Apolipoprotein A-I proteolysis in aortic valve stenosis: role of cathepsin S.

Aortic valve stenosis (AVS) is the most common valvular heart disease in the Western world. Therapy based on apolipoprotein A-I (apoA-I), the major protein component of high-density lipoproteins, results in AVS regression in experimental models. Nevertheless, apoA-I degradation by proteases might lead to suboptimal efficacy of such therapy. An activatable probe using a quenched fluorescently labeled full-length apoA-I protein was generated to assess apoA-I-degrading protease activity in plasma derived from 44 men and 20 women with severe AVS (age 65.0 ± 10.4 years) as well as from a rabbit model of AVS. In human and rabbit AVS plasma, apoA-I-degrading protease activity was significantly higher than in controls (humans: 0.038 ± 0.009 vs 0.022 ± 0.005 RFU/s, p < 0.0001; rabbits: 0.033 ± 0.016 vs 0.017 ± 0.005 RFU/s, p = 0.041). Through the use of protease inhibitors, we identified metalloproteinases (MMP) as exerting the most potent proteolytic effect on apoA-I in AVS rabbits (67%, p < 0.05 vs control), while the cysteine protease cathepsin S accounted for 54.2% of apoA-I degradation in human plasma (p < 0.05 vs control) with the maximum effect seen in women (68.8%, p < 0.05 vs men). Accordingly, cathepsin S activity correlated significantly with mean transaortic pressure gradient in women (r = 0.5, p = 0.04) but not in men (r = - 0.09, p = 0.60), and was a significant independent predictor of disease severity in women (standardized beta coefficient 0.832, p < 0.001) when tested in a linear regression analysis. ApoA-I proteolysis is increased in AVS. Targeting circulating cathepsin S may lead to new therapies for human aortic valve disease.

Beneficial Effects of High-Density Lipoproteins on Acquired von Willebrand Syndrome in Aortic Valve Stenosis.

BACKGROUND: Infusions of apolipoprotein A-I (apoA-I), the major protein component of high-density lipoproteins (HDL), result in aortic valve stenosis (AVS) regression in experimental models. Severe AVS can be complicated by acquired von Willebrand syndrome, a haemorrhagic disorder associated with loss of high-molecular-weight von Willebrand factor (vWF) multimers (HMWM), the latter being a consequence of increased shear stress and enhanced vWF-cleaving protease (ADAMTS-13) activity. Although antithrombotic actions of HDL have been described, its effects on ADAMTS-13 and vWF in AVS are unknown. METHODS AND RESULTS: We assessed ADAMTS-13 activity in plasma derived from a rabbit model of AVS (n = 29) as well as in plasma collected from 64 patients with severe AVS (age 65.0 ± 10.4 years, 44 males) undergoing aortic valve replacement (AVR). In both human and rabbit AVS plasma, ADAMTS-13 activity was higher than that in controls (p < 0.05). Accordingly, AVS patients had less HMWM than controls (66.3 ± 27.2% vs. 97.2 ± 24.1%, p < 0.0001). Both ADAMTS-13 activity and HMWM correlated significantly with aortic transvalvular gradients, thereby showing opposing correlations (r = 0.3, p = 0.018 and r = -0.4, p = 0.003, respectively). Administration of an apoA-I mimetic peptide reduced ADAMTS-13 activity in AVS rabbits as compared with the placebo group (2.0 ± 0.5 RFU/sec vs. 3.8 ± 0.4 RFU/sec, p < 0.05). Similarly, a negative correlation was found between ADAMTS-13 activity and HDL cholesterol levels in patients with AVS (r = -0.3, p = 0.045). CONCLUSION: Our data indicate that HDL levels are associated with reduced ADAMTS-13 activity and increased HMWM. HDL-based therapies may reduce the haematologic abnormalities of the acquired von Willebrand syndrome in AVS.

Improvement of aortic valve stenosis by ApoA-I mimetic therapy is associated with decreased aortic root and valve remodelling in mice.

