Human malignancy associated with Y chromosome: Genotypes and haplotypes associated with susceptibility to Colorectal Cancer patients

Journal of Chemical and Pharmaceutical Research, 2016, 8(4):919-927

Research Article

_{CODEN(USA) : JCPRC5}

ISSN : 0975-7384

Human malignancy associated with Y chromosome: Genotypes and

haplotypes associated with susceptibility to Colorectal Cancer patients

Ali H. Al-Marzoqi

, Hawraa J. Kadhim, Ammal R. Shimran, Nebras M. Al-Khafaji,

Sabreen A. Kamal, Rusul Adil Naji, Zubaida Amer Faisal, Sarah Abbas Fakhri and

Redaah Abd-Alrazzak

College of Science for Women, Babylon University, Iraq

_____________________________________________________________________________________________

ABSTRACT

Methylenetetrahydrofolate reductases (MTHFR) considered as a critical character in digestion system of folate furthermore collaborate with union of nucleic corrosive, repair arrangement of DNA and methylation. Our revision was meant to reveal essence concerning polymorphism in MTHFR gene in addition to the CRC danger. The study populace included 77 disease male patients with CRC (mean of age in years 64±8.7) separated as; (41 with colon malignancy and 36 with rectal growth) and 55 as controls admitted to the Merjan Healing center in span stretched out from October 2015 until March 2016. The outcomes uncovered that hereditary polymorphisms of all qualities incorporating into this study for MTHFR 677 quality in CRC patients; the recurrence of MTHFR C677 genotypes were as kindred TT 12.0 (15.6%), CT 24.0 (31.2%), and CC 41.0 (53.2%) in CRC, and in the control was 19.0 (34.5%), 14.0 (25.5%), and 22.0 (40.0%) individually. Amalgamation frequencies for MTHFR 677 TT homozygous and CT 677 heterozygous watched were 31.2% in CRC and 47.3% in the controls. The Genotype dissemination for MTHFR 1298 quality in CRC patients; the recurrence of MTHFR C677 genotypes were as kindred CC 9.0 (11.7%), CA 21.0 (27.3%), and AA 47.0 (61%) in CRC, while in the control was as individual 8.0 (14.5%), 21.0 (38.2%), and 26.0 (47.3%) separately. The recurrence for MTHFR 1298 quality AA homozygous and AC 1298 heterozygous watched was 74.7% in CRC and 66.4% in the controls. Spreading of genotype for XRCC1 399 quality in CRC patients; the Genotype conveyance for this quality in CRC patients; the recurrence of XRCC1 399 genotypes were as kindred TT 28.0 (36.4%), TG 13.0 (16.9%), and GG 36.0 (46.8%) in CRC, while in the control was as individual 27.0 (49.1%), 6.0 (10.9%), and 22.0 (40%) separately. The recurrence for XRCC1 399 quality TT homozygous and TG 399 heterozygous watched were 44.87% in CRC and 45.45% in the controls. MTHFR; XRCC1 SNPs is connected with expanded CRC hazard.

Keywords: Y-chromosome; CRC; Genotype; Haplotype; MTHFR; XRCC1.

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INTRODUCTION

Loss of Y chromosome [LOY] is a surely understood marvel that is connected with maturing and saw with fluctuating frequencies in bone marrow cells [1,2] or in fringe platelets [3,4]from sound more established male. The relationship of LOY with hematological growths has been subtle. Be that as it may, LOY has been accounted for in leukemia [5-8] and in patients anticipated to have poor reaction to tumor treatment [9]. Different studies have found the affiliation just in patients who demonstrated LOY in more than 75% [10] or 100% of the influenced cells [11].

2006 [25]. Be that as it may, the instruments in charge of such sex‑differences are still to a great extent obscure. The most noteworthy hereditary contrasts in the middle of men and ladies are qualities on their sex chromosomes, that is, XY for men and XX for ladies. Men are inclined to X-connected ailments brought on by transformations on qualities on their X chromosome while ectopic articulation of the qualities on their Y chromosome could have male‑specific impacts on ordinary advancement, physiology, and illnesses. The human Y chromosome can be grouped basically into three districts: the Y chromosome of male exact locale [MSY], pseudoautosomal areas [PAR1 and PAR2], and heterochromatin locale on Yq.26 PARs contain 20 protein‑coding qualities [16 qualities in PAR1 and 4 qualities in PAR2] that are additionally present on the X chromosome. The MSY contains 23 protein‑coding qualities and various pseudogenes [27-29]. While qualities in PARs are available in both X and Y chromosomes and experience meiotic recombination comparatively with autosomal qualities, qualities in MSY are avoided from meiotic recombination with a homologous chromosome accomplice. The MSY qualities advanced amid around 300 million years in the wake of start of X‑Y separation [30].

