Abstract
Objectives: To investigate the roles of insulin-like growth factor II (IGF2), myeloperoxidase (MPO), E-cadherin (CDH1), urokinase and xeroderma pigmentosum group A and D (XPA, XPD) polymorphisms upon leiomyoma susceptibility. Study Design: Women were divided into: group 1, leiomyoma (n=158); group 2, non-leiomyoma (n=156). Polymorphisms (IGF2 exon 9*A/G, MPO-463*A/G, CDH1-Pml I, urokinase-ApaL, XPA*A-23G, XPD*Lys751Gln) were amplified by polymerase chain reaction and detected by electrophoresis after restriction enzyme digestion. Genotype and allelic frequencies were compared between both groups. Results: Associations between leiomyoma with IGF2 and CDH1 polymorphism exist. Proportions of IGF2 exon 9*AA/AG/GG in and CDH1* CC/CT/TT in the groups were: group 1, 38/39.2/22.8% and 27.8/66.5/5.7%; group 2, 22.4/53.9/23.7% and 21.2/64.1/14.7. MPO, urokinase, XPA and XPD in both groups were non-significantly different. Proportions of MPO*AA/AG/GG, urokinase*CC/CT/TT, XPA*AA/AG/GG and XPD*AA/AC/CC were: group 1: 1.9/23.4/74.7%, 0.6/7/92.4%, 20.9/55.1/24%, 85.4/14.6/0%; group 2: 3.8/24.4/71.8%, 1.3/4.5/94.2%, 22.4/53.9/23.7%, 84.6/15.4/0%. Conclusion: IGF2*A allele and CDH1*C allele were correlated with leiomyoma susceptibility, which may be associated with leiomyoma development. MPO, urokinase, XPA and XPD polymorphisms are not related to leiomyoma susceptibilities.
- Cadherin
- insulin-like growth factor
- leiomyoma
- myeloperoxidase
- polymorphism
- urokinase
- xeroderma pigmentosum
Leiomyoma, the most common benign uterine neoplasma, occurs in around one in four women during their lifetime (1). The factors involved in the initiation and growth of leiomyoma remain unclear. Traditionally, oestrogen has been considered to be the major promoter of leiomyoma growth (2). However, formation of leiomyoma is viewed as a multi-step processes (3). The neoplastic transformation of myometrium to leiomyoma likely involves somatic mutations of normal myometrium and the complex interactions of sex steroids, cytokines, local growth factors, DNA mutations and the dysfunction of the apoptosis system (4).
Growth factors and their receptors play important roles in the pathogenesis of leiomyoma (5). Some polypeptide hormones, such as insulin-like growth factor II (IGF2), have been reported to influence the growth of leiomyoma (6). IGF2 could preferentially promote the growth of leiomyoma cells compared to normal myometrial cells (6). Hyperexpression of IGF2 and increased production of IGF2 peptide could act as autocrine growth factors or co-carcinogens (6).
Genomic instabilities of tumour suppressor genes play major roles in the development and progression of various tumours (7). The cadherin complex regulates cellular adhesion and motility, which functions as an invasion suppressor system. E (epithelial) cadherin (CDH1), a transmembrane adhesion molecule, is one of the key molecules in epithelial cell adhesion (8). CDH1 thus plays an important role in the construction of tissues and organs (8). Reduction of cell-cell adhesion is related to a reduced expression of CDH1 (4). CDH1 is associated with invasiveness, lymph node metastasis, distant metastasis, and other poor prognostic factors (8). Another potential factor in the local regulation of tumour cell invasion is the urokinase-type plasminogen activator (uPA) system. Urokinase is a plasminogen activator that cleaves plasminogen to plasmin and hence stimulates fibrinolysis (9). The concentrations of urokinase of leiomyoma were lower than those in normal myometrium (9). The plasminogen, fibrinogen, and fibrinogen products of menstrual fluid were higher in patients with leiomyoma than those in the normal population (10).
The DNA repair system is required to maintain the genetic integrity of all tissues. Reduced DNA repair capacity might increase the susceptibility to tumorigenesis. The DNA repair pathway also plays a critical role in protecting the genome from insult by cancer-causing agents. Polymorphisms of DNA repair are important in modulating genotoxic effects and tumour susceptibility. Xeroderma pigmentosum (XP) is a group of rare autosomal recessive conditions characterized by defective DNA repair. The XP genes are involved in nucleotide excision repair of DNA (11). Several XP genes, including XPA and XPD, play important roles in determining the efficiencies of the transcription-coupled repair pathway (11).