BACKGROUND AND PURPOSE: We have shown that infusions of apolipoprotein A-I (ApoA-I) mimetic peptide induced regression of aortic valve stenosis (AVS) in rabbits. This study aimed at determining the effects of ApoA-I mimetic therapy in mice with calcific or fibrotic AVS. EXPERIMENTAL APPROACH: Apolipoprotein E-deficient (ApoE(-/-) ) mice and mice with Werner progeria gene deletion (Wrn(Δhel/Δhel) ) received high-fat diets for 20 weeks. After developing AVS, mice were randomized to receive saline (placebo group) or ApoA-I mimetic peptide infusions (ApoA-I treated groups, 100 mg·kg(-1) for ApoE(-/-) mice; 50 mg·kg(-1) for Wrn mice), three times per week for 4 weeks. We evaluated effects on AVS using serial echocardiograms and valve histology. KEY RESULTS: Aortic valve area (AVA) increased in both ApoE(-/-) and Wrn mice treated with the ApoA-I mimetic compared with placebo. Maximal sinus wall thickness was lower in ApoA-I treated ApoE(-/-) mice. The type I/III collagen ratio was lower in the sinus wall of ApoA-I treated ApoE(-/-) mice compared with placebo. Total collagen content was reduced in aortic valves of ApoA-I treated Wrn mice. Our 3D computer model and numerical simulations confirmed that the reduction in aortic root wall thickness resulted in improved AVA. CONCLUSIONS AND IMPLICATIONS: ApoA-I mimetic treatment reduced AVS by decreasing remodelling and fibrosis of the aortic root and valve in mice.

Regression of aortic valve stenosis by ApoA-I mimetic peptide infusions in rabbits.

BACKGROUND AND PURPOSE: Aortic valve stenosis (AVS) is the most common valvular heart disease, and standard curative therapy remains open heart surgical valve replacement. The aim of our experimental study was to determine if apolipoprotein A-I (ApoA-I) mimetic peptide infusions could induce regression of AVS. EXPERIMENTAL APPROACH: Fifteen New Zealand White male rabbits received a cholesterol-enriched diet and vitamin D(2) until significant AVS was detected by echocardiography. The enriched diet was then stopped to mimic cholesterol-lowering therapy and animals were allocated randomly to receive saline (control group, n=8) or an ApoA-I mimetic peptide (treated group, n=7), three times per week for 2 weeks. Serial echocardiograms and post mortem valve histology were performed. KEY RESULTS: Aortic valve area increased significantly by 25% in the treated group after 14 days of treatment (P=0.012). Likewise, aortic valve thickness decreased by 21% in the treated group, whereas it was unchanged in controls (P=0.0006). Histological analysis revealed that the extent of lesions at the base of valve leaflets and sinuses of Valsalva was smaller in the treated group compared with controls (P=0.032). The treatment also reduced calcification, as revealed by the loss of the positive relationship observed in the control group (r=0.87, P=0.004) between calcification area and aortic valve thickness. CONCLUSIONS AND IMPLICATIONS: Infusions of ApoA-I mimetic peptide lead to regression of experimental AVS. These positive results justify the further testing of high-density lipoprotein (HDL)-based therapies in patients with valvular aortic stenosis. Regression of aortic stenosis, if achieved safely, could transform the clinical treatment of this disease.

Lipoic acid supplementation and endothelial function.

Endothelial dysfunction is caused by all the recognized cardiovascular risk factors and has been implicated in the complex processes leading to the initiation and progression of atherosclerosis. Short-term treatment with lipoic acid is shown in the current issue of the British Journal of Pharmacology to improve endothelial function of aortic rings of old rats. The age-related decrease in phosphorylation of nitric oxide synthase and Akt was improved by lipoic acid supplementation. The improved phosphorylation status may have been due to reduced activity of the phosphatase PPA2, associated with decreased levels of endothelial ceramide induced by lipoic acid. Neutral sphingomyelinase activity was also reduced by lipoic acid, which was due, at least in part, to increased glutathione levels in endothelial cells. The favourable antioxidant, anti-inflammatory, metabolic and endothelial effects of lipoic acid shown in rodents, in this and other recently published studies, warrant further assessment of its potential role for prevention and treatment of cardiovascular diseases.

Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation.

The protease granzyme B (GrB) plays a key role in the cytocidal activity during cytotoxic T lymphocyte (CTL)-mediated programmed cell death. Multiple caspases have been identified as direct substrates for GrB, suggesting that the activation of caspases constitutes an important event during CTL-induced cell death. However, recent studies have provided evidence for caspase-independent pathway(s) during CTL-mediated apoptosis. In this study, we demonstrate caspase-independent and direct cleavage of the 45 kDa unit of DNA fragmentation factor (DFF45) by GrB both in vitro and in vivo. Using a novel and selective caspase-3 inhibitor, we show the ability of GrB to process DFF45 directly and mediate DNA fragmentation in the absence of caspase-3 activity. Furthermore, studies with DFF45 mutants reveal that both caspase-3 and GrB share a common cleavage site, which is necessary and sufficient to induce DNA fragmentation in target cells during apoptosis. Together, our data suggest that CTLs possess alternative mechanism(s) for inducing DNA fragmentation without the requirement for caspases.

The role of caspases in T cell development and the control of immune responses.

Apoptosis is responsible for the removal of potentially autoreactive or useless T cells during thymic selection and activated T cells in the periphery. Specific families of receptors, kinases, transcription factors, and cysteine proteases, termed caspases, are involved in the apoptotic cascade leading to proteolysis of specific substrates and to morphological changes associated with programmed cell death. Although common members of the apoptotic cascade are shared between different cell types, it appears that cell-specific factors can influence the response to a given apoptotic stimuli. Characterization and understanding of the basic mechanisms involved in the different pathways protecting or leading to cell death may provide novel ways to control inappropriate apoptosis involved in several diseases.

The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during Fas-mediated apoptosis.

Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli. Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes. Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved. We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments. Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein. Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A. We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates. Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication. These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.

Structure-function relationships of 3 beta-hydroxysteroid dehydrogenase: contribution made by the molecular genetics of 3 beta-hydroxysteroid dehydrogenase deficiency.

The transformation of delta 5-3 beta-hydroxysteroids into the corresponding delta 4-3-keto-steroids is an essential step for the biosynthesis of all classes of active steroids: progesterone, mineralocorticoids, glucocorticoids, androgens, and estrogens. These steroid hormones play a crucial role in the differentiation, development, growth, and physiological function of most human tissues. The structures of several cDNAs encoding 3 beta-HSD isoenzymes have been characterized in human and several other vertebrate species: human types I and II; macaque; bovine; rat types I, II, III, and IV; mouse types I, II, III, IV, V and VI; hamster types I, II, and III; and rainbow trout. Their transient expression reveals that 3 beta-HSD and delta 5-delta 4-isomerase activities reside within a single protein. Distinct approaches have been used for a better understanding of the structure-function relationships of these 3 beta-HSD enzymes: i) affinity radiolabeling studies of the human type I 3 beta-HSD; ii) identification and the functional consequences of the human type-II 3 beta-HSD mutations detected in patients with 3 beta-HSD deficiency. Taken together, all of these data were examined to determine whether the relationship between the genotype and the phenotype of these patients were consistent with in vitro mutagenesis studies. 3 beta-HSD deficiency, transmitted in an autosomic recessive disorder, is characterized by varying degrees of salt wasting; in genetic males, fetal testicular 3 beta-HSD deficiency causes an undervirilized male genitalia (male pseudohermaphroditism); females exhibit either normal sexual differentiation or mild virilization. All mutations were detected in the type II 3 beta-HSD gene, which is expressed almost exclusively in the adrenals and gonads. No mutation was detected in the type I 3 beta-HSD gene, which is expressed in peripheral tissues. The finding of a normal type I 3 beta-HSD gene explains the elevated delta 5-steroids and mild virilization of affected girls at birth. To date, 24 mutations have been identified in 25 distinct families with 3 beta-HSD deficiencies. All nonsense and frameshift mutations introducing a premature termination codon were associated with the classical salt-losing form. The locations of these nonsense mutations suggest that at least the first 318 amino acids out of 371 are required for 3 beta-HSD activity. The consequences of the missense mutations on some domains of the 3 beta-enzyme, such as membrane-spanning domains, cofactor-binding site, and steroid-binding site, were reviewed. The future crystallization of the overexpressed normal and mutant-type II-3 beta-HSD enzymes should contribute to a better understanding of the structure-function relationships of this enzyme, especially for missense mutations located outside the putative functional regions.

