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Results for “CYP19 gene”

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At least 19 recordsLinked to original sources

PubMed2002

Association of the CYP17 gene and CYP19 gene polymorphisms with risk of endometriosis in Japanese women.

BACKGROUND: To investigate whether polymorphisms of CYP17 and CYP19 genes are associated with the risk of endometriosis, we analysed the frequency and distribution of a single nucleotide polymorphism at the 5' untranslated region of the CYP17 gene, and a tetranucleotide (TTTA) tandem repeat polymorphism and a 3 bp insertion (I)/deletion (D) polymorphism in intron 4 of the CYP19 gene. METHODS: We studied 140 patients with endometriosis, 67 with adenomyosis and/or leiomyomas and 177 healthy control women. RESULTS: The distribution of the genotypes of CYP17 and alleles of the TTTA repeat polymorphism of CYP19 were not significantly different between the groups. In contrast, an increased frequency of the D/D genotype was observed in the endometriosis group as compared with the control group (D/D genotype versus I/I plus I/D genotypes; corrected P = 0.024). This was more evident in the endometriosis subgroups with chocolate cysts (corrected P = 0.043) and at severe clinical stages (corrected P = 0.035). CONCLUSIONS: The results suggest that the 3 bp I/D polymorphism of the CYP19 gene may be weakly associated with the susceptibility of endometriosis in a Japanese population.

Adult↗

PubMed2000

Cis-acting elements regulating the placenta-specific promoter of the bovine Cyp19 gene.

Cyp19 encodes aromatase cytochrome P450, the key enzyme of oestrogen biosynthesis. In the bovine placenta, the majority of Cyp19 transcripts include a 5' untranslated region which is encoded by exon 1.1; this suggests that its 5'-flanking region is the predominant placental promoter. The aim of the present investigation was to examine the promoter activity of this region and to map cis-acting regulatory elements in order to improve our understanding of the complex regulation of this gene within the placenta. As an initial approach, human JEG-3 choriocarcinoma cells were transiently transfected with reporter-gene constructs consisting of different 5'-flanking sequences of exon 1.1 fused to the luciferase gene as a reporter. To localise and further characterise functional cis-acting elements, targeted point mutations and electrophoretic mobility-shift experiments were used. The data demonstrate, for the first time, (1) that the bovine exon 1.1 5'-flanking sequence is an active promoter, (2) that 404 bp of this region are sufficient for constitutive reporter-gene expression in JEG-3 cells and (3) that the region includes at least two different enhancer elements; the data also suggest (4) that one of these elements consists of the E-box motif CATGTG and that the second enhancer element includes the half-site hexameric sequence AGGTCA and additional nucleotides flanking this element upstream.

Animals↗

PubMed2002

Overexpression of aromatase P450 in leiomyoma tissue is driven primarily through promoter I.4 of the aromatase P450 gene (CYP19).

The CYP19 gene encoding aromatase P450 (estrogen synthetase) is expressed in several extragonadal sites and regulated in a tissue-specific fashion, which is achieved by alternative use of the seven different promoters (and corresponding exons 1) of the CYP19 gene. Previously, we demonstrated that aromatase P450 is overexpressed in leiomyoma tissue and that in situ estrogen synthesized in leiomyoma tissues possibly plays a role in leiomyoma growth. To elucidate the mechanism of overexpression of aromatase P450, we determined the promoter use of aromatase P450 in leiomyomas. 5'-Rapid amplification of cDNA ends analysis revealed that of six leiomyoma nodules tested, four nodules contained I.4-specific transcript of aromatase P450 alone, one nodule contained PII-specific transcript alone, and the remaining nodule contained both I.4- and PII-specific transcripts simultaneously. The levels of aromatase transcripts were then quantified by competitive RT-PCR assay. Among 21 leiomyomas, I.4-specific transcript and PII-specific transcript were predominant in 18 and 2 leiomyomas, respectively, whereas the remaining leiomyoma was negative for aromatase P450 expression. We next compared the aromatase activity of leiomyoma cells stimulated by promoter-specific regulatory factors. A combination of IL-1beta and dexamethasone, known as a potent inducer of promoter I.4-driven transcription, effectively increased aromatase activity. A combination of (Bt)(2)cAMP, 3-isobutyl-1-myethylxanthine, and PGE(2), known as inducers of promoter II-driven transcription, also increased aromatase activity, but the increases found were smaller than that induced by dexamethasone and IL-1beta. The transcriptional ability of the promoter I.4 sequence was confirmed by transient transfection assay using primary cells released from leiomyomas and established cells from normal myometrium (KW cells). Luciferase vectors containing promoter I.4 sequence (-340/+14 or longer) showed a significant increase in luciferase activity in response to dexamethasone. Deletion or mutation of a putative glucocorticoid-responsive element in the promoter I.4 sequence eliminated promoter activity. These results indicate that promoter I.4 is the major promoter responsible for overexpression of aromatase P450 in leiomyomas and that a glucocorticoid-responsive element within it plays a substantial role in the expression of aromatase P450.

