Cytochrome P2A13 and P1A1 gene polymorphisms are associated with the occurrence of uterine leiomyoma

Problem To investigate the association between the occurrence of uterine leiomyoma and two SNPs of the CYP 2A13 and CYP 1A1 genes.

of study Prospective case control study with 132 women with clinically and surgically diagnosed uterine leiomyoma and 260 controls. Genotyping was performed by polymerase chain reaction (PCR) based amplification of CYP 2A13 and CYP 1A1 genes, and restriction fragment length polymorphism (RFLP) analysis.

Comparing women with uterine leiomyoma and controls, we demonstrate statistical significant differences of allele frequency and genotype distribution for the CYP 1A1 polymorphism (P = 0.025 and P = 0.046, respectively). Furthermore, for the CYP 2A13 polymorphism we found a significant difference concerning allele frequency (P = 0.033). However, for the genotype distribution, only borderline significance was observed (P = 0.064).

The CYP 2A13 and CYP 1A1 SNPs are associated with uterine leiomyoma in a Caucasian population and may contribute to the understanding of the pathogenic mechanisms of uterine leiomyoma.

Leiomyoma are the most common gynecological tumors in women, but very little is known about their molecular pathology. Uterine leiomyoma are benign smooth-muscle tumors affecting about 22% of women during their lifetime [1]. Uterine leiomyoma are often asymptomatic, but may cause symptoms ranging in severity from mild abdominal discomfort to uterine bleeding, pelvic pain, and infertility [2]. Thus, the leiomyoma are the most common indication for hysterectomy representing a significant public health problem. The leiomyoma develop by proliferation of a single smooth-muscle cell [3]. The growth and the development of leiomyoma is estrogen dependent [4]. It was found that the majority of muscle cells contributing to leiomyoma express both the estrogen receptor ERalpha and ERbeta [5]. However, exact mechanisms explaining the occurrence of leiomyoma remain unclear. Because of the common occurrence of this tumor and the potential severity of associated symptoms, further investigation about its pathophysiology is needed. In two different randomly selected human genomes, 99.9% of the DNA sequence is identical. The remaining 0.1% of DNA contains sequence variations [6]. The most common type of such variation is called a single-nucleotide polymorphism (SNP) [6]. In a certain population mutations with an allele frequency of at least 1% are called polymorphisms [7]. Determination of the SNPs which are highly abundant, stable, and distributed throughout the genome is a new way to investigate the etiology of polygenetic disorders with complex inheritance patterns, such as schizophrenia, diabetes, or development of tumors [6]. Identification and cataloguing of the SNPs represents an important task for molecular biology in future [8]. The available evidence is not conclusive but it is consistent in pointing to an association or a heritable etiology of genetic disorders, which will hamper the success of SNP-based association studies [9]. In this regard, the identification of susceptibility factors predisposing individuals for uterine leiomyoma could provide further understanding concerning the development of this benign tumor. Cytochrome P 450 encompasses a family of enzymes which is involved in metabolic activation of carcinogens [10] as well as in steroid hormone biosynthesis [11]. The CYP P450 profile of a cell indicates the capacity to form reactive metabolites [12]. The CYP P450 isoenzymes are predominantly expressed in the liver. Conclusively the genotype of various premenopausal women appears to influence their circulating estrogen levels. However, studies of relationship of hormone levels and genotype in men have produced inconclusive results [13]. Furthermore, it has been shown that human endometrial epithelium has the potential of producing CYP P450 enzymes known to generate genotoxic intermediates that affect the estrogen receptor [14]. Since growth and development of leiomyoma is estrogen dependent, we hypothesised that polymorphisms in the CYP P450 genes 2A13 and 1A1 may be associated with leiomyoma by influencing CYP P450 mediated conversion of circulating androstenedione into estrone, and then via 17beta-hydroxysteroid dehydrogenase into estradiol. We thus investigated the allele and genotype frequencies of CYP 2A13 exon 5 and CYP 1A1 exon 7 SNPs in a Middle-European white population of women with clinically and surgically diagnosed uterine leiomyoma in comparison to a healthy male control population.