Colorectal Cancer (CRC) has known a complex polygenic issue which would be a standout amongst the most known reasons for mortality in men [31]. Albeit late studies have distinguished various variations, quality combinations, and expression marks have partnered with prostate tumor, then ID and portrayal of qualities that have included in this growth, has stayed as a considerable test [32]. The many-sided quality and multigenic nature of growth has brought about different extensive studies, have been accomplishing a frameworks level comprehension of the key hereditary arbiters, included in prostate malignancy [33]. One point of convergence in growth examination would be the reproduction of co-expression systems. Whenever exact, co-expression systems have spoken to the key middle people that have included in a particular procedure. The accessibility of the far reaching quality expression information has helped the advancement of different condition of-workmanship co-expression systems reproduction strategies [34-36].

Methylenetetrahydrofolate reductase (MTHFR), an essential compound in folate digestion system majorly affects instruction of folic corrosive passageway because of the alteration of methylene-THF to the 5-methyl-THF. The most well-known polymorphisms of the MTHFR quality are C677T and A1298C, which are thought to diminish the catalyst movement prompting a reduction in methyl-THF and to be connected with hyperhomocysteinemia, especially in folate lack. The 677 C→T move (exon 4) causes an amino corrosive substitution from alanine to valine at codon 222 inside of the reactant district of the protein, bringing about MTHFR with diminished action [37]. A few studies have reported that MTHFR variations assume a critical part in infection forms and the weakness to a few issues, including vascular maladies, neural tube imperfections and tumor. The relationship between the MTHFR quality polymorphisms and hereditary powerlessness to disease has been broadly assessed in late concentrates, yet the conclusions are questionable. A few studies have conveyed MTHFRC677T gene variation of genotype homozygous SNPs was connected to the expanded CRC danger [38]. In other case, MTHFR 677TT genotype was describe in different studies and report that people could diminished CRC danger, while others watch on no account relationship between the genotype and hereditary vulnerability of MTHFRC677T to CRC and gastric cancer [39, 40].

EXPERIMENTAL SECTION

Subjects

The study populace included 77 growth male patients with CRC (mean of age in years 64±8.7) separated as; (41 with colon malignancy and 36 with rectal disease) and 55 as controls admitted to the Merjan Hospital in term reached out from October 2015 until March 2016. All patients and control were from the Babil with complete demographic and behavioral data. The subjects were coordinated for age and sex. Neurotic affirmation aimed at growth subjects was done, in addition to that the subjects selected as control had no history of tumor.

DNA isolation

DNA segregated from entire blood utilizing Genomic DNA Purification Kit; The Wizard® Genomic DNA Purification Kit is intended for detachment of DNA from white platelets, tissue society cells and creature tissue, plant tissue, yeast, and Gram positive and Gram negative microscopic organisms. The Wizard® Genomic DNA Purification Kit depends on a four-stage process. The initial phase in the sanitization method lyses the cells and the cores. For separation of DNA from white platelets, this stride includes lysis of the red platelets in the Cell Lysis Solution, trailed by lysis of the white platelets and their cores in the Nuclei Lysis Solution. After lysis, the leukocytes were pelleted and processed with proteinase-K and nucleic corrosive was removed after the standard convention.

Genotyping

Genotyping was performed by PCR-RFLP; the genotyping convention for PCR-RFLP was done as taking after; for MTHFR C677T 20 µL response blend containing 5 µL PCR sections and 2 µL 10X cushion at 37°C overnight. The items were processed by 10 U HinfI limitation catalysts (BioLabs Inc., New England) and imagined after electrophoresis on 10% polyacrylamide gels with ethidium bromide. The PCR result of MTHFR A1298C was handled with 10 U MboII limitation catalyst (BioLabs Inc., New England) in a 20-µL response blend containing 5

µL PCR pieces and 2 µL 10X support at 37°C overnight.

Assimilation items were envisioned after electrophoresis on 10% polyacrylamide gels with ethidium bromide. PCR enhancement was performed with 25 µL response blends encompassing 2 µL of genomic nucleic acid, Go Taq polymerase with 12.5 µL, 1 µL groundwork for the preliminary and 6.5 µL ddH2O.

Response circumstances encompassed starting denaturation stage at 95 ℃ for 5 min, after that 30 cycles of denaturation at 95 ℃ _{for 1 min, toughening for 1 min in 66} ℃ _{(XRCC1 399), 62} ℃ _{(MTHFR A1298C), 58 for}

(MTHFR C677T) and prolongation at 71℃ _{for 1 min. PCR items were examined by agarose gel electrophoresis and}

all groundworks utilized as a part of this study was portray underneath in the table 1, figure 4 and 5.