Adult↗

PubMed2001

A loss of aggressive behaviour and its reinstatement by oestrogen in mice lacking the aromatase gene (Cyp19).

Aromatase P450 (CYP19) is an enzyme responsible for conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeting disruption of the CYP19 gene and observed that the ArKO males exhibited a complete loss of aggressive behaviour against intruder mice when examined using a resident-intruder paradigm. The defect in the behaviour of ArKO males was reinstated when the mice received supplements of 17beta-oestradiol soon after birth. Nevertheless, the cumulative duration of the behaviour displayed by the treated mice during the test period of 15 min was 19+/-10 s, which was much shorter than that displayed by wild-type males, 90+/-17 s. When the supplementation was started at 7 days after birth, the defect was not restored. These findings illustrate an absolute requirement for oestrogen during the neonatal stage of a male's life for the development of the potential for aggression observed in adulthood. Furthermore, the present study demonstrates that ArKO males are a useful model in which to investigate the neural mechanisms by which aggressive behaviour is controlled.

Age Factors↗

PubMed2005

Are the T/C polymorphism of the CYP17 gene and the tetranucleotide repeat (TTTA) polymorphism of the CYP19 gene genetic markers for premature coronary artery disease in Caucasians?

Gender differences in CAD have been clearly documented, and sex hormones have been recognized to influence the risk of CAD. The cytochrome P450c17alpha gene (CYP17) and the CYP19 gene influence concentrations of sex hormones. In this cross-sectional association study we tested the hypothesis whether the T/C polymorphism of the CYP17 gene and the tetranucleotide repeat (TTTA) polymorphism of the CYP19 gene are genetic markers for CAD in Caucasians. The TT genotype of the CYP17 gene polymorphism was not associated with premature CAD in men and women combined (OR 0.9; 95% CI = 0.6-1.4; P = 0.7), in men only (OR 1; 95% CI = 0.6-1.8; P = 0.7), and in women only (OR 0.8; 95% CI = 0.5-1.4; P = 0.4). The tetranucleotide repeat (TTTA) CYP19 gene polymorphism was not associated with premature CAD. Moreover, the genotypes containing the longer alleles (A6 or A7) were not associated with a lower incidence of CAD, and the genotypes containing the shorter alleles (A1 or A2) were not over-represented in the CAD patients. We may conclude that in Caucasian subjects neither the T/C CYP17 gene polymorphism nor the tetranucleotide repeat (TTTA) polymorphism of the CYP19 gene contributes to the genetic susceptibility to CAD, therefore they may not be used as genetic markers for CAD risk assessment.

Age of Onset↗

PubMed2006

The T/C polymorphism of the CYP17 gene and G/A polymorphism of the CYP19 gene in endometrial cancer.

Endogenous estrogen exposure is an important determinant of endometrial cancer risk. The CYP17 and CYP19 genes encode 17 hydroxylase/17,20-lyase and aromatase, respectively, both involved in sex hormone synthesis. The gene CYP17 and CYP19 are polymorphic and gene variability could contribute to the level of protein biosynthesis. In the present work the distribution of genotypes and frequency of alleles of the C/T polymorphism in promoter region of CYP17 and G/A polymorphism at position Val80 in CYP19 in subjects with endometrial cancer were investigated. Paraffin embedded tumour tissues were obtained from 100 women with endometrial cancer. DNA from normal endometrial tissue (n = 106) served as control. The polymorphisms were determined by PCR-RFLP. The distribution of the genotypes of the C/T polymorphism of CYP17 and G/A polymorphism of CYP19 in both control and patients did not differ significantly (p > 0.05) from those predicted by the Hardy-Weinberg distribution. There were no significant differences (p > 0.05) in genotype distributions and allele frequencies between subgroups assigned to histological stage. The results suggest that C/T polymorphism of the CYP17 gene as well as G/A polymorphism of CYP19 may not be linked with onset and development of endometrial cancer.