Unlike mutations, polymorphisms are not directly linked to certain diseases. However, they are useful in studying multifactorial disorders. Most single nucleotide polymorphisms (SNPs) do not alter the transcript levels of related genes. These SNPs might be in disequilibrium with other unidentified polymorphisms, which influences the susceptibility of individual diseases cooperatively. Furthermore, DNA stability is strongly dependent on the surrounding DNA sequence environment, complexity, first and second DNA architecture. The mutant alleles for these SNPs may serve as markers of a functional variant in a nearby gene.
Genetic studies of multifactorial diseases such as leiomyoma are difficult to undertake due to the uncertainties of polygenic traits. The identification of related genes is essential for the understanding of related pathogenesis for leiomyoma. This study aimed to evaluate whether gene polymorphisms are useful markers for predicting the susceptibility of leiomyoma and whether these SNPs are attractive markers for leiomyoma susceptibility. To the best of the Authors' knowledge, this is the first study in this field.
Materials and Methods
Pre-menopausal Taiwanese women with surgically diagnosed leiomyoma and non-leiomyoma were recruited. All patients were divided into two groups: group 1, leiomyoma (n=158); group 2, non-leiomyoma (n=156). All individuals with leiomyoma accepted laparoscopy or laparotomy management and disease was confirmed pathologically. No individuals had been treated with exogenic hormone in the two years preceding recruitment. The studies were approved by the ethical committee and institutional review board of China Medical University Hospital. All informed consents were obtained from all participants in the study.
All women underwent peripheral blood sampling for genotype analyses. The genomic DNA was isolated from peripheral blood leukocytes by use of a genomic DNA isolation kit (Blossom, Taipei, Taiwan). A total of 50 ng genomic DNA was mixed with 20 pmol of each polymerase chain reaction (PCR) primer in a total volume of 25 μl containing 10 mM Tris-HCl (pH 8.3), 50 mM potassium chloride, 2.0 mM magnesium chloride, 0.2 mM each deoxyribonucleotide triphosphate, and 1 U DNA polymerase (Amplitag; Perkin-Elmer, Foster City, CA, USA). A total of five gene polymorphisms were surveyed, including IGF2 exon 9, MPO-463, CDH1, urokinase, XPA A-23G, and XPD Lys751Gln. The SNP information for the genes involved was obtained through the internet (http://www.ncbi.nlm.nih.gov/locuslink/).
The PCR primer sequences and conditions were designed as given in Table I. In brief, the PCR amplification was performed in a programmable thermal cycler GenAmp PCR system 2400 (Perkin Elmer Applied Biosystems, Foster City, CA, USA). After PCR amplification, the individual gene polymorphisms were analysed by restriction digestion with restriction enzymes, including Pml I and ApaL I (New England Biolabs, Inc, Beverly, MA, USA) (Table I). The subsequent reaction buffer was used according as manufacturer's instructions (Perkin Elmer Applied Biosystems, Foster City, CA, USA). The reaction was incubated for 3 hours at 37°C. The PCR products were mixed together and 10 μl of this solution were loaded into 3% agarose gel containing ethidium bromide for electrophoresis. Genotypes and allelic frequencies for each gene polymorphism in both groups were compared. Correlation of these SNPs and leiomyoma was evaluated. Allelic frequencies are expressed as a percentage of the total number of alleles. The SAS system (Version 8.1; SAS Institute Inc., Cary, NC, USA) with χ2 test was utilized for statistical analyses. A p-value of <0.05 was considered statistically significant.
