A novel mutation in the HSD17B10 gene of a 10-year-old boy with refractory epilepsy, choreoathetosis and learning disability

Hydroxysteroid (17beta) dehydrogenase 10 (HSD10) is a mitochondrial multifunctional enzyme encoded by the HSD17B10 gene. Missense mutations in this gene result in HSD10 deficiency, whereas a silent mutation results in mental retardation, X-linked, syndromic 10 (MRXS10). Here we report a novel missense mutation found in the HSD17B10 gene, namely c.194T>C transition (rs104886492), brought about by the loss of two forked methyl groups of valine 65 in the HSD10 active site. The affected boy, who possesses mutant HSD10 (p.V65A), has a neurological syndrome with metabolic derangements, choreoathetosis, refractory epilepsy and learning disability. He has no history of acute decompensation or metabolic acidosis whereas his urine organic acid profile, showing elevated levels of 2-methyl-3-hydroxybutyrate and tiglylglycine, is characteristic of HSD10 deficiency. His HSD10 activity was much lower than the normal control level, with normal β-ketothiolase activity. The c.194T>C mutation in HSD17B10 can be identified by the restriction fragment polymorphism analysis, thereby facilitating the screening of this novel mutation in individuals with intellectual disability of unknown etiology and their family members much easier. The patient's mother is an asymptomatic carrier, and has a mixed ancestry (Hawaiian, Japanese and Chinese). This demonstrates that HSD10 deficiency patients are not confined to a particular ethnicity although previously reported cases were either Spanish or German descendants.     

Regulation of 17-beta hydroxysteroid dehydrogenase type 2 in human placental endothelial cells

17-beta hydroxysteroid dehydrogenase type 2 (HSD17B2) oxidizes estradiol to estrone, testosterone to androstenedione, and 20 alpha-dihydroprogesterone to progesterone. HSD17B2 is highly expressed in human placental tissue where it is localized to placental endothelial cells lining the fetal compartment. The aim of this study was to investigate the effects of potential regulatory factors including progesterone, estradiol, and retinoic acid (RA) onHSD17B2 expression in primary human placental endothelial cells in culture.HSD17B2 mRNA expression was not regulated by progesterone, the progesterone agonist R5020, or estradiol treatment. RA significantly induced HSD17B2 mRNA levels and enzyme activity in a dose- and time-dependent manner. Maximal stimulation occurred at Hour 48 at an RA concentration of 10(-6) M. Both retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA) were readily detected by immunoblotting in isolated placental endothelial cells. RNA interference directed against RARA or RXRA led to reduced basal levels of HSD17B2 mRNA levels and significantly abolished RA-stimulated HSD17B2 expression. Together, these data indicate that regulation of HSD17B2 mRNA levels and enzymatic activity by RA in the placenta is mediated by RARA and RXRA.     

Characterization of the HSD17B4 gene: D-specific multifunctional protein 2/17beta-hydroxysteroid dehydrogenase IV.

The HSD17B4 gene codes for a 80 kDa multifunctional enzyme containing three distinct functional domains and is localized in peroxisomes. The N-terminal part exhibits 3-hydroxyacyl-CoA dehydrogenase and 17beta-hydroxysteroid dehydrogenase activity whereas the central part shows enoyl-CoA hydratase activity. The carboxy-terminal part of the protein has sterol-carrier-protein activity. The protein is widely expressed, however in several tissues like brain, uterus and lung its expression is limited to specific cells like Purkinje cells or luminal epithelium. The HSD17B4 gene consist of 24 exons and 23 introns with classical intron-exon junctions spanning more than 100 kbp. The importance of the HSD17B4 protein is stressed by the identification of patients with severe clinical abnormalities due to mutations in the HSD17B4 gene. We have now checked the consequences of one frequent mutation, G16 S, which results in inactivation of the enzyme due to loss of interaction with NAD+.     

Evidence for expression of 11β-hydroxysteroid dehydrogenase type3 (HSD11B3/HSD11B1L) in neonatal pig testis