Adenocarcinoma↗

PubMed1996

Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients.

Estrogen biosynthesis in adipose tissue has assumed great significance in terms of a number of estrogen-related diseases. Recent evidence suggests that estrogen synthesized locally in the breast is of singular significance in the development of breast cancer in elderly women. The biosynthesis of estrogen from C19 steroids is catalyzed by a specific form of cytochrome P450, namely aromatase cytochrome P450 (P450arom; the product of the CYP19 gene). The human CYP19 gene comprises nine coding exons, II-X, and its transcripts are expressed in the ovary, placenta, testes, adipose tissue, and brain. Tissue-specific expression of the CYP19 gene is determined, at least in part, by the use of tissue-specific promoters, which give rise to transcripts with unique 5'-noncoding termini. Transcripts in adipose tissue contain 5'-termini derived from specific untranslated exons, corresponding to expression derived from the proximal promoter II and its splice variant I.3, as well as a distal promoter, I.4. The object of the present study was to determine the distribution of these various exon-specific transcripts in breast adipose tissues from cancer-free women undergoing reduction mammoplasty and from patients with breast cancer, because this would provide important clues as to the nature of the factors regulating aromatase expression in these sites. To achieve this, we employed competitive RT-PCR, utilizing an internal standard for each exon-specific transcript of the CYP19 gene, as well as for the coding region, to evaluate total CYP19 gene transcripts. In cancer patients (n = 18), total CYP19 gene transcript levels were significantly higher in adipose tissue proximal to a tumor in comparison with adipose tissue distal to a tumor, in agreement with previous findings. Moreover, total transcript levels were higher in breast adipose tissue of cancer patients in comparison with those of cancer-free individuals (n = 9), even when the adipose tissue from the cancer patient was taken from a quadrant with no detectable tumor. We observed that exon I.4-specific transcripts were predominant in breast adipose obtained from cancer-free women. In this tissue, promoter-II-specific and exon I.3-specific transcripts were present in low copy number. On the other hand, in breast cancer patients, CYP19 gene transcripts from breast adipose tissue had primarily promoter-II-specific and exon I.3-specific sequence, whereas comparatively few transcripts had exon I.4-specific sequence at the 5'-terminus. We conclude that CYP19 gene transcription in breast adipose tissue of cancer-free individuals uses preferably promoter I.4, implicating a role of glucocorticoids and members of the IL-6 cytokine family in the regulation of this expression. On the other hand, the increased expression in breast adipose tissue bearing a carcinoma results from expression from promoters II and I.3, which are regulated by unknown factors acting via increased cAMP formation, which are presumably secreted by the tumor or associated cells.

Adipose Tissue↗

PubMed2000

Polymorphisms in the human aromatase cytochrome P450 gene (CYP19) and breast cancer risk.

The aromatase enzyme catalyses the conversion of androgens to oestrogens in the oestrogen biosynthesis pathway. Because increased exposure to oestrogens is considered to be a risk factor for breast cancer, the human aromatase gene (CYP19) is a plausible candidate for low penetrance breast cancer susceptibility. Preliminary reports have suggested that specific alleles of a TTTA repeat may be associated with differences in breast cancer risk. We have identified two new polymorphisms in the CYP19 gene: a TCT insertion/deletion in intron 4 and a G-->T substitution in intron 6, which have rare allele frequencies of 0.35 and 0.45, respectively, in the British population. Comparison was made between the frequencies of these alleles and those of the TTTA repeat in up to 599 breast cancer cases and 433 normal controls from the East Anglian, British population. We found strong linkage disequilibrium between the alleles of these three loci, but no significant association of any alleles with breast cancer risk. The maximum odds ratios observed were: 1.03 (95% CI 0.68-1.55) for the intron 4 TCT insertion/deletion polymorphism [del/del versus ins/ins]; 1.56 (95% CI 0.63-3.83) for the intron 4 [TTTA](10) allele; 1.29 (95% CI 0. 75-2.21) for the intron 6 G-->T polymorphism [TT versus GG]. We conclude that the CYP19 gene has no major role in common breast cancer incidence in the British population.