TABLE 1: PCR PRIMERS SPECIFIC OLIGONUCLEOTIDE

Locus Mutation PCR primers bp. MTHFR C677T 5'-GAAGCAGGGAGCTTTGAGG-3'

5'-ACGATGGGGCAAGTGATG-3'

152

Digested with 10 U HinfI restriction enzyme;

TT homozygotes showed two fragments of 98 and 54 bp; CT heterozygotes showed three fragments of 152, 98, and 54 bp; wild-type homozygotes (CC) showed only one band of 152 bp.

MTHFR A1298C 5'-AGAGCAAGTCCCCCAAGGA-3' 5'-CTTT GTGACCATTCCGGTTTG-3'

123

Digested with 10 U MboIIrestriction enzyme

CC genotype showed a band of 95 bp and a residue of 28 bp, AC heterozygotes showed two bands of 95 and 67 bp and three residues of 28 bp, and AA homozygotes showed a band of 67 bp and two 28-bp

XRCC1 399 5'-TCCCTGCGCCGCTGCAGTTTCT-3' 5'-TGGCGTGTGAGGCCTTACCTCC-3'

447

RESULTS

TABLE 2: Common demographic physiognomies including the study

Criteria

Cancer cases Control cases

(total: 77) (total: 55) No. % No. %

Age

<64 41.0 53.2 34.0 61.8 >64 36.0 46.8 21.0 38.2 Sex

Male 52.0 67.5 23.0 41.8 Female 25.0 32.5 22.0 40.0 Smoking condition

Smoking 68.0 88.3 18.0 32.7 Not 9.0 11.7 37.0 67.3 Clinicopathological of colorectal Proximal 17.0 22.1 0.0 0.0 Distal 21.0 27.3 0.0 0.0 Rectal 33.0 42.9 0.0 0.0 No data 6.0 7.8 0.0 0.0

The consequences of RFLP-PCR uncovered the genotype circulations for MTHFR C677T, MTHFR A1298C and XRCC1 in patients with CRC. The appropriation quality polymorphisms in both patients and controls were planned with Hardy-Weinberg balance.

Hereditary polymorphisms of all qualities incorporating into this study were portrayed in figure 1, 2 and 3 and table 3. The Genotype appropriation for MTHFR 677 quality in CRC patients; the recurrence of MTHFR C677 genotypes were as kindred TT 12.0 (15.6%), CT 24.0 (31.2%), and CC 41.0 (53.2%) in CRC, and in the control was 19.0 (34.5%), 14.0 (25.5%), and 22.0 (40.0%) individually. Amalgamation frequencies for MTHFR 677 TT homozygous and CT 677 heterozygous watched were 31.2% in CRC and 47.3% in the controls as appeared in table3.

TABLE 3: Genotype distributions for CRC cases and control participants

MTHFR 677 Cancer cases Control cases

No. % No. %

CC 41.0 53.2 22.0 40.0 CT 24.0 31.2 14.0 25.5 TT 12.0 15.6 19.0 34.5 T allele 48.0 31.2 52.0 47.3 C allele 106.0 68.8 58.0 52.7

MTHFR1298 Cancer cases Control cases

No. % No. %

AA 47.0 61.0 26.0 47.3 AC 21.0 27.3 21.0 38.2 CC 9.0 11.7 8.0 14.5 A allele 115.0 74.7 73.0 66.4 C allele 39.0 25.3 37.0 33.6

XRCC1 399 Cancer cases Control cases

No. % No. %

TT 28.0 36.4 27.0 49.1 TG 13.0 16.9 6.0 10.9 GG 36.0 46.8 22.0 40.0 T allele 69.0 44.8 60.0 54.5 G allele 85.0 55.2 50.0 45.5

The Genotype dispersion for MTHFR 1298 quality in CRC patients; the recurrence of MTHFR C677 genotypes were as kindred CC 9.0 (11.7%), CA 21.0 (27.3%), and AA 47.0 (61%) in CRC, while in the control was as individual 8.0 (14.5%), 21.0 (38.2%), and 26.0 (47.3%) separately. The recurrence for MTHFR 1298 quality AA homozygous and AC 1298 heterozygous watched were 74.7% in CRC and 66.4% in the controls as appeared in table 3 and figure 2, 4 and 5.