Structure-function relationships and molecular genetics of the 3 beta-hydroxysteroid dehydrogenase gene family.

The isoenzymes of the 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene-isomerase (3 beta-HSD) gene family catalyse the transformation of all 5-ene-3 beta-hydroxysteroids into the corresponding 4-ene-3-keto-steroids and are responsible for the interconversion of 3 beta-hydroxy- and 3-keto-5 alpha-androstane steroids. The two human 3 beta-HSD genes and the three related pseudogenes are located on the chromosome 1p13.1 region, close to the centromeric marker D1Z5. The 3 beta-HSD isoenzymes prefer NAD+ to NADP+ as cofactor with the exception of the rat liver type III and mouse kidney type IV, which both prefer NADPH as cofactor for their specific 3-ketosteroid reductase activity due to the presence of Tyr36 in the rat type III and of Phe36 in mouse type IV enzymes instead of Asp36 found in other 3 beta-HSD isoenzymes. The rat types I and IV, bovine and guinea pig 3 beta-HSD proteins possess an intrinsic 17 beta-HSD activity specific to 5 alpha-androstane 17 beta-ol steroids, thus suggesting that such "secondary" activity is specifically responsible for controlling the bioavailability of the active androgen DHT. To elucidate the molecular basis of classical form of 3 beta-HSD deficiency, the structures of the types I and II 3 beta-HSD genes in 12 male pseudohermaphrodite 3 beta-HSD deficient patients as well as in four female patients were analyzed. The 14 different point mutations characterized were all detected in the type II 3 beta-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3 beta-HSD gene predominantly expressed in the placenta and peripheral tissues. The mutant type II 3 beta-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact transfected cells, at the exception of L108W and P186L proteins, which have some residual activity (approximately 1%). Mutations found in nonsalt-loser patients have some residual activity ranging from approximately 1 to approximately 10% compared to the wild-type enzyme. Characterization of mutant proteins provides unique information on the structure-function relationships of the 3 beta-HSD superfamily.

Nonsalt-losing male pseudohermaphroditism due to the novel homozygous N100S mutation in the type II 3 beta-hydroxysteroid dehydrogenase gene.