Adult↗

PubMed1997

Alternatively spliced transcripts of the aromatase cytochrome P450 (CYP19) gene in adipose tissue of women.

Estrogen biosynthesis in adipose tissue has assumed great significance in terms of a number of estrogen-related diseases. The biosynthesis of estrogens from C19 steroids is catalyzed by a specific form of cytochrome P450, namely aromatase cytochrome P450 (P450arom; the product of the CYP19 gene). The human CYP19 gene comprises nine coding exons, II-X, and its transcripts are expressed in the ovary, placenta, testes, adipose tissue, and brain. Tissue-specific expression of the CYP19 gene is determined at least in part by the use of tissue-specific promoters, which give rise to transcripts with unique 5'-noncoding termini. Thus, the distal promoter I.1 is responsible for expression uniquely in placenta. On the other hand, the proximal promoter II, which regulates expression via a cAMP-dependent signaling pathway, is responsible for expression in the gonads. Transcripts in breast adipose tissue contain 5'-termini corresponding to expression derived from promoters I.4, II, and I.3, with I.4-specific termini predominating. The latter are derived from promoter I.4, which contains a glucocorticoid response element and an interferon-gamma activation site element and is responsible for expression in the presence of glucocorticoids and members of the class I cytokine family. The object of the present study was to determine the distribution of these various transcripts in adipose tissue from abdomen, buttocks, and thighs of women, as this would provide important clues to the factors regulating aromatase expression in these sites. To achieve this, we employed competitive reverse transcription-PCR to amplify unique 5'-ends of each of the transcripts of the CYP19 gene that are expressed in adipose tissue as well as for the coding region to evaluate total CYP19 gene (P450arom) transcript levels. We observed that exon I.4-specific transcripts were predominantly present in adipose tissue samples obtained from women regardless of the tissue site or the age of the individual. In these tissues, promoter II- and exon I.3-specific transcripts were present in lower copy numbers. We also demonstrated that in these sites total or exon-specific P450arom transcripts levels increased in direct proportion to advancing age and that transcript levels were the highest in buttocks, followed by thighs, and lowest in the abdomen. These results suggest that in normal human adipose tissue, aromatase expression is mainly under local control by a number of cytokines via paracrine and autocrine mechanisms in the presence of systemic glucocorticoids.

Adipose Tissue↗

PubMed2000

A polymorphic tetranucleotide repeat in the CYP19 gene and male breast cancer.

The CYP19 gene codes for the aromatase enzyme that is involved in the synthesis of oestrogens. This case-control study examines the relationship between a tetranucleotide repeat sequence in the CYP19 gene and the development of male breast cancer. No significant differences were found between male breast cancer cases and controls.

Aromatase↗

PubMed2004

Organization of the human aromatase p450 (CYP19) gene.

The human CYP19 (p450arom) gene is located in the 21.2 region on the long arm of chromosome 15 (15q21.2). This gene spans a region that consists of a 30 kb coding region and a 93 kb regulatory region ( approximately 123 kb total length). Its regulatory region contains at least 10 distinct promoters regulated in a tissue- or signaling pathway-specific manner. The Human Genome Project data published in 2000 enabled us to accurately align these promoters within the 93 kb regulatory region of the p450arom gene. Each promoter is regulated by a distinct set of regulatory sequences in DNA and transcription factors that bind to these specific sequences. In most vertebrates, p450arom expression is under the control of gonad- and brain-specific promoters. In humans, however, there are at least eight additional promoters that were apparently recruited throughout evolution, possibly via alterations in DNA. A critical mechanism that permits the use of such a large number of promoters seems to be the extremely promiscuous nature of the common splice acceptor site because, with activation of each promoter, an untranslated first exon is spliced onto this common junction immediately upstream of the translation start site in the coding region. These partially tissue-specific promoters are used in the gonads, bone, brain, vascular tissue, adipose tissue, skin, fetal liver, and placenta for estrogen biosynthesis necessary for human physiology. Ovary and testis use promoter II, which is located immediately upstream of the coding region. The adipose tissue in general, including adipose tissue of the disease-free breast, on the other hand, maintains low levels of aromatase expression primarily via promoter I.4, which lies 73 kb upstream of the common coding region. Promoters I.3 and II are used only minimally in normal breast adipose tissue. Promoter II and I.3 activities in breast cancer tissue, however, are strikingly increased. Additionally, the endothelial-type promoter I.7 is also upregulated in breast cancer. Therefore, breast tumor tissue takes advantage of four promoters (II, I.3, I.7, and I.4) for aromatase expression and estrogen production. The sum of p450arom mRNA species arising from these four promoters contributes significantly to elevated levels of total p450arom mRNA in breast cancer in contrast to the normal breast that uses promoter I.4. Because each mRNA species contains the identical coding region regardless of the variable untranslated first exon, the encoded protein functions as the aromatase enzyme in each case.

