Association of miR-146α gene polymorphism and expression levels of its target protein RhoA in Pakistani breast cancer patients.

OBJECTIVE: To assess the association of miR-146a and its target protein RhoA expression levels in breast cancer. METHODS: The case-control study was conducted at Riphah International University, Islamabad, Pakistan, from March 2017 to November 2018, and comprised confirmed breast cancer cases and controls who were matched for age and ethnicity. Genotyping and expression profiling of archived samples was performed. Data was analysed using SPSS 22. RESULTS: Of the 590 subjects, 295(50%) each were cases and controls. Among the cases, there were 195(66%) Punjabis, 59(20%) Pathans and 41(14%) Kashmiris. The corresponding numbers among the controls were 198(67%), 58(19.7%) and 39(13.2%). The association between genotypes of the cases and controls was significant (p<0.05). Strong association was seen in dominant, recessive and allelic models (p=0.05). In Punjabi group the association was (p<0.00) significant, but this association was not significant in Kashmiri and Pathan groups (p>0.05). No association was found with the receptor status and miR-146a polymorphism. CONCLUSIONS: The miR-146a gene polymorphism rs2910164 G/C was found to have increased susceptibility to breast cancer at genotype and allelic levels.

Association study of interleukin-1 receptor associated kinase 1 rs3027898 A/C gene polymorphism and preeclampsia in Pakistani population.

OBJECTIVES: To find the association between interleukin-1 receptor-associated kinase 1 rs3027898 gene polymorphism and preeclampsia. METHODS: The case-control study was conducted from October, 2018 to September, 2019 at the Railway General Hospital and the Department of Biochemistry, Islamic International Medical College, Rawalpindi, Pakistan, and comprised patients diagnosed with preeclampsia and healthy controls. The interleukin receptor-associated kinase-1 polymorphism was determined using multiplex tetra primer amplification refractory mutation system polymerase chain reaction. Outcomes were determined in terms of association of interleukin receptor-associated kinase-1 with preeclampsia. Data was analysed using SPSS 22. RESULTS: Of the 160 subjects, 80(50% were cases with a mean age of 30±5.3 years and 80(50%) were controls with a mean age of 27±3.7 years. AC genotype was seen in 45(56.25%) cases and 30(37.5%) controls, AA genotype in 25(31.25%) cases and 30(37.5%) controls, while CC genotype was seen in 10(12.5%) cases and 20(25%) controls (p>0.05). CONCLUSIONS: There was no significant association of interleukin receptor-associated kinase-1 genotypes with preeclampsia.

Association of single nucleotide polymorphism of transforming growth factor ß1 (T29C) in breast cancer patients: a case control study in Rawalpindi.

OBJECTIVE: To determine the association of single nucleotide polymorphism in three CC, TT and TC genotypes of transforming growth factor ß1 T29C in breast cancer patients. METHODS: The case-control study was conducted from April 2017 to April 2018 at the Islamic International Medical College, Rawalpindi, Pakistan, in collaboration with Nuclear Oncology Medicine and Radiotherapy Institute and Holy Family Hospital, Rawalpindi. Using convenience sampling, breast cancer cases and healthy controls were enrolled. All investigations were done using standardized laboratory protocols. The outcomes were determined in terms of association of single nucleotide polymorphism of transforming growth factor ß1with breast cancer. Data was analysed using SPSS 21. RESULTS: Of the 150 subjects, 80(53.3%) were cases and 70(47.7%) were healthy controls. Among the cases, the most frequent genotype was CC 38(47.5%) followed by TC 26(32.5%) and TT 16(20%). Among the controls, the corrsesponding values were 50(71.42%), 13(18.5%) and 7(10%). Transforming growth factor ß1 TC genotype was strongly associated with the increased risk of developing breast cancer (odds ratio: 3.79). CONCLUSIONS: The incidence of breast cancer was markedly lower among women with CC genotype compared to those with CT or TT genotypes.

Comparison of asymmetric dimethylarginine levels between pre- and post-menopausal women - a cross-sectional study from Rawalpindi.