Results
No significant differences between leiomyoma and control groups were found in terms of age (30.4±3.5 vs. 31.7±4.4) and body mass index (21.5±2.3 vs. 21.6±2.1). Genotype and allelic proportions of IGF2 exon 9 and CDH1 polymorphisms in both groups were significantly different (Table II). Dominant percentages of IGF2*A and CDH1*C homozygotes presented in leiomyoma group. Proportions of IGF2*A homozygote/heterozygote/G homozygote and A/G alleles in both groups were: (group 1) 38/39.2/22.8% and 57.6/42.4%; (group 2) 22.4/53.9/23.7% and 49.4/50.6%, respectively (Table II). Proportions of CDH1*C homozygote/heterozygote/T homozygote and C/T alleles in both groups were: (group 1) 27.8/66.5/5.7% and 61.1/38.9%; (group 2) 21.2/64.1/14.7% and 54.5/45.5%, respectively. In contrast, genotype and allelic proportions of MPO-463 and urokinase in both groups were not significantly different (Table II). Dominant presentations of MPO*G and urokinase*T related genotypes or alleles presented in all individuals. Proportions of MPO*A homozygote/heterozygote/G homozygote and A/G alleles in both groups were: (group 1) 1.9/23.4/74.7% and 13.6/86.4%; (group 2) 3.8/24.4/71.8% and 16/84%, respectively (Table II). Proportions of urokinase*C homozygote/heterozygote/T homozygote and C/T alleles in both groups were: (group 1) 0.6/7/92.4% and 4.1/95.9%; (group 2) 1.3/4.5/94.2% and 3.5/96.5%, respectively.
Furthermore, genotype proportions of different XPA and XPD polymorphisms in both groups were not significantly different (Table II). The XPA*A and G genotypes and alleles appear with equal distributions. Proportions of XPA*A homozygote/heterozygote/G homozygote and A/G alleles in both groups were: (group 1) 20.9/55.1/24% and 48.4/51.6%; (group 2) 22.4/53.9/23.7% and 49.4/50.6%, respectively (Table II). All individuals in both groups had the XPD*A-related genotypes and alleles. Proportions of XPD*A homozygote/heterozygote/C homozygote and A/C alleles in both groups were: (group 1) 85.4/14.6/0% and 92.7/7.3%; (group 2) 84.6/15.4/0% and 92.3/7.7%, respectively.
Discussion
Despite the high prevalence of uterine leiomyoma, or fibroids, its etiology remains obscure. Several growth factors, including IGF, epidermal GF, fibroblast GF and platelet-derived GF, are present in the uterus (12). IGF2 is involved in the growth of uterine smooth muscle tumours (5). Leiomyoma contains more IGF2 mRNA than does myometrium, which might be related to its genesis and progression (13). IGF2 functions as autocrine or paracrine mediator of oestrogen action in maintaining, differentiating, and enhancing tumour growth (6). IGF is a potent mitogen which may act as a mediator of sex steroid-induced cell growth and differentiation. The mRNA expression of IGF2 is regulated by ovarian hormone (14). The hypooestrogenism induced by the gonadotrophin-releasing hormone analogue is also related to a decreased level of IGF (14). The changes in IGF binding to myometrium may play a role in the pathogenesis of leiomyoma (14).
IGF2, a 67-amino acid mitogenic peptide, plays an immunohistochemical role in leiomyoma development (14). The regulation of human IGF2, which is located at chromosome 11p15.5, is extremely complex. IGF2 is involved in the growth regulation of mixed mesodermal tumours of the uterus (14). Uterine leiomyosarcoma has been shown to oversecrete IGF2 (5). IGF2 is responsible for the differentiation of skeletal muscle and the down-regulation of IGF receptor gene expression (15). Alternation or loss of imprinting of IGF2 is related to oncogenesis through either inactivation of tumour suppressor genes or activation of growth-promoting genes (5). Biallelic expression of the gene leads to overexpression of IGF2 peptide and increased mitogenic activity (5). The appearance of biallelic expression of IGF2 may be an early event in tumourigenesis. The two alleles of IGF2 that are expressed in tumour tissue may be associated with different production of IGF2 mRNA and protein (16).
The presence of an abnormal CDH molecule may result in the dysfunction of the cell-cell adhesion system, further triggering cancer invasion and metastasis (17). CDH1, a member of the cadherin family of cell surface glycoproteins, plays a tumour suppressor role. CDH1, which is found in most epithelial tissues (18), consequently plays an important role in the development of epithelial structures. Furthermore, CDH1 is recognised as a useful tumour marker since its altered expression correlates with increased tumour aggressiveness and dedifferentiation (18). CDH is associated with embryogenesis, polarization, differentiation, and cell migration in inflamed tissue (17). Genetic alterations in any component of the cadherin complex may induce the loss of adhesion function.
