CHROMOSOMAL DEFECTS IN UTERINE FIBROIDS

Journal of Cell and Tissue Research Vol. 12(1) 2985-2990 (2012) ISSN: 0974- 0910 (Available online at www.tcrjournals.com) Original Article CHROMOSO...
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Journal of Cell and Tissue Research Vol. 12(1) 2985-2990 (2012) ISSN: 0974- 0910 (Available online at www.tcrjournals.com)

Original Article

CHROMOSOMAL DEFECTS IN UTERINE FIBROIDS VERONICA, M.,1 LAKSHMI, R. K.,2 VENKATESHWARI, A.,3 MAMATA, D.4 ? AND PRATIBHA, N.1 1

Department of Genetics, Osmania University, Hyderabad, 500 007; 2Center for Cellular and Molecular Biology, Habsiguda, Hyderabad 500 007; 3Institute of Genetics and Hospital for Genetic Diseases, Ameerpet, Hyderabad 500016; 4Infertility Institute and Research Center, Secunderabad 500 003, Andhra Pradesh, India. Received: February 19, 2012; Accepted: March 11, 2012 Abstract: Uterine fibroids are benign (noncancerous) masses of muscle tissue that enlarge and/or distort the uterus and originate from the smooth muscle cells within the myometrium. Clinically apparent in 25% of women, several predisposing factors have been identified that include age (late reproductive years), nulliparity, and obesity. Fibroids cause excessive menorrhagia, severe abdominal pain, urinary incontinence and constipation; others develop as subclinical (asymptomatic) pelvic masses. Fibroids are associated with infertility and if present during pregnancy may contribute to spontaneous abortion, premature labor or dystocia. About 40% of Uterine Leiomyomas show non- random cytogenetic abnormalities. In the present article we report 3 different cytogenetic abnormalities which have led to the disease symptoms. 1) A translocation involving 12 and 7 chromosome may lead to the dysregulation of decorin gene expression leading to fibrillar instability thus ensuring unchecked growth of connective tissue; 2) Another case involves 15p arm duplication. 15th chromosome is an acrocentric chromosome and harbors genes responsible for RNA expression. It is also worth noting that this region is rich in tandem repeats. Gene localized at this area is the RNR3 gene wherein its overexpression can render it cancerous, 3) The third case represents a mosaic karyotype wherein a set of cells harvested showed the complete set of chromosomes and another set showed deletion of one of the X chromosomes.

Key words: Chromosomal defects, Uterine fibroids

INTRODUCTION Uterine leiomyomata (UL) commonly known as fibroids are the most common pelvic tumors of the female genital tract with a peak onset in the third to fourth decade of life. Symptomatic disease occurs in 20–25% of reproductive-age women, although systematic histologic examinations of hysterectomy specimens have shown a prevalence of UL as high as 77% with an average of 6.5 tumors per uterus. Despite their benign nature, UL have a significant level of morbidity manifesting as a spectrum of clinical symptoms related to the size and anatomic location of the tumors. Symptoms include urinary incontinence, constipation, menorrhagia, abdominal pain, difficult and extended menstrual periods and impairment of

fertility (1). Their incidence seems to be higher in women of African origin than women of other ethnic groups. Risk factors for UL include early age of menarche, nulliparity, oral contraceptive use and BMI (2). Uterine fibroids are independent, monoclonal tumors derived presumably from a single mutated myometrial cell (3) and involve abnormal karyotypes. Cytogenetic studies have shown that 40% of tumors are karyotypically abnormal, suggesting that genetic aberrations at the submicroscopic level may be present in karyotypically normal fibroids. This has allowed classification of UL into well defined subgroups which include translocations between 12 and 14 chromosomes e.g. t(12;14)(q15;q23–24) predicts a