We included 132 women with clinically diagnosed uterine leiomyoma undergoing surgical intervention at the Department of Gynecology of the University of Freiburg School of Medicine between 2002 and 2004. Local institutional review board approval and informed consent from all subjects were obtained. Leiomyoma were diagnosed by detailed sonographic examination and confirmed by histological examination after hysterectomy or myomectomy. Blood was taken from all participants by peripheral antecubital venous puncture and stored at −18°C until analysis. Population controls were recruited from ambulatory male patients. Patients with any systemic diseases, such as malignancies or any kind of genetic diseases, were excluded from the control group. Genotyping To determine the CYP 2A13 genotypes, we amplified a genomic DNA fragment by polymerase chain reaction (PCR). Oligonucleotide primers flanking exon 5 of CYP 2A13 were used. The sequence of the forward primer was 5′-CCT GGA CAG ATG CCT TTA ACT CCG-3′. The reverse sequence was 5′-TGG CTT TGC ACC TGC CTG CAC T-3′. PCR amplification was carried out with 0.2 mM dNTP, 10 μM forward primer, 10 μM reverse primer, 1.5 mM MgCl2, 1 U Taq polymerase (Quiagen, Hilden, Germany) in the recommended buffer. Cycling conditions were: 45 cycles in an Eppendorf Thermocycler at 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s after the initial denaturation step (94°C for 5 min). A 7 min final extension step at 72°C was added. The total volume of the PCR reaction was 25 μl. For genotyping of the CYP 2A13, a fragment of 332 bp was amplified, including exon 5 and containing a recognition site for Hha I (recognition motif: 5′...GCGC...3′; 3′...CGCG...5′). The sequence at this position can be determined by a single restriction digest with Hha I. The digestion generates two fragments of 99 and 223 bp [15]. Fragments were visualised after electrophoresis on a 1% agarose gel by staining with ethidium bromide. Pictures were taken with a Raytest digital camera system (Raytest, Straubenhardt, Germany). For analysis of the CYP 1A1 genotypes a genomic DNA fragment was amplified by polymerase chain reaction (PCR). The sequence of the forward primer was 5´-CTG TCT CCC TCT GGT TAC AGG AAG C-3´. The reverse sequence was 5´-TTC CAC CCG TTG CAG CAG GAT AGC C-3´. PCR amplification was again carried out with 0.2 mM dNTP, 10 μM forward primer, 10 μM reverse primer, 1.5 mM MgCl2, 1 U Taq polymerase (Quiagen, Hilden, Germany) in the recommended buffer. Cycling conditions were: 45 cycles in an Eppendorf Thermocycler at 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s after the initial denaturation step (94°C for 5 min). A 7 min final extension step at 72°C was added. The total volume of the PCR reaction was 25 μl. For genotyping of the CYP 1A1, a fragment of 204 bp was amplified including a C4887A polymorphism in exon 7, and containing a recognition site for BsaI (recognition motif: 5′...GGTCTC(N)1...3′; 3′....CCAGAG(N)5...5′). The sequence at this position can be determined by a single restriction digest with BsaI [16]. The digestion generates two fragments of 150 and 54 bp. The fragments were visualised after electrophoresis on a 1% agarose gel by staining with ethidium bromide. Statistical analysis Differences in frequencies of the CYP 1A1 and the CYP 2A13 alleles in study and control groups were analysed using Chi square test. The odds ratio (OR) was used as a measure of strength of the association between allele frequencies and uterine leiomyoma. For calculation we used a total of 369 patients. A P < 0.05 was considered to be significant.

The 126 women with uterine leiomyoma and 243 controls were examined. Characteristics of women with uterine leiomyoma are shown in Table 1. The CYP 2A13 allele and genotype frequencies of women with uterine leiomyoma and controls are shown in Table 2. The CYP 2A13 allele frequencies among women with uterine leiomyoma and controls were 3.6 and 1.2%, respectively, for the T allele, and 96.4 and 98.8%, respectively, for the C allele. The 3375C > T allele corresponds to an Arg257Cys change in the predicted amino acid sequence. This difference in allele frequency among women with leiomyoma and controls is statistically significant (P = 0.033; odds ratio [OR] 2.96). The difference between the prevalence of the different genotypes (T/T, C/T and C/C) is of borderline significance using a recessive genotype model (C/C vs. T/T and C/T: P = 0.064; OR 2.68). However, using a dominant genotype model the difference is not statistically significant (T/T vs. C/T and C/C: P = 0.16; OR = 0.017). The CYP 1A1 allele and genotype frequencies of women with uterine leiomyoma and controls are shown in Table 3. With respect to this examination blood samples of 8 women with leiomyoma and 21 controls could not be analysed due to technical reasons. CYP 1A1 allele frequencies among women with uterine leiomyoma and controls were 2.5 and 6.5%, respectively, for the A allele, and 97.5 and 93.5%, respectively, for the C allele. There is a statistical significant association between carriage of the C allele and the occurrence of uterine leiomyoma (P = 0.025, odds ratio [OR] 2.68). The homozygous C/C genotype was found more often among women with leiomyoma (94.9%) compared to controls (88.2%). This difference was significant (P = 0.046; OR 2.48) using a recessive genotype model (C/C vs. A/A and C/A). However, using a dominant genotype model (A/A vs. C/C and C/A) this difference was not significant (P = 0.2; OR 0.026).