DISCUSSION

(CRC) is viewed as a noteworthy wellbeing issue: it is the most common disease and the second biggest reason for malignancy demise in Western nations [48]. Customarily, CRC investigation was concentrated for chromosomal insecurity (CIN) passageway, wherein APC quality transformation consider as main pathogenic occasion which can prompts misfortunes of allele associated to translocation and physical quality intensification and that established model of carcinogenetic is in charge of more than 75% from all cases of CRC [49]. The following cancer-causing trail is portrayed with most recent many years in twentieth century, which is identified with deactivation the befuddle reparation quality framework, that thusly prompts inactivation of changed tumor silencer qualities, which is called MSI or MMR passageway. Lynch disorder is the worldview for that option cancer model; a diploid tumor prompts by these disorders that have microsatellite flimsiness (MSI) phenotype. Developing proof recommends that some polymorphisms in the MTHFR quality add to sporadic CRC [50].

Numerous studies done on CRC, have reported a defensive relationship among the genotype of MTHFR 677TT associated with the jeopardy of other disease [51, 52]. Moreover, other concentrate likewise proposed that people with folate sufficient prominence which are consider homozygous for previous gene SNPs require diminished the danger of CRC. Also, others watched that the TT genotype was defensive in folate-loaded subjects, while the blend of TT and low folate status presented no insurance, or even demonstrated an expanded danger. These outcomes recommend that the malignancy hazard connected with MTHFR polymorphisms may show a quality supplement communication that relies on upon the level of folate admission or plasma folate levels. Be that as it may, we couldn't assess the quality supplement collaboration in our study because of an absence of information with respect to the plasma folate levels of case gathering. Despite the fact that we had data on the plasma folate levels of numerous solid people in the control bunch, which depended on the all-inclusive community [53, 54].

The aftereffects of this study displayed an on a very basic level balanced spread of a couple of alleles like (T allele in MTHFR C677T, C allele in MTHFR A1298C) between CRC patients diverged from control. We record a foremost relationship of two polymorphisms in MTHFR with powerlessness to CRC. The affiliation saw between the SNPs in the MTHFR quality and helplessness to CRC has been conflicting. There is a constrained study on that subject which discovered MTHFR polymorphism C677T does not influence the danger of CRC. In any case, the lion's share of studies distributed gave great confirmation that homozygosity to the T-allele is connected with an

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

No. % No. %

Cancer cases Control cases

Figure 3: Genotype distribution for XRCC1 399 gene in colorectal

cancer patients

TT TG GG

0.0 10.0 20.0 30.0 40.0 50.0 60.0

No.

%

No.

% Cancer cases

Control cases

Figure 1: Genotype distribution for MTHFR 677 gene in colorectal cancer patients

10,131 CRC patients and 15,362 controls, recommended that the 677T allele gives a defensive impact against CRC hazard [56]. Also, others distributed a HuGE audit on the MTHFR C677T polymorphism, in which the 677TT genotype gave off an impression of being connected with a lessened danger for CRC [57].

The 19q13.2 site which containing XRCC1 is comprises of many exons encoding a numerous amino acids corrosive enzymatic free atomic protein platform which communicates with enzymatic components, for example, PDRP, DNA polymerase β DNA and ligase III to encourage protein/protein reactions and proficient repair of SSBs DNA [58]. Notwithstanding the point that the polymorphism within XRCC1 Arg399Gln was performed to protect affectability to the DNA repair imperfection and alkylating operators methyl methane sulfonate in the cell line EM9 lacking XRCC1, the conflicting reports in relation to variations of the XRCC1[59]. The fact that conceivable that SNPs of additional reparation potentials, XRCC2for example, which is considered as linkage disequilibrium with XRCC1 Arg399Gln, may possibly adjust impact of this SNPs with the danger of colorectal cancer[60, 61, and 62].

The outcomes on fruitlessness evaluation demonstrated that a large portion of the semen tests from those barren male patients have a place with 20-36 years of age classification have cancellation inside of Y-chromosome area, the thought that that erasure in charge of male barrenness emerges from the clinical perception of the patients' male regenerative framework 63, 64 and 65].

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0

No. % No. %

Cancer cases Control cases

Figure 2: Genotype distribution for MTHFR 1298 gene in colorectal cancer patients

Figure 4: Gel electrophoresis of MTHFR A1298C PCR products. Lane No. 1-6 shows the 123 bp for the gene product, (Ladder) lanes contain the 100 bp DNA Step Ladder, 3% NuSieve® 3:1 agarose gel in 1X TBE buffer containing 0.8µg/ml ethidium bromide

Figure 5: Gel electrophoresis of MTHFRA C677T and XRCC1 PCR products. Lane No. 1-6 shows the MTHFRA C677T 152 bp (down), while Lane No. 1-5 shows the XRCC1 447bp (up) PCR products for the gene, (Ladder) lanes contain the 100 bp DNA Step Ladder, 3%

NuSieve® 3:1 agarose gel in 1X TBE buffer containing 0.8µg/ml ethidium bromide

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