OBJECTIVE: To compare serum levels of asymmetric dimethylarginine between premenopausal and postmenopausal women, and to evaluate its relationship with time since onset of menopause. METHODS: The cross-sectional comparative study was conducted at Islamic International Medical College, Rawalpindi, Pakistan, from April 2017 to March 2018, and comprised individuals recruited from the Gynaecology and Obstetrics outpatient department of the Pakistan Railway Hospital in the city. The subjects were divided into two groups; pre-menopausal women in Group I and postmenopausal in Group II. Group II was subdivided into three categories according to time since onset of menopause. Serum asymmetric dimethylarginine levels were estimated in each subject. Data was analysed using SPSS 21. RESULTS: Of the 80 subjects, there were 40(50%) in Group I with a mean age of 36.25}6.8 years, 40(50%) in Group II with a mean age of49.83}4.35 years. There was a significant increase in mean asymmetric dimethylarginine levels in Group II compared to Group I (p<0.05). There was also a significant increase in asymmetric dimethylarginine levels with increase in duration since the onset of menopause (p<0.001). CONCLUSIONS: Levels of asymmetric dimethylarginine increased significantly after menopause.

Genetic polymorphism of miRNA-196a and its target gene annexin-A1 expression based on ethnicity in Pakistani female breast cancer patients.

OBJECTIVE: To evaluate the association of miR-196a rs11614913 C/T genetic variation and its target gene annexin A1 mRNA expression with breast cancer risk in Pakistani female ethnicities. METHODS: This case control study, conducted from March 2017 to November 2018 included 295 breast cancer patients, 295 controls of three Pakistani ethnicities and archived 100 samples of cohort group for genotyping and expression profiling. Genotyping of miR-196a (rs11614913 C/T) was done by ARMS PCR technique. Annexin-A1 (ANXA1) mRNA expression was measured with qRT-PCR and detection of protein expression of ANXA1 was done by immunohistochemistry. RESULTS: CC homozygous genotype of miR-196a rs11614913 was present in 81.4% of cases and 73.9% controls. C/T polymorphism was found to be significantly associated with decrease risk of breast cancer (OR=0.25 (0.11- 0.58, p <0.05). Similar trend was seen with the minor T allele (OR=0.55 (0.39-0.77, p <0.05, and both dominant and recessive models (OR=0.64; p=0.02 and OR=0.26, p=0.00). In the KPK ethnic group significant decrease association with breast cancer risk was observed (OR= 0.22 (0.09-0.53, p < 0.05). Immunohistochemical staining showed loss of ANXA1 protein expression in 72 samples, and significant association was observed with pathological type p=0. 00 and triple negative receptor status p=0.03 and with genotypes of miR-196a p=0.00. Increase relative expression of 2.81± .88 by qPCR analysis of ANXA1 mRNA was noted with TT genotype. CONCLUSIONS: Our results demonstrate that miR-196a rs11614913 C/T polymorphism is associated with a decreased risk and loss of protein expression in breast cancer in the Pakistani population.

Noninvasive prenatal testing of beta-thalassemia for common Pakistani mutations: a comparative study using cell-free fetal DNA from maternal plasma and chorionic villus sampling.

BACKGROUND: The discovery of circulating cell-free fetal DNA (cff-DNA) in maternal plasma has inspired the noninvasive prenatal testing (NIPT) approaches for various genetic fetal screening including rhesus D typing, sex determination, aneuploidies, and single-gene disorders. OBJECTIVE: Noninvasive determination of paternally inherited beta-thalassemia mutations in maternal total cell-free DNA (cf-DNA) by using allele-specific amplification refractory mutation system (ARMS) real-time PCR (RT-PCR) in concordance with the conventional invasive method. METHODS: An observational study was conducted at the Armed Forces Institute of Blood Transfusion in collaboration with the genetics resource center from March 2021 to August 2021. A total number of 26 couples were selected having a history of previously affected children with beta-thalassemia. A routine chorionic villus sampling (CVS) invasive procedure was carried out, and the mutation analysis was done using conventional PCR. To assess NIPT, a total cf-DNA was also extracted from maternal plasma and analyzed using allele-specific ARMS RT-PCR. RESULTS: Based on conventional PCR testing, 13 of 26 couples were found having beta-thalassemia carriers with homozygous mutation, and 13 couples were carriers with heterozygous mutations. Further to assess NIPT, the cf-DNA of 13 pregnant females among the couples with different mutational patterns was analyzed by allele-specific ARMS RT-PCR to detect paternally inherited mutations. In comparison with conventional PCR, 11 cases (84.6%) were matched successfully, while two cases (15.4%) had no concordance with conventional invasive prenatal testing (IPT). CONCLUSION: NIPT using maternal cf-DNA by allele-specific ARMS RT-PCR can be feasible to screen paternal inherited mutant alleles to rule out pregnant women from invasive procedures where the test would be negative for paternal inheritance. However, a low amount of fetal DNA in maternal plasma is a limiting factor and required further improvement to enrich fetal cf-DNA for complete concordance with conventional IPT.

Effect of smoke on PAPP-A and lipid profile in normal rats.

