br CYP gene is located in
CYP19 gene is located in the chromosome 15q21.2 contains ten
Abbreviations: PCR, polymerase chain reaction; CYP19, cytochrome P45 19 gene; EC, endometrial cancer; OC, ovarian cancer; OR, odds ratio
E-mail address: [email protected] (A.I. Al-Badran).
exons (Chen et al., 1988), many reports showed that variety of nu-cleotide polymorphisms in CYP19 gene were resulting variations an-drogen level in the serum (Polymeropoulos et al., 1991).CYP19gene have many polymorphisms including [TTTA] repeat polymorphism in G418 4 (Kristensen et al., 2000; Healey et al., 2000), estrogen-de-pendent usually associated with CYP19 gene polymorphisms include breast cancer, endometrial tumor, endometriosis and uterine myoma (Paynter et al., 2005; Bulun et al., 2005).
2. Material and methods
Sixty two of Blood Samples were collected from Endometrial and Ovarian Cancer Patients from Al- Sader Teaching Hospital-Basrah Center for Oncology and Hematological Disease included twenty three samples with endometrial cancer and thirty nine samples with Ovarian Cancer. Their ages range between (37 to 80) years old. On other hand sixty blood samples of female with no cancer were collected as a control group their ages between (27 to 75) years old. Two ml of peripheral blood was drawn by sterilized syringe from the two groups then they have been kept in sterilized EDTA tubes for DNA extraction. Genomic DNA was extracted by using Genomic DNA Mini Kit (Geneaid, Taiwan), then detected by Agarose Gel Electrophoresis,0.8% agarose gel con-taining Ethidium bromide, visualized by (UV) light (300 nm) (Sambrook and Russell, 2001).
2.1. Genotyping analysis of the CYP19 gene
For detection the tetra nucleotide (TTTA) repeat polymorphism in intron 4 of CYP19 gene.PCR was done by using pair of primers Forward, 5′-GCAGGTACTTAGTTAGCTAC3′; Reverse, 5′TTACAGTGAGCCAAGGT CGT-3′ (Kurosaki et al., 1997). The reaction was mixed according to instructions of Manufacturer Company for PCR PreMix (Bioneer, Korea), and the PCR condition was initial denaturation at 95 °C for 5 min, then 35 cycles of denaturation at 95 °C for 1 min, annealing at 59 °C for 30 s, and extension at 72 °C for 30 s. The final extension was done at 72 °C for 5 min.
Thirty five μl of PCR products were sent to Macrogen Company “http://dna.macrogen.com” for sequencing. The sequences were pro-cessed and analyzed using Basic Local Alignment Search Tool ʻBLASTʼ to search for homologous sequences in the National Center for Biotechnology Information database (NCBI). “http://www.blast.ncbi. nlm.nih.gov.”
2.2. Statistical analysis
Descriptive statistic has been used to describe patient's character-istics by using percentage. ORs and the 95% CLs were calculated by using SPSS program. OR were considered significant if OR ≥ 1.5 ac-cording to (Sheskin, 2004).
The extracted genomic DNA was electrophoreses on 0.8% agarose gel as shown in Fig. 1.
The PCR products were detected by using 2% agarose gel electro-phoresis (Fig. 2).
From the result of sequencing the tetra repeats (TTTA) of CYP19 gene was calculated (Figs. 3, 4).
While the risk to develop Ovarian Cancer patients have increased
CYP19 gene has important role in the production of the estrogen. In our study, (TTTA) polymorphism of the CYP19 gene revealed six alleles (7–12) repeats as in Figs. 3 and 4. The common allele frequencies of the CYP19 (TTTA) allele repeat polymorphism in the healthy control women was seven, Tables 2, 3. The women have two CYP19 alleles. Homozygosity was defined as two repeats of the same length, different lengths for two repeats indicated heterozygosity to analyse the asso-ciation of this polymorphism with EC and OC.
TheCYP19 (TTTA) n alleles were divided into two subgroups using the (TTTA)9 allele as a cut-off point (based on the median number of TTTA repeats): short CYP19 alleles with nine or fewer TTTA repeats and long CYP19 alleles with more than nine TTTA repeats. The same cut-off allele has been used in previous studies exploring the distribution of the CYP19 (TTTA)n polymorphism (Baghaei et al., 2003; Xita et al., 2008; Lazaros et al., 2011).
The CYP19(TTTA), 11 allele presence in all members of a family with aromatase, has suggested the association of long CYP19 alleles with an enhanced aromatase activity (Lazaros et al., 2012). In contract to Kado et al. found no significant differences in TTTA repeat poly-morphisms (Kado et al., 2002). Some studies revealed that longer allele such as (TTTA)10, (TTTA)11, (TTTA)12 were reported to be associated with the risk of breast cancer (Haiman et al., 2000; Miyoshi et al., 2000)