Recently, the structure of two genes encoding isoenzymes responsible for 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) activity in the human was elucidated. This activity is an essential step in the biosynthesis of all classes of steroid hormones. In the classic severe form of 3 beta HSD deficiency, patients present with adrenal insufficiency, various degrees of salt loss, and incomplete masculinization in males. Here we report the characterization of the molecular basis of congenital adrenal hyperplasia due to 3 beta HSD deficiency in a male pseudohermaphrodite born from consanguineous parents and having no clinical salt loss. To analyze the structure of the type I and II 3 beta HSD genes of the patient, DNA fragments, generated by polymerase chain reaction amplification of the four exons and the exon-intron boundaries of these genes, were directly sequenced. The patients carried a homozygous missense mutation converting Asn100 to Ser in exon 3 of his type II 3 beta HSD gene. His parents were heterozygous for the same point mutation. The absence of clinical salt loss associated with a male pseudohermaphroditism suggested that 3 beta HSD activity was impaired to different levels in the testes and adrenal. To elucidate whether this N100S missense mutation affected preferentially a steroidogenic pathway, enzymatic activity was analyzed by in vitro analysis of mutant recombinant enzyme generated by site-directed mutagenesis after its transient expression in COS-1 cells. Using homogenates from transfected cells, the N100S 3 beta HSD enzyme showed a Km value for pregnenolone of 25 +/- 3 mumol/L compared with 3.5 +/- 0.2 mumol/L for the normal human type II 3 beta HSD enzyme. Similar results were obtained using dehydroepiandrosterone as substrate. In addition to decreasing apparent affinity, the N100S mutation decreased the relative specific activity (Vmax), leading to a relative specificity (relative Vmax/Km) 2.7% and 11% that of normal type II 3 beta HSD using pregnenolone or dehydroepiandrosterone as substrate, respectively. Moreover, the mutant N100S protein had an apparent decreased affinity for NAD+, with a Km value of 650 +/- 66 mumol/L compared with 20 +/- 2 mumol/L for normal type II 3 beta HSD. Except for the hypothetical effect of local factors, these findings suggest that a very weak residual activity of the normal type II 3 beta HSD enzyme could prevent salt loss, but it was insufficient for normal male sex differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular basis of human 3 beta-hydroxysteroid dehydrogenase deficiency.

The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) catalyses an essential step in the biosynthesis of all classes of steroid hormones. Classical 3 beta-HSD deficiency is responsible for CAHII, a severe form of congenital adrenal hyperplasia (CAH) that impairs steroidogenesis in both the adrenals and gonads. Newborns affected by 3 beta-HSD deficiency exhibit signs and symptoms of adrenal insufficiency of varying degrees associated with pseudohermaphroditism in males, whereas females exhibit normal sexual differentiation or mild virilization. Elevated ratios of 5-ene-to 4-ene-steroids appear as the best biological parameter for the diagnosis of 3 beta-HSD deficiency. The nonclassical form has been suggested to be related to an allelic variant of the classical form of 3 beta-HSD as described for steroid 21-hydroxylase deficiency. To elucidate the molecular basis of the classical form of 3 beta-HSD deficiency, we have analysed the structure of the highly homologous type I and II 3 beta-HSD genes in 12 male pseudohermaphrodite 3 beta-HSD deficient patients as well as in four female patients. The 14 different point mutations characterized were all detected in the type II 3 beta-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3 beta-HSD gene predominantly expressed in the placenta and peripheral tissues. The finding of a normal type I 3 beta-HSD gene provides the explanation for the intact peripheral intracrine steroidogenesis in these patients and increased androgen manifestations at puberty. The influence of the detected mutations on enzymatic activity was assessed by in vitro expression analysis of mutant enzymes generated by site-directed mutagenesis in COS-1 cells. The mutant type II 3 beta-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact transfected cells, whereas those with mutations found in nonsalt-loser index cases have some residual activity ranging from approximately 1-10% compared to the wild-type enzyme. Although in general, our findings provide a molecular explanation for the enzymatic heterogeneity ranging from the severe salt-losing form to the clinically inapparent salt-wasting form of the disease, we have observed that the mutant L108W or P186L enzymes found in a compound heterozygote male presenting the salt-wasting form of the disease, has some residual activity (approximately 1%) similar to that observed for the mutant N100S enzyme detected in a homozygous male patient suffering from a nonsalt-losing form of this disorder.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification and characterization of the G15D mutation found in a male patient with 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency: alteration of the putative NAD-binding domain of type II 3 beta-HSD.