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J. Cell Tissue Research gain-of-function mutation in the form of a fusion protein, or potential dysregulation of a particular gene product. A deletion (eg, del(7)(q22q32)) is consistent with a loss-of-function mutation, typified by tumor suppressor genes. A trisomy (eg, trisomy 12) predicts gene dosage mechanism. Thus, it appears that there are probably many different genetic pathways by which a fibroid can grow and develop (4). Chromosomes other than these are also found to be involved in the etiopathogenesis of UL. A tendency for karyotypically abnormal leiomyomas to be more cellular and to have a higher mitotic index than do chromosomally normal tumors has also been reported (5). The present study deals with cytogenetic analysis of 60 uterine fibroid samples which revealed 3 abnormal karyotypes. The abnormal karyotypes pertaining to the patient cohort have been discussed in a case wise manner, which may pin-point towards the various genes that may be involved in uterine fibroids via the disruption of specific/non-random chromosomes. Methodology: 60 uterine fibroid samples collected from Infertility Institute and Research Center (IIRC), Secunderabad were subjected to lymphocyte culturing and karyotyping. 2ml of the patient’s blood was collected in a heparinized tube for lymphocyte culturing (6) followed by GTG banding [7]. Case Presentation Case 1: A patient aged 42 years was referred for karyotyping analysis. She was diagnosed with uterine fibroids. No other information was available upon consultation with the patient. 2ml of the patient’s blood was collected in a heparinized tube for lymphocyte culturing (6) and GTG banding was carried out as per standard protocols (7). Twenty five metaphase plates were analyzed using the Cytovision software to deduce the karyotype. The chromosomes were identified and arranged according to the guidelines issued by the International System for Human Cytogenetic Nomenclature (ISCN, 1995) (8). Chromosomal analysis revealed an abnormality in the patient with 46, XX, t(7;12)(q32;q21) chromosomal constitution. The karyotype revealed 46 chromosomes with a shortened chromosome which did not belong to any group; as part of the 12th chromosome had been lost. This was revealed by

banding pattern comparison. Careful examination of the karyotype showed that the missing part of the 12th chromosome (12q21) had been translocated to the 7th chromosome (7q32) as shown in (Fig 1). Analysis of the 12q21 band at the genetic level showed the presence of a gene; Decorin. It is a component of connective tissue which primarily binds to several types of collagen predominantly to type I, II and VI. Binding of Decorin to collagen tissues promotes fibrillar stability. It also influences cell adhesion in concert with fibronectin and thrombospondin. It also plays a role in matrix assembly interacting with type I collagen. Apart from having the capability of suppressing the growth of various tumor cell lines, it also activates the epidermal growth factor receptor in some type of cells, thereby triggering a signaling cascade that leads to phosphorylation of mitogen-activated protein kinase, to the induction of p21, and to growth suppression (9). Case 2: A patient with a history of primary infertility was referred for cytogenetic analysis. She was 31 years old and diagnosed with uterine fibroid. Familial history of patient revealed parental consanguinity wherein parents are first cousins. Lymphocyte culturing and GTG banding was carried out (6 and 7). Cytogenetic analysis of the patient using GTG banding showed the presence of extra chromosomal material on the short arms of chromosome 15 (Fig 2). PCR analysis was carried out to confirm whether the extra material is from the Y chromosome, the result being negative. Observations using GTG banding have shown the probability of satellite stalks being duplicated; which mainly comprises of beta satellite DNA, NOR regions and a possibility of tandemly repeated DNA which lies just below NOR regions. Gene localized at this region was found to be Ribosomal (RNR3) wherein its overexpression can lead to neoplastic transformation making RNR a target for cancer therapy (10). Case 3: A patient aged 31 with recurrence of fibroids and primary infertility was referred for cytogenetic analysis. Cytogenetic analysis revealed two different cell lines existing as a mosaic on the analysis of fifty metaphase plates using the Cytovision Software. Two cells of a cell line depicted mosaic 45,X karyotype, out of the 50 nuclei screened and the rest showed 46,XX karyotype (Fig 3).

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Fig. 2: Presence of extra material on the p arms of the 15th chromosome.

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J. Cell Tissue Research RESULTS AND DISCUSSION Approximately 40% of UL have non-random and tumor-specific chromosome abnormalities. This has allowed classification of some UL into well-defined subgroups which include deletion of portions of 7q, trisomy 12 or rearrangements of 12q15, 6p21 or 10q22. Additional abnormalities, which appear consistently but not as frequent, include rearrangements of chromosomes X, 1, 3 and 13 respectively. The variety of chromosomal rearrangements, including but not limited to translocation, deletion and trisomy, predict different molecular genetic mechanisms in UL growth and proliferation (1). The precise molecular mechanisms by which chromosomal abnormalities regulate myoma growth still remain elusive. The wide variety of cytogenetic abnormalities associated with fibroids may represent the biological basis for the differential growth responsiveness of individual tumors (3). We report a case (Case 1) with 46,XX,t(7;12) (q32;q21). Though chromosome 12 translocations involving 14 and deletions of chromosome 7 are known to be associated with fibroids but the translocation involving chromosomes 7 and 12 is known to be a rare and unique defect in the present study. We have observed the regions of translocation to be between q32 of chromosome 7 and q21 of chromosome 12 respectively. Analysis of the 12q21 band at the genetic level showed the presence of a gene; decorin. Decorin is a member of the small leucine-rich proteoglycans gene family, which consists of a core protein and a single covalently linked glycosaminoglycan chain. Decorin is known to bind to several types of collagen but types I, II, and VI, are predominant which promotes fibril stability. It is a component of the connective tissue and binds to type I collagen fibrils, and plays a role in matrix assembly. On interaction with ECM molecules especially fibronectin and thrombospondin, it influences cell adhesion. Decorin is capable of suppressing the growth of various tumor cell lines. Additionally, at least in some types of cells, decorin activates the epidermal growth factor receptor, thereby triggering a signaling cascade that leads to phosphorylation of mitogen-activated protein kinase, to the induction of p21, and to growth suppression. Alternatively its core protein also interacts with TGFβ which are independent to type I collagen binding sites. The addition of decorin to osteoblasts resulted in an increased biological activity of TGF-β (9) hence

decorin may act as a growth factor reservoir in the ECM, modulating the biological processes, such as cell proliferation and differentiation. There is also a strong evidence that collagen fibril-associated decorin is able to arrest TGF-β in the ECM, as TGFβ is a inhibitor of proliferation (11). Disruption of decorin gene due to the rearrangements may alter modulation of the ECM proteins which in turn may influence growth and proliferation (9). The translocation t(7;12) (q32;q21) leads to disruption of the 7q32 band. Close to this region (7q31.1) lies the CAV1 gene. It plays a role as a tumor suppressor gene and a negative regulator of the Ras-p42/44 MAP kinase cascade (12). Loss of heterozygosity analysis implicates 7q31.1 in the pathogenesis of multiple types of cancer, including breast, ovarian, prostate, and colorectal carcinoma, uterine sarcomas and leiomyomas (13). Yang et. al. 1998 (14) found elevated caveolin-1 levels associated with lymph node metastasis in prostate cancer raising the possibility that CAV1 may also act as an oncogene. Thus the rearrangements between 12th and 7th chromosomes may alter the signal transducing pathway involved in proliferation and growth control of the smooth muscle cells of the uterus. A 15 p arm duplication identified, the gene revealed a ribosomal RNA 3 gene (RNR3) (15p12; OMIM Database). Sequences on the p arm of chromosome 15, may relate to the nucleolar organizing genes, which have an important role in the maturation of benign and malignant cells of hemato-lymphopoietic origin (15). The upregulation/overexpression of the ribosomal gene during proliferative changes may be one of the possibilities of UL being malignant. RNR activity is crucial for rapidly dividing cells; ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleoside diphosphates (NDPs) into deoxyribonucleotides (dNDPs), one of the most specific and rate-limiting steps in DNA synthesis. Given the huge energetic investment of generating the necessarily large pool of dNTPs for DNA replication and repair, it is not surprising that RNR expression is carefully regulated and restricted to the late G1 and S (and G2) phases of the cell cycle or to conditions of DNA damage . In mammals, only the expression of RNR2 is regulated, reportedly at the transcriptional level. In contrast, RNR1 is constitutively expressed throughout the cycle. Overexpression of RNR can lead to neoplastic

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Fig. 3: Depicting two different cell lines, one showing the mosaic pattern and the other with a normal set of chromosomes.

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J. Cell Tissue Research transformation, thus making it a target for cancer therapy (10). In mammalian cells, the RNR small subunit is the site of action of several antitumor agents, including hydroxyurea and 4-methyl-5-amino1-formylisoquinoline thiosemicarbazone (MAIQ) (16 and 17). In case 3 we report a rare case of mosaicism 46,XX;45,X which is usually seen in patients with premature ovarian failure(POF) and is associated with infertility. Deletions of the tandem 5(IV) and 6(IV) type IV collagen gene located on the X chromosome were recently found to cause familial systemic fibroid tumors, indicating that the matrix surrounding smooth muscle cells can influence their proliferation. In addition, abnor malities in programmed cell death have been described in such fibroid tumors with mosaicism. However this needs to be substantiated/revalidated on abnormal mosaic karyotypes (18). CONCLUSION Cytogenetic analysis plays an important role in preliminary identification of genetic pathways leading to disease manifestation. One such area of interest is uterine fibroids, wherein cytogenetic abnormalities may elucidate various genetic pathways in the etiology of the condition. In the pr esent study novel cytogenetic abnormalities in uterine fibroids may promote identification of putative/candidate genes and their role in signal transduction involving growth and proliferation of smooth muscle cells of the uterus. With the advent of microarray technology, one can think of developing cytochips in order to pinpoint the exact cause of the condition. The information so obtained can be utilized for the genotype-phenotype correlation. Further the identification of putative genes involved may aid in genetic diagnosis of the condition, genetic risk prediction in the immediate female family members and management of the condition by appropriate therapeutic measures to avert infertility in reproductive age females. ACKNOWLEDGEMENTS Financial support fr om University Gr ants Commission, New Delhi is acknowledged. I would also like to acknowledge the support lent by the Chromosome Diagnostic Group, CCMB.

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