MPO is a 150-kDa hemoprotein stored exclusively in the azurophilic granules of monocytes and neutrophils. MPO produces strong oxidants and procarcinogens, such as benzo(a)pyrene and aromatic amine intermediates, which may contribute to endothelial dysfunction and damage (19). MPO may act as a co-carcinogen by generating free radicals and activating aromatic amines and carcinogens (20). MPO-463*A allele is related to lower MPO expression, as well as reduced risk of individual cancers (20). The uPA system is associated with cell differentiation, migration, cell-associated plasminogen activation, pericellular proteolysis, extracellular matrix degradation and intracellular signal transduction, which further influences angiogenesis, invasion and metastasis of cancer (21). Urokinase levels in endometrial cancer or endometriosis have been shown to be significantly higher than those in normal endometrium (22). uPA is associated with progression of clinical stage in endometrial carcinoma (22), and elevated uPA is correlated with myometrial invasion and unfavorable prognosis (22).
Variation in DNA repair capacity is linked to a risk of certain types of cancer. Around 40% of leiomyomas appear to have an abnormal karyotype (23). Specific chromosomal abnormalities, such as chromosome deletion, translocation, rearrangement, or trisomy, are seen in approximately 30% of uterine leiomyomas (23). The XP-related genes may play an essential role in DNA repair, both in the global genomic repair and in the transcription-coupled repair pathways (24). Among the XP gene family, the XPA 23*GG and XPD polymorphism have been associated with reduced repair proficiency and cancer susceptibility (11).
SNPs are the most abundant variation of the DNA sequence in the human genome. More than 1.4 million SNPs have been identified in the human genome, allowing the possibility of a genome-wide linkage disequilibrium mapping of the genes in the human population. SNPs markers have gained popularity due to the ability to analyse them quickly, accurately and inexpensively in different diseases. Such SNP analysis provides a new way for the identification of complex gene-associated diseases, such as leiomyoma. Reviewing the MEDLINE database, some studies observed the correlation or non-correlation statuses of these polymorphisms with individual diseases (Table III). However, few investigators demonstrated their correlation with leiomyoma. Vu et al. (5) demonstrated that the incidence of IGF2*heterozygosity is significantly lower in patients with uterine leiomyosarcoman than in healthy individuals. They also observed that the IGF2 of myometrium and leiomyoma was always expressed monoallelically. In this study, we observed that the genotype distributions for IGF2 were significantly different between the individuals with leiomyoma and the normal population. IGF2*A homozygosity is related to a higher risk of leiomyoma development. Heterozygosity or G homozygosity are related to lower risk of leiomyoma development, in agreement with Vu et al. (5).
This study also showed that the genotype distributions and allelic frequencies for CDH1 were significantly different between individuals with leiomyoma and the normal population. C homozygotes possessed a higher risk of developing leiomyoma, whereas the C/T heterozygote or the T homozygote had a lower risk. In contrast, no-association of another four polymorphisms (MPO, urokinase, XPA*A-23G, XPD*Lys751Gln) with leiomyoma was observed. These findings indicated these gene variations may not be good candidates as genetic markers for susceptibility to leiomyoma.
In conclusion, an association between leiomyoma and IGF2 and CDH1 polymorphisms exists. The IGF2*A and CDH1*C alleles were correlated with leiomyoma susceptibility, which may be associated with leiomyoma development. Therefore, the IGF2 and CDH1 polymorphisms may contribute to the pathogenesis of leiomyoma. Although the real roles of these gene variations have not yet been clarified, they should be further investigated in order to more fully understand their roles in leiomyoma development. In so doing, it will be possible to ascertain their suitability as predictive markers of leiomyoma susceptibility, and possibly develop new early therapeutic interventions in women at high risk for leiomyoma. Furthermore, the effects of other growth factors or cadherin polymorphisms upon leiomyoma development should be investigated further. In contrast, there is no correlation between leiomyoma and the other four polymorphisms. Therefore, it can be concluded that MPO*-463A/G, urokinase, XPA*A-23G and XPD*Lys751Gln polymorphisms do not appear to be related to leiomyoma susceptibility, and are thus not useful as predictive markers of leiomyoma susceptibility. However, the mapping of the nucleotide excision repair pathway and related genes may contribute to the understanding of their roles upon tumorigenesis. After the clarification of these issues, certain DNA repair gene polymorphisms may become useful markers for predicting the future development of leiomyoma as well as providing the valuable insight into the pathogenesis of leiomyoma.
Acknowledgements
All authors fulfilled the condition for authorship. There was no commercial support in the process of performing this study and submitting this manuscript.
Footnotes
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↵* Both authors contributed equaly to this work.
- Received February 11, 2010.
- Revision received April 16, 2010.
- Accepted April 23, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved