel insights into molecular mechanisms of reduced CYP21 activity, eventually explaining the patient's phenotype.

Introduction: 3beta-Hydroxysteroid dehydrogenase (3beta-HSD) deficiency is a rare cause of congenital adrenal hyperplasia caused by inactivating mutations in the HSD3B2 gene. Most mutations are located within domains regarded crucial for enzyme function. The function of the C terminus of the 3beta-HSD protein is not known., Objective: We studied the functional consequences of three novel C-terminal mutations in the 3beta-HSD protein (p.P341L, p.R335X and p.W355X), detected in unrelated 46,XY neonates with classical 3beta-HSD type II deficiency showing different degrees of under-virilization., Methods and Results: In vitro expression of the two truncated mutant proteins yielded absent conversion of pregnenolone and dehydroepiandrosterone (DHEA), whereas the missense mutation p.P341L showed a residual DHEA conversion of 6% of wild-type activity. Additional analysis of p.P341L, including three-dimensional protein modeling, revealed that the mutant's inactivity predominantly originates from a putative structural alteration of the 3beta-HSD protein and is further aggravated by increased protein degradation. The stop mutations cause truncated proteins missing the final G-helix that abolishes enzymatic activity irrespective of an augmented protein degradation. Genital appearance did not correlate with the mutants' residual in vitro activity., Conclusions: Three novel C-terminal mutants of the HSD3B2 gene are responsible for classical 3beta-HSD deficiency. The C terminus is essential for the enzymatic activity. However, more studies are needed to clarify the exact function of this part of the protein. Our results indicate that the genital phenotype in 3beta-HSD deficiency cannot be predicted from in vitro 3beta-HSD function alone.

Background: Sufficient androgen receptor (AR) activity is crucial for normal male sexual differentiation. Here we report on two unrelated 46, XY patients suffering from undervirilization and genital malformations. Both patients had a short polyglycine (polyG) repeat of 10 residues and a relatively long polyglutamine (polyQ) repeat of 28 and 30 residues within the transactivation domain of the AR. In addition, they also harbor a rare A645D substitution., Objective: We made a set of AR expression plasmid constructs with varying polyQ and polyG tract sizes in context with or without the A645D substitution and analyzed their in vitro transactivation capacity in transfected CHO cells., Results: We found that a short polyG repeat downmodulated AR activity to approximately 60-65% of the wild-type receptor. This effect was aggravated by A645D in context of a long polyQ repeat to less than 50% activity. In contrast, in the context of a short polyQ and a short polyG repeat, the A645D mutation rescues AR activity to almost wild-type levels, demonstrating a contradictory effect of this mutation, depending on the size of the polymorphic repeats., Conclusions: A combination of a short polyG repeat with a long polyQ repeat and an A645D substitution might contribute to the development of virilization disorders and explain the observed phenotypes of our patients as a form of androgen insensitivity. The whole recreation of AR sequence variations including individual polymorphic repeat sizes could unravel possible interference of mutations and variations on AR activity by in vitro transfection.

Steroid 5alpha-reductase deficiency is a rare autosomal recessive disorder caused by mutations in the SRD5A2-gene, resulting in diminished dihydrotestosterone (DHT) formation and, hence, in a severe virilization deficit of the external genitalia in patients with 46,XY karyotype. The phenotype of affected individuals is variable and has been reported to range from completely female over genital ambiguity to normal male, depending on the type of mutation and its effect on enzyme activity. Here we report an adolescent 46,XY patient with predominantly female appearance, who had been gonadectomized in early infancy. Genital status revealed a urogenital sinus equivalent to Prader stage III. Molecular genetic analysis demonstrated a homozygous point mutation in exon 2 of the SRD5A2-gene, leading to a premature termination in codon position 111 of the 5alpha-reductase 2 enzyme, and not allowing formation of a functional 5alpha-reductase type 2 enzyme. This case demonstrates that even despite a complete loss of function of 5alpha-reductase type 2, marked virilization is possible, most likely the result of a testosterone (T) effect during foetal life.

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.

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.

Various mutations within the human androgen receptor gene have been documented to cause defective sexual differentiation in karyotypic male individuals. In this study, we report a previously undescribed point mutation at the donor splice-site of the second intron of the androgen receptor gene in a patient with a completely female phenotype. The sequence alteration was detected by single-strand-conformation-analysis-PCR and genomic sequencing. Applying competitive reverse transcribed PCR, cDNA sequencing and Western blotting, we could demonstrate considerable aberrations of structure and concentration of the transcript and its translation product in the patient's fibroblasts from the genital region. (1) In the transcript, exon 1 and 3 are directly linked to each other, the complete second exon is skipped. The mRNA predictively suffers a codon frame-shift in exon 3 associated with a premature termination between codons 598 and 599, leading to a truncated androgen receptor protein lacking any in vivo function. (2) Steady-state concentration levels of transcript and protein are abnormally low. Our observations highlight the influence of exon-flanking intron sequences on proper expression and function of gene products.

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.