We report the detection of a homozygous G to A mutation converting codon Gly15 into Asp15 in the type II 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) gene in a male pseudo-hermaphrodite born from consanguineous parents and suffering from severe salt-losing 3 beta-HSD deficiency. To investigate further the potential involvement of residue 15 in the beta alpha beta dinucleotide-binding fold, we have studied the effect of substituting Gly15 for Ala15. We assessed the effect of the G15D and G15A missense mutations on enzymatic activity by analyzing mutant enzymes generated by site-directed mutagenesis of type II 3 beta-HSD cDNA after their transient expression in COS-1 cells. In intact transfected cells, after a 2-h incubation, the percentage of conversion of [3H]pregnenolone (PREG) into [3H]progesterone (PROG) was 35% and 50% for the G15A and native type II 3 beta-HSD enzymes, respectively, whereas no detectable activity was observed in cells expressing the G15D protein. This finding is in agreement with the severity of the disease in the homozygote G15D index case. On the other hand, in homogenates from cells transfected with the normal pCMV-type II 3 beta-HSD plasmid or with the mutated pCMV-G15D or pCMV-G15A plasmid, the Km values for PREG were 0.72 microM, 3.2 microM, and 3.4 microM, respectively, when incubated for 1 h in the presence of excess (1 mM) NAD+. Moreover, the expressed G15D and G15A proteins had decreased affinities for NAD+ with Km values of 113 microM and 148 microM, respectively, compared with 22 microM for normal type II 3 beta-HSD.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic linkage mapping of HSD3B1 and HSD3B2 encoding human types I and II 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase close to D1S514 and the centromeric D1Z5 locus.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) catalyses an essential step in the biosynthesis of all steroid hormones. Consequently, classical 3 beta-HSD deficiency is responsible for a severe form of congenital adrenal hyperplasia. The HSD3B1 and HSD3B2 genes encoding the types I and II 3 beta-HSD isoenzymes, respectively, have been previously assigned by in situ hybridization to the chromosome 1p13.1 region. To determine the physical distance between these two genes, NotI and SacII digests of genomic DNA were resolved by pulse-field gel electrophoresis and hybridized with type I and type II 3 beta-HSD cDNAs used as probes. The detection of a single band under low stringency conditions indicates that HSD3B1 and HSD3B2 are located within an approximately 0.29 megabase SacII DNA fragment. We constructed a high resolution genetic map of the region flanking the polymorphic HSD3B1 and HSD3B2 genes including ten Généthon markers and the two NIH/CEPH markers AMY2B and D1Z5. The HSD3B1A and HSD3B2A markers were mapped relative to other reference markers through eight CEPH reference families. The order of polymorphic genes and markers is: pter-[AMY2B-D1S239]-D1S457-D1S502-D1S250-+ ++D1S252-[HSD3B1A -HSD3B2A-D1S514]-[D1Z5-D1S442]-D1S305-D 1S303-D1S484-qter. The D1S514 marker was thus closely linked to HSD3B1A (theta < 0.001; lod = 14.13) and HSD3B2 (theta = 0.008; lod = 35.36). The HSD3B loci are located 1-2 cM of the centromeric marker D1Z5.

No evidence of mutations in the genes for type I and type II 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in nonclassical 3 beta HSD deficiency.

Nonclassical 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase deficiency (NC3 beta HSDD) has been diagnosed in hyperandrogenic women with an increasing frequency during the last 14 yr. Fifteen menarcheal women with androgen excess syndrome, diagnosed with NC3 beta HSDD previously were restudied, in 12 after discontinuation of glucocorticoid treatment, in 2 patients never treated with glucocorticoids, and in 1 both before and after glucocorticoid therapy. Each of the 15 patients underwent ACTH stimulation testing, in some cases on multiple occasions. Although some (very few) patients seem to have improved with time, others remained the same or got worse. Molecular DNA analysis was also performed in 6 of the patients, using the strategy successfully used to detect point mutations in the type II 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) gene, which are responsible for classical 3 beta HSD deficiency. This strategy consists of the direct sequencing of polymerase chain reaction-amplified DNA fragments corresponding to the complete coding sequence and all intron-exon junctions and to the 5'- and 3'-noncoding region of this gene. We were unable to demonstrate any mutation of the type II 3 beta HSD gene in these 6 patients. To gain additional information about potential mutations, direct sequencing of the type I 3 beta HSD gene was also performed using this same strategy, and no mutations were found. The present study strongly suggests that unlike the salt-losing and nonsalt-losing forms of classical 3 beta HSD deficiency, NC3 beta HSDD is not due to a mutant type II 3 beta HSD enzyme. However, the possibility remains of a mutation(s) in the unsequenced regions of the type II 3 beta HSD gene or elsewhere, such as in a gene for modulatory protein, playing a specific role in the expression of the type II 3 beta HSD gene. On the other hand, knowing the multiple hormonal controls to which 3 beta HSD activity is subject, it cannot be excluded that at least in some cases, NC3 beta HSDD may be an acquired defect, the result of endogenous or environmental factors.