Aromatase↗

PubMed2006

Promoter activity and chromosomal location of the Rana rugosa P450 aromatase (CYP19) gene.

Sex is determined genetically in amphibians, but is reversed occasionally by steroid hormones. The phenotypic sex of some amphibian species can be reversed from male to female by estrogens. Estrogens, which are synthesized from testosterone irreversibly by the enzyme P450 aromatase (CYP19), are essential for ovarian development in vertebrates. CYP19 expression is reportedly regulated by steroidogenic factor-1 (SF-1), also designated as Ad4BP, in fish and mammals. However, it is unknown if this is also the case in amphibians. Thus, to elucidate the role of SF-1 in CYP19 gene expression in the gonad of amphibians, it is necessary to isolate and characterize the promoter region of the CYP19 gene of amphibians. For this purpose, we first cloned the promoter region of CYP19 from genomic DNA fragments of the frog Rana rugosa. As a result, a potential binding site of SF-1 was found in the region. When a luciferase promoter assay in HEK 293 cells was carried out to examine the ability of SF-1 as a transcriptional regulator, we found that R. rugosa SF-1 stimulated the expression of the CYP19 gene of the tilapia Oreochromis niloticus, but not that of the frogs R. rugosa and Xenopus laevis. RT-PCR analysis revealed that CYP19 mRNA was expressed at a higher level in the indifferent gonads of females than in those of males. This was also true to SF-1 mRNA In addition, FISH analysis showed that the CYP19 gene was located on chromosome 3 of R. rugosa. Taken together, our data suggest that CYP19, an autosomal gene, is expressed in the undifferentiated gonads to an extent greater in females than in males, but its expression probably is not regulated by SF-1 alone. Another factor(s) may be required if SF-1 promotes the expression of the CYP19 gene in R. rugosa as it does in fish and mammals.

Animals↗

PubMed1999

Constitutional genetic variation at the human aromatase gene (Cyp19) and breast cancer risk.

The activity of the aromatase enzyme, which converts androgens into oestrogens and has a major role in regulating oestrogen levels in the breast, is thought to be a contributing factor in the development of breast cancer. We undertook this study to assess the role of constitutional genetic variation in the human aromatase gene (Cyp19) in the development of this disease. Our genotyping of 348 cases with breast cancer and 145 controls (all Caucasian women) for a published tetranucleotide repeat polymorphism at intron 4 of the Cyp19 gene revealed the presence of six common and two rare alleles. Contingency table analysis revealed a significant difference in allelic distribution between cases and controls (chi2 5df = 13.52, P = 0.019). The allele measuring 171 bp was over-represented in cases; of 14 individuals homozygous for this allele, 13 were cases. These individuals had a higher incidence of cancer in family members and an earlier age at diagnosis than other cases. In sequencing Cyp19's coding exons and regulatory regions, we discovered a perfect association between a silent polymorphism (G-->A at Val80) and the high-risk genotype. Our conclusion is that constitutional genetic variation at the Cyp19 locus is associated with the risk of developing breast cancer, with the 171-bp allele serving as the high-risk allele.

Adult↗

PubMed2004

Effects of endocrine disrupting chemicals on the expression of CYP19 genes in zebrafish (Danio rerio) juveniles.

