[Show abstract][Hide abstract] ABSTRACT: Inactivating mutations of the 5α-steroid reductase type-2 (SRD5A2) gene result in a broad spectrum of masculinization defects, ranging from a male phenotype with hypospadias to a female phenotype with Wolffian structures. Molecular studies of the SRD5A2 revealed a new heterozygous gene variant within the coding region that results in phenotypic expression. A c.92C>T transition changing serine to phenylalanine at codon 31 of exon 1 (p.Ser31Phe) was identified in a patient with 46,XY disorder of sexual development who displayed glandular hypospadias with micropenis and bilateral cryptorchidism. The restoration of the p.Ser31Phe mutation by site-directed mutagenesis and transient expression assays using cultured HEK-293 cells showed that this novel substitution does not abolish but does deregulate the catalytic efficiency of the enzyme. Thus, the maximum velocity (Vmax) value was higher for the mutant enzyme (22.5 ± 6.9 nmol DHT mg protein−1 h−1) than for the wild-type enzyme (9.8 ± 2.0 nmol DHT mg protein−1 h−1). Increased in vitro activity of the p.Ser31Phe mutant suggested an activating effect. This case provides evidence that heterozygous missense mutations in SRD5A2 may induce the abnormal development of male external genitalia.
[Show abstract][Hide abstract] ABSTRACT: In hamsters, the Harderian glands (HGs) exhibit a marked sexual dimorphism which is thought to depend on dihydrotestosterone (DHT); however, it is unclear whether hamster HGs contain one or more 5 alpha-reductases and whether these enzymes are differentially expressed in males and females. In this study, we isolated specific cDNAs for 5 alpha-reductase 1 (Srd5a1) and 5 alpha-reductase 2 (Srd5a2), determined their sequences and investigated their expression in the HG of both sexes. Isozyme 1, cloned from liver mRNA, encodes a protein of 255 amino acids (aa); isozyme 2 cDNA, isolated from the epididymis encodes a 254-aa protein. When assayed in transfected HEK-293 cells, the type 1 isozyme displayed activity over a broad pH range (6.5-8), while isozyme 2 had a pH optimum of 5.5. Both isoenzymes efficiently catalyzed the in vitro transformation of T into DHT, with apparent K(m) values of 7.1 and 1.9 micromol/L for Srd5a1 and Srd5a2, respectively. Real-time PCR analysis revealed higher mRNA levels for Srd5a1 than for Srd5a2. Expression of both isoenzymes increased slightly in HGs of castrated males and showed variations during the estrous cycle in females. Hormonal replacement with 17beta-estradiol administered to spayed females induced the up-regulation of Srd5a2 mRNA levels. Altogether, our results demonstrated that both Srd5a1 and Srd5a2 are expressed in HGs without clear differences between males and females. The biochemical characteristics and relative expression of these 5 alpha-reductases support the view that both isozymes may play a relevant role in modulating androgen signaling in HG.
General and Comparative Endocrinology 12/2009; 166(2):388-95. · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inactivating mutations of the SRD5A2 gene result in steroid 5α-reductase 2 deficiency, an autosomal recessive disorder expressed as a male-limited disorder of sex development. Herein, genomic DNA was isolated from 11 new patients with apparent steroid 5α-reductase 2 deficiency. Coding sequence abnormalities in SRD5A2 were assessed by exon-specific polymerase chain reaction, single-stranded conformation polymorphism, and direct sequencing. Likewise, enzymatic activity of the P212R gene variant of SRD5A2 was assessed. DNA analysis revealed mutations in all patients (G115D, R171S, N193S, E197D, G203S, P212R). Three individuals were compound heterozygotes, 6 were homozygotes, and 2 more were single heterozygotes for SRD5A2 mutations; remarkably, 40% of the mutant alleles (9/22) contained the gene variant P212R. The results described in this study represent, along with our previous reports, the largest number of patients with steroid 5α-reductase 2 deficiency belonging to nonrelated families. Regarding the frequency of the p.P212R mutation in our population and its presence throughout all of our country, it allows us to hypothesize that the presence of this mutation may constitute a founder gene effect.
Journal of Andrology 12/2009; 31(4):358-64. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of estradiol on the Harderian gland (HG) are believed to be partially regulated by the transcriptional regulation of the estrogen-related genes via estrogen receptor (ER). In reptiles, however, it has not been well established whether the HG contains or expresses steroid nuclear receptors. As a first step toward investigating the molecular mechanisms of estrogen signalling in the HG, we isolated the cDNA for ERalpha in the sea turtle Lepidochelys olivacea. ERalpha was cloned using RT-PCR coupled with 5' and 3' RACE procedures. The cDNA contains a complete open reading frame encoding 588 amino acid residues. Comparative analysis of this amino acid sequence showed moderate to strong conservation of the ERalpha (Esr1) gene within divergent vertebrate groups. In transfection studies, the cloned ER displayed high affinity K(d)=0.25nM and high specificity for 17beta-estradiol. Binding assays using sucrose density gradients demonstrated a specific 7-7.5 S binding component in the HG cytosolic fractions. RT-qPCR analysis showed significant ERalpha mRNA expression in the liver, HG, lung and brain. Altogether, these results provide evidence for the expression of intracellular ERs in the HG of the sea turtle and suggest that ERalpha may be an important modulator of the estrogen-mediated response in the HG of reptiles.