Molecular basis of congenital adrenal hyperplasia in two siblings with classical nonsalt-losing 3 beta-hydroxysteroid dehydrogenase deficiency.

We report mutations of the type II 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) gene in two siblings, male and female, with congenital adrenal hyperplasia caused by classical nonsalt-losing 3 beta HSD deficiency. During childhood, the male sibling, born with ambiguous genitalia, and the female sibling, born with normal genitalia, both manifested symptoms of mild androgen excess; both apparently had normal zona glomerulosa function. Gonadal dynamic study at puberty showed the presence of partial gonadal 3 beta HSD deficiency in both siblings despite their spontaneous pubertal maturation. The 5'-region as well as exons I-II, III, and IV and portions of the adjacent introns of the type II 3 beta HSD gene were amplified by polymerase chain reaction and sequenced. In both siblings and their mother, an identical single nucleotide substitution mutation in intron III, six bases up-stream from exon IV, was identified in one allele. This mutation, G to A at nucleotide 6651, may create a new splicing junction and affect the normal splicing of the messenger ribonucleic acid. In the other allele of both siblings, a missense mutation from GGG (Gly) to AGG (Arg) at codon 129 (G129R) in exon IV was found. We assessed the effect of the G129R missense mutation on enzymatic activity by in vitro analysis of the mutant recombinant enzyme generated by site-directed mutagenesis after its transient expression in COS-1 cells. Using homogenates from transfected cells, the G129R 3 beta HSD enzyme showed a Km value for pregnenolone of 10 +/- 2 mumol/L compared with 1.00 +/- 0.03 mumol/L for the wild-type type II 3 beta HSD enzyme. When dehydroepiandrosterone was used as substrate, the Km value for G129R3 beta HSD was 14 +/- 2 mumol/L compared with 2.1 +/- 0.2 mumol/L for the wild-type II 3 beta HSD enzyme. In addition to an apparent decrease in affinity, the G129R mutation caused a marked decrease in the apparent relative specific activity, thus leading to apparent relative specific efficiencies (relative specific activity/Km) of 2.0% and 4.7% that of the normal type II 3 beta HSD using pregnenolone or dehydroepiandrosterone as substrate, respectively. It appears likely that this low level of activity is sufficient to prevent salt loss, but it is also possible that part of the enzymatic activity comes from the putative remaining percentage of correctly spliced n6651 allele in these patients.

Functional characterization of the novel L108W and P186L mutations detected in the type II 3 beta-hydroxysteroid dehydrogenase gene of a male pseudohermaphrodite with congenital adrenal hyperplasia.