Cytochrome P450 aromatase (CYP19) is the key steroidogenic enzyme responsible for conversion of androgens to estrogens which play a critical role in developmental sex differentiation and adult reproductive cycles in vertebrates. To evaluate the potential roles of endocrine disrupting chemicals (EDC) on reproductive physiology of fish, the influence of multiple classes of EDC on the transcript abundance of two CYP19 isoforms, CYP19A1 and A2, were investigated in zebrafish juveniles. The pharmaceutical, clofibrate, and the pesticide, atrazine, did not influence the expression of either CYP19 gene. Estrogenic compounds, nonylphenol (NP) and a pharmaceutical estrogen, ethinylestradiol (EE), strongly enhanced the expression of CYP19A2 gene in dose-dependent manner. Exposure to benzo[a]pyrene (BaP) significantly increased CYP19A2 transcript abundance. Furthermore, BaP when co-treated with EE partially suppressed EE-induced upregulation of CYP19A2. In contrast, the expression of CYP19A1 was basically resistant to EDC treatment although EE at high concentration (1-100 nM) downregulated its expression. These findings suggest that multiple classes of EDC may potentially perturb developmental and reproductive physiology in fish through differential transcriptional modulation of the CYP19 genes with the most evident disruption in neural tissue.

Animals↗

PubMed2001

Identification of the regulatory regions of the human aromatase P450 (CYP19) gene involved in placenta-specific expression.

Expression of the human CYP19 gene in placental syncytiotrophoblast, ovarian granulosa and luteal cells and adipose stromal cells is regulated by tissue-specific promoters which lie upstream of unique untranslated first exons. In placenta, the majority of CYP19 mRNA transcripts contain 5'-sequences encoded by exon I.1 which lies >35 kb upstream of the translation initiation sequence in exon II. Mononuclear cytotrophoblasts isolated from midterm human placenta spontaneously fuse in culture to form multinucleated syncytiotrophoblast. These morphological changes are associated with a marked induction of CYP19 gene expression. To functionally define genomic regions required for placenta-specific expression, fusion genes containing various amounts of exon I.1 5'-flanking sequence linked to the human growth hormone (hGH) structural gene, as reporter, were introduced into human trophoblast cells in primary monolayer culture and into transgenic mice. Our findings using transfected cells and transgenic mice suggest that sequences between -501 and -42 bp upstream of exon I.1 contain a positive enhancer element(s) that mediates the actions of trophoblast-specific transcription factors, as well as a negative element(s) that binds inhibitory transcription factors in other cell types. Our findings from transgenic studies further indicate that mouse placenta contains the necessary transcription factors required to activate the human CYP19 promoter although mouse placenta does not express endogenous aromatase.

Animals↗

PubMed2025

Association of CYP19 gene SNPs (rs7176005 and rs6493497) with polycystic ovary syndrome susceptibility in Northern Chinese women.

PURPOSE: The objective of this study was to elucidate the relationship between two single nucleotide polymorphisms (SNPs) rs7176005 and rs6493497 in CYP19 gene and the risk of polycystic ovary syndrome (PCOS) in Northern Chinese women. METHODS: In this case-control study, a total of 340 women with PCOS and 340 matched healthy controls were recruited. Polymerase chain reaction ligase detection reaction (PCR-LDR) method was used to investigate two SNPs (rs7176005 and rs6493497) in the 5'-flanking region of CYP19 gene exon 1. RESULTS: We observed a significant association of rs7176005 and rs6493497 with reduced risk of PCOS. Compared with CC genotype, a significant association of CT genotype (p = 0.019), TT genotype (p < 0.001) and combined CT + TT genotype (p < 0.001) with reduced risk of PCOS was observed. The result of linkage disequilibrium analysis showed that these two SNPs are in complete linkage disequilibrium (r2 = 1). For rs7176005 SNP, compared with CC genotype, CT, TT and CT + TT genotypes reduced the risk of PCOS. The age, BMI-adjusted OR were 0.650 (95% CI = 0.460-0.917), 0.158 (95% CI = 0.066-0.376) and 0.545(95% CI = 0.391-0.759), respectively. CONCLUSIONS: These findings highlight a significant association between CYP19 gene polymorphisms and PCOS susceptibility, implying potential protective effects of T and A alleles. Of course, the major limitation of this study is the sample size of the case-control study. Larger cohort studies are needed to confirm these findings and investigate the underlying causes.

Adult↗

PubMed1995

Quantitative detection of alternatively spliced transcripts of the aromatase cytochrome P450 (CYP19) gene in aromatase-expressing human cells by competitive RT-PCR.