General and Comparative Endocrinology 04/2009; 162(2):203-9. · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dihydrotestosterone is crucial for normal development of external genitalia and prostate in the male embryo. Autosomal recessive mutations in the 5 alpha-reductase type 2 (SRD5A2) gene disrupt the synthesis of dihydrotestosterone in the urogenital tract and give rise to genetic males with undervirilized external genitalia that may be female-like or ambiguous. In this study, three unrelated 46,XY children (0.5, 3, and 8 years old) who presented severe undermasculinization at birth were examined for genetic abnormalities in the SRD5A2 gene. Coding sequence abnormalities were ascertained by exon-specific polymerase chain reaction (PCR), single-stranded conformational polymorphism (SSCP), and sequencing analysis. Functional properties of the mutant alleles were investigated by means of site-directed mutagenesis assays. DNA molecular studies showed that all three patients were compound heterozygotes for SRD5A2 mutations. Patient 1 had a point mutation 547G --> A in exon 3 (G183S) and a novel dinucleotidic mutation 634,635CC --> TG in exon 4 (P212X). This double change results in premature termination signal (TGA) at codon 212, which predicts the expression of a truncated 211-amino acid protein. Patient 2 was the carrier of mutations G115D in exon 3 and S210F in exon 4. Patient 3 had two substitution mutations in exon 1, including a novel G --> C transversion at nucleotide 169 (E57Q) and a G --> A transition at nucleotide 254 (G85D). In transitory transfection assays, the recombinant cDNAs harboring mutations E57Q and G85D showed residual 5 alpha-reductase activity, whereas those with mutations G115D, S210F, and P212X were devoid of activity. In contrast, the G183S substitution affected the catalytic activity of the enzyme by decreasing its affinity for testosterone substrate. We describe six different mutations of the SRD5A2 gene detected in three children with genital ambiguity. These genotypes are consistent with the clinical phenotype of steroid 5 alpha-reductase 2 deficiency. Our data suggest that the combined gene variants (E57Q/G85D, G115D/S210F, and G183S/P212X) result in subfunctional or nonfunctional enzymes, causing masculinization defects in these patients. This further underscores that exon 4 of SRD5A2 may be a site prone to inactivating mutations.
Journal of Human Genetics 01/2008; 53(5):401-6. · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ferrochelatase (protohaem ferrolyase, EC 22.214.171.124), the terminal enzyme of the haem biosynthetic pathway, catalyses the insertion of ferrous iron into protoporphyrin IX to form protohaem. The Syrian hamster Harderian gland (HG) is known for its ability to produce and accumulate large amounts of protoporphyrins. In this species, the female gland contains up to 120 times more porphyrin than the male gland. Data from biochemical studies suggest that this gland possesses the enzymatic complex for haem biosynthesis but lacks ferrochelatase activity. The abundance of intraglandular haem proteins does not support this idea. To gain more insight into this process, we isolated cDNA for ferrochelatase from hamster liver, using the 5'- and 3'- rapid amplification of complementary DNA ends (RACE), and investigated its expression in HG from males and females. The full-length cDNA comprises an open reading frame of 1269 bp encoding a polypeptide of 422 amino-acid residues. Hamster DNA sequence exhibits 92% identity to mouse and 87% identity to human sequences. The predicted hamster enzyme was shown to have structural features of mammalian ferrochelatase, including a putative NH2- terminal presequence, a central core of about 330 amino-acid residues and an extra 30-50-amino-acid stretch at the carboxyl-terminus. RNA blotting experiments indicated that this cDNA hybridized to a liver mRNA of about 2.1 kb, while a weak hybridization signal was observed with mRNA from HG preparations. RT-PCR assays confirmed the expression of specific transcripts in both tissues. Male glands contained approximately twofold more enzyme mRNA than female glands. Likewise, the intraglandular content of mRNA varied during the oestrous cycle, with the highest levels found in the oestrous phase. These cyclic variations were less evident in liver. Ovariectomy plus treatment with progesterone or 17beta-oestradiol plus progesterone increased ferrochelatase mRNA of the gland. In HG of short- or long-term castrated males, the administration of testosterone did not affect the ferrochelatase mRNA concentration. Based on mRNA expression levels, we conclude that Harderian ferrochelatase may play an active role in maintaining the physiological pool of haem required for processing cytochromes and other glandular haem proteins. Likewise, the sex-steroid hormones appear to have only a modest influence upon Harderian ferrochelatase.
Journal of Endocrinology 05/2006; 189(1):103-12. · 4.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Androgen insensitivy syndrome (AIS) is the most frequent cause of male pseudohermaphroditism resulting from target-organ resistance to androgen action. Individuals bearing the complete form of the disease (CAIS) present a female phenotype and a lack of pubic and axillary hair. In the present study, four 46,XY patients born in two generations from a kindred with a history of AIS were examined for genetic abnormalities in the androgen receptor gene (AR). All eight exons encoding the AR protein were individually amplified from genomic DNA followed by a mutation screening with single-strand conformation polymorphism analysis. Sequencing of the mutant AR revealed a novel insertion/deletion mutation in exon 5. A deletion of 7 bp is replaced by an insertion of 11 nucleotides, which represents a duplication of the adjacent downstream sequence. The mutation g.2640_2646delAGGATGC/2652_2662insTTCGCCCCTGA, results in a frameshift that introduces a premature termination signal TGA, nine codons downstream. Such a rearrangement predicts a truncation of the AR, thereby deleting a large portion of the ligand-binding domain (amino acid position 768-919). Furthermore, although this mutation breaks the translational reading frame starting from codon 760, examination of the complementary DNA suggested that it does not disturb mRNA splicing. These changes have been found in all the patients and appear to account for the observed absence of detectable androgen binding to the AR in cultured fibroblasts and for the CAIS phenotype in the kindred. This disorder represents the first insertion/deletion mutation of the AR that probably arose by a slipped-strand mispairing mechanism.
Journal of Human Genetics 02/2003; 48(7):346-51. · 2.37 Impact Factor