11β-hydroxysteroid dehydrogenase (HSD11B) catalyzes the interconversion between active and inactive glucocorticoid, and is known to exist as two distinct isozymes: HSD11B1 and HSD11B2. A third HSD11B isozyme, HSD11B1L (SCDR10b), has recently been identified. Human HSD11B1L, which was characterized as a unidirectional NADP⁺-dependent cortisol dehydrogenase, appears to be specifically expressed in the brain. We previously reported that HSD11B1 and abundant HSD11B2 isozymes are expressed in neonatal pig testis and the Km for cortisol of NADP⁺-dependent dehydrogenase activity of testicular microsomes obviously differs from the same activity catalyzed by HSD11B1 from pig liver microsomes. Therefore, we hypothesized that the neonatal pig testis also expresses the third type of HSD11B isozyme, and we herein examined further evidence regarding the expression of HSD11B1L. (1) The inhibitory effects of gossypol and glycyrrhetinic acid on pig testicular microsomal NADP⁺-dependent cortisol dehydrogenase activity was clearly different from that of pig liver microsomes. (2) A highly conserved human HSD11B1L sequence was observed by RT-PCR in a pig testicular cDNA library. (3) mRNA, which contains the amplified sequence, was evaluated by real-time PCR and was most strongly expressed in pig brain, and at almost the same levels in the kidney as in the testis, but at lower levels in the liver. Based on these results, neonatal pig testis appears to express glycyrrhetinic acid-resistant HSD11B1L as a third HSD11B isozyme, and it may play a physiologically important role in cooperation with the abundantly expressed HSD11B2 isozyme in order to prevent Leydig cell apoptosis or GC-mediated suppression of testosterone production induced by high concentrations of activated GC in neonatal pig testis.     

Prolactin receptor-associated protein/17beta-hydroxysteroid dehydrogenase type 7 gene (Hsd17b7) plays a crucial role in embryonic development and fetal survival

Our laboratory has previously cloned and purified a protein named PRAP (prolactin receptor-associated protein) that was shown to be a novel 17beta-hydroxysteroid dehydrogenase (HSD) enzyme with dual activity. This enzyme, renamed HSD17B7 or PRAP/17beta-HSD7, converts estrone to estradiol and is also involved in cholesterol biosynthesis. The major site of its expression is the corpus luteum of a great number of species including rodents and humans. To examine the functional significance of HSD17B7 in pregnancy, we generated a knockout mouse model with targeted deletions of exons 1-4 of this gene. We anticipated a mouse with a severe fertility defect due to its inability to regulate estrogen levels during pregnancy. The heterozygous mutant mice are normal in their development and gross anatomy. The females cycle normally, and both male and female are fertile with normal litter size. To our surprise, the breeding of heterozygous mice yielded no viable HSD17B7 null mice. However, we found HSD17B7 null embryo alive in utero on d 8.5 and d 9.5. By d 10.5, the fetuses grow and suffer from severe brain malformation and heart defect. Because the brain depends on in situ cholesterol biosynthesis for its development beginning at d 10, the major cause of fetal death appears to be due to the cholesterol synthetic activity of this enzyme. By ablating HSD17B7 function, we have uncovered, in vivo, an important requirement for this enzyme during fetal development.     

Closing the gap: identification of human 3-ketosteroid reductase,the last unknown enzyme of mammalian cholesterol biosynthesis

The protein encoded by the HSD17B7 gene was originally described as a prolactin receptor-associated protein and as 17beta-hydroxysteroid dehydrogenase (HSD) type 7. Its ability to synthesize 17beta-estradiol in vitro has been reported previously. However, we demonstrate that HSD17B7 is the ortholog of the yeast 3-ketosteroid reductase Erg27p and converts zymosterone to zymosterol in vitro, using reduced nicotinamide adenine dinucleotide phosphate as cofactor. Expression of human and murine HSD17B7 in an Erg27p-deficient yeast strain complements the 3-ketosteroid reductase deficiency of the cells and restores growth on sterol-deficient medium. A fusion of HSD17B7 with green fluorescent protein is located in the endoplasmic reticulum, the site of postsqualene cholesterogenesis. Further critical evidence for a role of HSD17B7 in cholesterol metabolism is provided by the observation that its murine ortholog is a member of the same highly distinct embryonic synexpression group as hydroxymethyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme of sterol biogenesis, and is specifically expressed in tissues that are involved in the pathogenesis of congenital cholesterol-deficiency disorders. We conclude that HSD17B7 participates in postsqualene cholesterol biosynthesis, thus completing the molecular cloning of all genes of this central metabolic pathway. In its function as the 3-ketosteroid reductase of cholesterol biosynthesis, HSD17B7 is a novel candidate for inborn errors of cholesterol metabolism.     

2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency is caused by mutations in the HADH2 gene

2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency is a novel inborn error of isoleucine degradation. In this article, we report the elucidation of the molecular basis of MHBD deficiency. To this end, we purified the enzyme from bovine liver. MALDI-TOF mass spectrometry analysis revealed that the purified protein was identical to bovine 3-hydroxyacyl-CoA dehydrogenase type II. The human homolog of this bovine enzyme is a short-chain 3-hydroxyacyl-CoA dehydrogenase, also known as the "endoplasmic reticulum-associated amyloid-beta binding protein" (ERAB). This led to the identification of the X-chromosomal gene involved, which previously had been denoted "HADH2." Sequence analysis of the HADH2 gene from patients with MHBD deficiency revealed the presence of two missense mutations (R130C and L122V). Heterologous expression of the mutant cDNAs in Escherichia coli showed that both mutations almost completely abolish enzyme activity. This confirms that MHBD deficiency is caused by mutations in the HADH2 gene.     