C19 steroids are converted to oestrogens in a number of tissues by a specific form of cytochrome P450, namely aromatase P450 (P450arom; the product of the CYP19 gene). The human CYP19 gene comprises nine coding exons, II-X. The tissue-specific expression is determined by the use of tissue-specific promoters, which give rise to P450arom transcripts with unique 5'-untranslated sequences. The majority of the transcripts present in the ovary contain promoter-II-specific sequences, while transcripts in the placenta contain exon I.1. Transcripts in adipose tissue possess exon I.3 and exon I.4. Also, the distribution of alternative transcripts in adipose stromal cells depends on the culture conditions. Therefore, a competitive RT-PCR method was designed to quantitatively detect alternatively spliced transcripts present in various tissues and cells maintained in different culture conditions. Specific synthetic transcripts with different 5' termini (exon I.3, exon I.4 and promoter-II-specific sequences) and the coding region were used as internal standards. This competitive RT-PCR method was used to quantitatively detect three 5' termini, i.e. promoter-II-specific sequence, exon I.3 and exon I.4, in transcripts in human adipose stromal cells and ovarian granulosa cells in primary culture. The quantity of total P450arom transcripts was judged by amplifying the coding region. We were also able to quantify rare transcripts which could not be detected previously by Northern analysis.

Adipose Tissue↗

PubMed2002

Impact of estrogen replacement therapy in a male with congenital aromatase deficiency caused by a novel mutation in the CYP19 gene.

Recent reports of the impact of estrogen receptor alpha and aromatase deficiency have shed new light on the importance of estrogen for bone formation in man. We describe a novel mutation of the CYP19 gene in a 27-yr-old homozygous male of consanguinous parents. A C to A substitution in intron V, at position -3 of the splicing acceptor site before exon VI of the CYP19 gene, is the likely cause of loss of aromatase activity. The mRNA of the patient leads to a frameshift and a premature stop codon 8 nucleotides downstream the end of exon V. Both parents were shown to be heterozygous for the same mutation. Apart from genua valga, kyphoscoliosis, and pectus carniatus, the physical examination was normal including secondary male characteristics with normal testicular size. To substitute for the deficiency, the patient was treated with 50 micro g transdermal estradiol twice weekly for 3 months, followed by 25 micro g twice weekly. After 6 months estrogen levels (<20 at baseline and 45 pg/ml at 6 months; normal range, 10-50) and estrone levels (17 and 34 ng/ml; normal range, 30-85) had normalized. Bone maturation progressed and the initially unfused carpal and phalangeal epiphyses began to close within 3 months and were almost completely closed after 6 months. The bone age, assessed by roentgenographic standards for bone development by Gruelich and Pyle, was 16.5 at baseline and 18-18.5 yr after 6 months of treatment. Bone density of the distal radius (left), assessed by quantitative computed tomography, increased from 52 to 83 mg/cm(3) (normal range, 120-160) and bone mineral density of the lumbar spine, assessed by dual-energy x-ray-absorptiometry, increased from 0.971 to 1.043 g/cm(2) (normal range, >1.150). Osteocalcin as a bone formation parameter increased from 13 to 52 micro g/l (normal range, 24-70) and aminoterminal collagen type I telopeptide as a bone resorption parameter increased from 62.9 to 92.4 nmol/mmol creatinine (normal range, 5-54). Semen analysis revealed oligoazoospermia (17.4 million/ml; normal >20) at baseline. After 3 months of treatment, the sperm count increased (23.1 million/ml) and decreased rapidly (1.1 million/ml) during the following 3 months. The sperm motility was reduced at baseline and decreased further during treatment. Area under the curve of insulin, C-peptide, and blood glucose levels during oral glucose tolerance test decreased after 6 months (insulin: 277 vs. 139 micro U/ml.h; C-peptide 52 vs. 15 ng/m.h; area under the curve glucose: 17316 vs. 12780 mg/d.min). Triglycerides (268 vs. 261 mmol/liter) and total cholesterol levels (176 vs. 198 mmol/liter) did not change significantly, but the low-density lipoprotein/high-density lipoprotein ratio decreased from 5.37 to 3.56 and lipoprotein (a) increased from 19.9 to 60.0 mg/dl (normal range, <30). In this rare incidence of estrogen deficiency, estrogen replacement demonstrated its importance for bone mineralization and maturation and glucose metabolism in a male carrying a novel mutation in the CYP19 gene.

Adult↗