Genetic analysis leading to new clinical applications could deeply change our knowledge concerning mechanisms through which different neoplasms develop and progress. In this regard, analysis of SNPs represents a powerful approach for genomic screening of candidate genes that may be associated with the development of multifactorial disorders. In this study we investigated the association of an SNP in the coding region of the CYP 2A13 gene as well as an SNP in the CYP 1A1 gene and the occurrence of uterine leiomyoma in a large Caucasian population. We demonstrated that polymorphisms of the CYP 2A13 gene and the CYP 1A1 gene are significantly associated with the development of uterine leiomyoma. The rationale to test CYP 2A13 and CYP 1A1 genes as candidate genes for uterine leiomyoma was based on data about estrogen dependent growth of leiomyoma [4]. In their study about the role of sex hormones in the pathogenesis of uterine leiomyoma, the authors investigated estrogen receptor contents of both normal uterine myometrium and leiomyoma. They concluded that high estrogen receptor contents in leiomyoma may be the result of local high estradiol concentrations, and cause growth and development of leiomyoma. Although there are substantial data about associations between polymorphisms of the cytochrome P 450 enzymes and the capacity to metabolise estrogen [17–22], so far little information is available about the effect of these polymorphisms on the development and growth of uterine leiomyoma. Interindividual differences of enzyme activities causing different rates of metabolization and bioactivation of estrogen may be associated with polymorphisms of the CYP 450 enzymes [23]. Allelic variants of these enzymes may change enzyme kinetics, leading to accumulation of different estrogen metabolites. Our study demonstrates a significant association between the investigated CYP 2A13 and CYP 1A1 gene polymorphisms and the occurrence of uterine leiomyoma in a Caucasian population. These data are in accordance to a study about the role of another cytochrome P450 polymorphism in the pathobiology of uterine leiomyoma from black South African women. The authors were able to show a significant association between the cytochrome P450c17alpha gene (CYP17) and the occurence of uterine leiomyoma in black women [24]. Although growth and development of leiomyoma is estrogen dependent, there is no data on the estrogen metabolic profile in women with different cytochrome P 450 polymorphisms. Our findings may open the intriguing possibility to fill the gap between genetic predisposition for leiomyoma and estrogen metabolization by demonstrating a significant association of CYP 2A13 and CYP 1A1 polymorphisms and women with uterine leiomymoma. It has to be acknowledged that the design of our study has limitations. The selection of the control group, namely healthy males with proven fertility, may cause selection bias since this group is not a representative of a solely female disorder. However, due to the logistic difficulty of finding a sufficient number of clinically and histologically proven healthy females without leiomyoma, our approach to use males as controls seems justified. This approach is in accordance with several previously published studies [25–29]. Nevertheless, this shortcoming has to be taken into account when interpreting our results. In summary, for the first time our findings show evidence of an association between cytochrome P450 2A13 (CYP 2A13) gene SNP and a cytochrome P450 1A1 (CYP 1A1) gene SNP among women with uterine leiomyoma. We demonstrate, that the investigated CYP 2A13 SNP of exon 5 as well as the SNP of CYP 1A1 exon 7 are associated with uterine leiomyoma in a large Caucasian population. The identification of a context between leiomyoma as benign uterine neoplasm and specific gene variants involved in estrogen metabolism allows further insights concerning pathophysiology and etiology of the development of uterine leiomyoma and may contribute to a genetic characterisation of susceptible women.

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Arch Gynecol Obstet 274, 367–371 (2006). https://doi.org/10.1007/s00404-006-0201-8 Received: Accepted: Published: Issue date: DOI: https://doi.org/10.1007/s00404-006-0201-8