Two isoenzymes are responsible for 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) activity in humans. We analyzed the structure of types I and II 3 beta-HSD genes in a male pseudohermaphrodite suffering from a severe salt-losing form of congenital adrenal hyperplasia. We did not detect any mutation in the type I 3 beta-HSD gene, but we found two different missense mutations in exon IV of the type II 3 beta-HSD gene of the patient; a conversion of codon Leu108 into a Trp (L108W) inherited from his mother and a conversion of codon Pro186 into a Leu (P186L) inherited from his father. We assessed the effect of the L108W and P186L mutations on 3 beta-HSD activity by in vitro analysis of mutant enzymes expressed in heterologous COS-1 cells. Using homogenates from transfected cells, the Km values for PREG were 7 +/- 2 and 8 +/- 2 microM for the recombinant L108W and P186L enzymes, respectively, compared with 2.2 +/- 0.2 microM for the normal type II 3 beta-HSD enzyme. Moreover, Km values for NAD+ were much higher for the L108W and P186L proteins, being 678 +/- 166 and 920 +/- 351 microM, respectively, compared with 24 +/- 3 microM for the normal type II 3 beta-HSD enzyme. Vmax values for PREG and NAD+ were lower for both mutant enzymes; thus, the in vitro overall efficiency, relative to the normal enzyme, is approximate as 0.3% and 0.2% for the L108W and P186L enzymes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection and functional characterization of the novel missense mutation Y254D in type II 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) gene of a female patient with nonsalt-losing 3 beta HSD deficiency.

Three beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) deficiency is a form of congenital adrenal hyperplasia characterized by severe impairment of steroid biosynthesis in the adrenals and gonads. To better understand the molecular basis of the phenotypic heterogeneity found in 3 beta HSD deficiency, we analyzed the structure of type I and II 3 beta HSD genes in a female patient with nonsalt-losing 3 beta HSD deficiency diagnosed at puberty. We directly sequenced DNA fragments generated by polymerase chain reaction amplification of the four exons, the exon-intron boundaries, and the 5'-flanking regions of each gene. No mutation was detected in the type I 3 beta HSD gene, which is the predominant species expressed in the placenta and peripheral tissues. We detected a novel missense mutation, Y254D, in one allele of the patient's type II 3 beta HSD gene, which is the almost exclusive type expressed in the adrenals and gonads. The influence of the Y254D mutation on enzymatic activity was assessed by analyzing the recombinant mutant enzyme generated by site-directed mutagenesis after its transient expression in COS-1 monkey kidney cells. Recombinant mutant type II 3 beta HSD enzyme carrying the Y254D substitution exhibits no detectable activity with C21 delta 5-steroid pregnenolone or C19 delta 5-steroid dehydroepiandrosterone used as substrate. The absence of restriction fragment length polymorphism by Southern blot analysis and the finding that all of the amplified DNA fragments possess the expected length suggest the absence of deletions, duplications, or re-arrangements in the other allele. A putative second mutation could be located farther than 1427 basepairs upstream of the initiation codon, thus potentially affecting the normal expression of this gene or within intronic regions, generating an alternative aberrant splicing site. These are possibilities that remain to be elucidated. The present findings, which describe the novel missense mutation Y254D in the human type II 3 beta HSD gene, provide useful information on the structure-activity relationships of the 3 beta HSD superfamily.

Congenital adrenal hyperplasia caused by a novel homozygous frameshift mutation 273 delta AA in type II 3 beta-hydroxysteroid dehydrogenase gene (HSD3B2) in three male patients of Afghan/Pakistani origin.

Classical 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency is an autosomal recessive form of congenital adrenal hyperplasia caused by mutations in the type II 3 beta-HSD (HSD3B2) gene. The sequence of the type II 3 beta-HSD gene was determined by direct sequencing of asymmetric PCR products in three male infants suffering from a severe salt-losing form of 3 beta-HSD deficiency and belonging to three families originating from Afghanistan and Pakistan. The three patients were homozygous for the frameshift mutation 273 delta AA resulting from deletion of two adenosines at codon 273, thus leading to a premature termination codon at position 279. This mutation was detected in the heterozygous state in all the relatives studied. The observation that all three patients share the same haplotype for HSD3B1A, HSD3B1C, HSD3B2A, and the microsatellite marker D1S252 indicates that a founder effect is responsible for the severe form of 3 beta-HSD deficiency found in these three families.