Peroxisome targeting of porcine 17beta-hydroxysteroid dehydrogenase type IV/D-specific multifunctional protein 2 is mediated by its C-terminal tripeptide AKI

The product of the porcine HSD17B4 gene is a peroxisomal 80 kDa polypeptide containing three functionally distinct domains. The N-terminal part reveals activities of 17beta-estradiol dehydrogenase type IV and D-specific 3-hydroxyacyl CoA dehydrogenase, the central part shows D-specific hydratase activity with straight and 2-methyl-branched 2-enoyl-CoAs. The C-terminal part is similar to sterol carrier protein 2. The 80 kDa polypeptide chain ends with the tripeptide AKI, which resembles the motif SKL, the first identified peroxisome targeting signal PTS1. So far AKI, although being similar to the consensus sequence PTS1, has neither been reported to be present in mammalian peroxisomal proteins, nor has it been shown to be functional. We investigated whether the HSD17B4 gene product is targeted to peroxisomes by this C-terminal motif. Recombinant human PTS1 binding protein Pex5p interacted with the bacterially expressed C-terminal domain of the HSD17B4 gene product. Binding was competitively blocked by a SKL-containing peptide. Recombinant deletion mutants of the C-terminal domain lacking 3, 6, and 14 amino acids and presenting KDY, MIL, and IML, respectively, at their C-termini did not interact with Pex5p. The wild-type protein and mutants were also transiently expressed in the HEK 293 cells. Immunofluorescence analysis with polyclonal antibodies against the C-terminal domain showed a typical punctate peroxisomal staining pattern upon wild-type transfection, whereas all mutant proteins localized in the cytoplasm. Therefore, AKI is a functional PTS1 signal in mammals and the peroxisome targeting of the HSD17B4 gene product is mediated by Pex5p.     

Genetic diversity and admixture patterns in Indian populations

The clinical, biochemical and genetic features of a Cypriot origin male of non-consanguineous parents due to 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD-3) deficiency are presented. The patient, currently a 10 old male, was referred to our clinic because of ambiguous genitalia at birth. Gonads were palpable in the inguinal canal bilaterally and no Müllerian structures identified on pelvic ultrasound. Chromosomal analysis showed an apparently normal male 46,XY karyotype. Diagnosis of 17β-HSD-3 deficiency in the newborn was suspected based on biochemical findings, following human chorionic gonadotrophin (hCG) stimulation test. Sequence analysis and real time PCR along with MLPA identified the patient with a novel 11.96 kb duplication that spans exons 3-10 of the HSD17B3 gene and extends from intron 2 to intron 10 in compound heterozygosity with the known p.R80Q missense mutation leading to 17β-HSD-3. In conclusion, 17β-HSD-3 deficiency was diagnosed in this patient based on endocrinologic evaluation and confirmed with genetic analysis of the HSD17B3 gene. The novel large duplication spanning exons 3-10 of the HSD17B3 gene that we report here in compound heterozygosity with the known p.R80Q leads to 17β-HSD-3 deficiency presenting as 46,XY Disorder of Sex Development. Following diagnosis and appropriate genetic counselling, the patient was raised a boy and successfully underwent surgical correction of crytptorchidism and hypospadias.     

Placenta defects and embryonic lethality resulting from disruption of mouse hydroxysteroid (17-beta) dehydrogenase 2 gene

Hydroxysteroid (17-beta) dehydrogenase 2 (HSD17B2) is a member of aldo-keto reductase superfamily, known to catalyze the inactivation of 17beta-hydroxysteroids to less active 17-keto forms and catalyze the conversion of 20alpha-hydroxyprogesterone to progesterone in vitro. To examine the role of HSD17B2 in vivo, we generated mice deficient in Hsd17b2 [HSD17B2 knockout (KO)] by a targeted gene disruption in embryonic stem cells. From the homozygous mice carrying the disrupted Hsd17b2, 70% showed embryonic lethality appearing at the age of embryonic d 11.5 onward. The embryonic lethality was associated with reduced placental size measured at embryonic d 17.5. The HSD17B2KO mice placentas presented with structural abnormalities in all three major layers: the decidua, spongiotrophoblast, and labyrinth. Most notable was the disruption of the spongiotrophoblast and labyrinthine layers, together with liquid-filled cysts in the junctional region and the basal layer. Treatments with an antiestrogen or progesterone did not rescue the embryonic lethality or the placenta defect in the homozygous mice. In hybrid background used, 24% of HSD17B2KO mice survived through the fetal period but were born growth retarded and displayed a phenotype in the brain with enlargement of ventricles, abnormal laminar organization, and increased cellular density in the cortex. Furthermore, the HSD17B2KO mice had unilateral renal degeneration, the affected kidney frequently appearing as a fluid-filled sac. Our results provide evidence for a role for HSD17B2 enzyme in the cellular organization of the mouse placenta.     

Lack of association between common polymorphisms in the 17beta-hydroxysteroid dehydrogenase type V gene (HSD17B5) and precocious pubarche

BACKGROUND: 17beta-Hydroxysteroid dehydrogenase (type V; HSD17B5) is a key enzyme involved in testosterone production in females. A single nucleotide polymorphism (SNP) in the promoter region of its gene was recently found to be associated with polycystic ovary syndrome (PCOS) and its related hyperandrogenaemia. Precocious pubarche (PP) is a clinical entity pointing to adrenal androgen excess from mid-childhood onward and is associated with ovarian androgen excess from puberty onward. It is therefore a strong risk factor for PCOS. METHODS: To investigate associations between this promoter SNP along with three exonic SNPs (one non-synonymous and two synonymous) from the same gene, and PP, a case-control study was performed in 190 girls with PP (84 of which were also tested for functional ovarian hyperandrogenism) from Barcelona, Spain and 71 healthy controls. Clinical features and hormone concentrations relevant to hyperandrogenism were compared by HSD17B5 genotype and haplotype. RESULTS: Neither HSD17B5 genotypes nor haplotype were associated with PP, or subsequent androgen excess in girls from Barcelona (all P>0.05). CONCLUSIONS: HSD17B5 SNPs predicted to have functional effects do not appear to be a risk factor for PP in girls from Barcelona, despite these girls being at high risk of developing androgen excess in adulthood.     

A 5-methylcytosine hotspot responsible for the prevalent HSD17B10 mutation

Approximately half of the cases of hydroxysteroid (17β) dehydrogenase X (HSD10) deficiency are due to a missense C>T mutation in exon 4 of the HSD17B10 gene. The resulting HSD10 (p.R130C) loses most or all catalytic functions, and the males with this mutation have a much more severe clinical phenotype than those carrying p.V65A, p.L122V, or p.E249Q mutations. We found that the mutated cytosine which is + 2259 nucleotide from the ATG of the gene, is > 90% methylated in both the active and inactive X chromosomes in two normal females as well as in the X chromosome of a normal male. Since 5-methylcytosine is prone to conversion to thymine by deamination, the methylation of this cytosine in normal X chromosomes provides an explanation for the prevalence of the p.R130C mutation among patients with HSD10 deficiency. The substitution of arginine for cysteine eliminates several hydrogen bonds and reduces the van der Waals interaction between HSD10 subunits. The resulting disruption of protein structure impairs some if not all of the catalytic and non-enzymatic functions of HSD10. A meta-analysis of residual HSD10 activity in eight patients with the p.R130C mutation showed an average 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) activity of only 6 (± 5) % of the normal control level. This is significantly lower than in cells of patients with other, clinically milder mutations and suggests that the loss of HSD10/MHBD activity is a marker for the disorder.     

环腺苷一磷酸对人Ⅰ型17β羟类固醇脱氢酶在绒癌细胞系中表达的调节作用

由HSD17B1基因编码的人Ⅰ型17β-羟类固醇脱氢酶(17β-hydroxysteroid dehydrogenasetype 1,简称Ⅰ型17HSD)催化雌酮与雌二醇之间的转化。本文研究环腺苷一磷酸简称(cAM-P)对该酶在培养的绒癌细胞系(JAR和JEG-3)中表达的调节作用。用8-bromo-cAMP处理两种绒癌细胞后,观察到在伴随1.3 kbⅠ型17 HSDmRNA表达的同时,Ⅰ型17 HSD蛋白浓度也显著上升。标记基因分析表明,cAMP可诱导HSD 17 B1基因启动子在JAR和JEG-3细胞系中的转录活性,参与调节这一诱导作用的区域位于HSD 17 B1基因编码区上游-659至-550处。凝胶阻滞实验显示这一区域可同JAR、JEG-3、T-47 D和HeLa细胞核抽提物形成特异的DNA-蛋白复合物。本结果首次证实cAMP激活HSD 17 B1基因启动子在绒癌细胞中的转录。