Cigarette smoking is a major risk factor for atherosclerosis and cardiovascular disease. Active and passive cigarette smoke exposure predisposes to cardiovascular events. Pregnancy associated plasma protein-A is a metalloproteinase of the metzincin super family. It cleaves specific insulin like growth factor binding proteins thereby regulating local insulin like growth factor bioavailability which is a mediator of atherosclerosis. In this study we wanted to establish if Pregnancy associated plasma protein-A levels are increased in normal rats and if the Lipid profile was affected, if exposed to passive smoking. Sixty albino rats of Sprague-Dawley strain were used, weighing 200-250 gm. Both the groups were kept in identical chambers. One group of 30 rats was further exposed to cigarette smoke. Blood triglycerides were determined by enzymatic colorimetric method. In this animal study it was seen that smoking had no effect on (triglycesides), cholesterol, LDL and HDL levels. Smoke exposure has no effect on pregnancy associated plasma protein-A (PAPP-A) levels.

To assess the levels of zinc in serum and changes in the lens of diabetic and senile cataract patients.

OBJECTIVE: To assess the levels of zinc in serum and changes in the lens of diabetic and senile cataract patients METHODS: A comparative cross sectional study was done at LRBT (Layton Rehmatullah Benevolent Trust) free eye hospital Mandra from July 2010 to August 2010. Twenty Type 2 diabetic patients with cataracts and 20 patients without diabetes of senile cataract were included in the study. Glucose levels were determined by glucose oxidase method and zinc levels measured by colorimeteric method (RANDOX). RESULTS: The statistical analysis showed a statistically significant decrease in zinc levels in diabetic patients with cataract when compared to individuals with senile cataract (P <0.05). In senile lenses multilayering of cells, wrinkling of the lens capsule, and apoptotic cell death were seen, but in diabetic lenses number of cells decreased and cells were smaller as compared to senile lenses. The results were not statistically significant P >0.05. CONCLUSION: Our findings in this study show a relationship between zinc deficiency and increased occurrence of cataract among people with diabetes.

Glutathione S-Transferase Theta 1 / Mu 1 Null Genotype and Risk of Development of Actinic Keratosis in Pakistani Population.

OBJECTIVE: To assess the frequency and association of Glutathione S-Transferase Theta 1 and Glutathione S-Transferase Mu 1 null genotypes in development of actinic keratosis (AK) in a group of Pakistani population. STUDY DESIGN: Case-control analytical study. PLACE AND DURATION OF STUDY: Department of Biochemistry, Islamic International Medical College, Rawalpindi in collaboration with Department of Dermatology, Railway Hospital and Rural Health Center, District Health Office, Rawalpindi from September 2018 to September 2019.  Methodology: A total of 86 participants were included in this study with 27 biopsy proven cases of AK and 59 matched controls. Blood samples were collected after obtaining written informed consent; and DNA was extracted by Chelex™ method. Multiplex PCR (M-PCR) was done to find respective allelic frequencies of GSTM1 and GSTT1 genes in both cases and controls.  Results: Mean age of participants in cases and controls was 62.93 ±10.29 years and was 61.42 ±9.96 years, respectively. There were 18 males (66.7%) and 9 females (33.3%); and 43 males (72.9%) and 16 females (27.1%) in cases and controls, respectively. There was a significant association of GSTT1 null genotype with AK (OR: 2.72, 95% CI: 1.05-7.05, p = 0.037). There was a positive correlation between GSTT1 null genotype and AK (r = 0.225, p = 0.037). CONCLUSION: GSTT1 null genotype has a significant association for AK development in the studied Pakistani population. Key Words: Actinic keratosis, Glutathione S-Transferase Mu 1, Glutathione S-Transferase Theta 1, Polymerase chain reaction, Squamous cell carcinoma.

Serum Albumin and C3 Complement Levels in Endometriosis.

OBJECTIVE: To compare the efficacy of albumin and C3 complement as serum marker for the early diagnosis of endometriosis in women of reproductive age. STUDY DESIGN: Cross-sectional comparative study. PLACE AND DURATION OF STUDY: Biochemistry Department, Islamic International Medical College, Rawalpindi, in collaboration with the Gynecology and Obstetrics Department, Pakistan Railways Hospital, Rawalpindi, from March 2017 to February 2018. METHODOLOGY: Forty-four patients with endometriosis and 44 controls of reproductive age group were enrolled for the study. Those having other comorbid systemic or endocrine diseases were excluded from the study. Levels of albumin and C3 complement were measured by using Micro Lab 300 and ELISA, respectively; and the results were compared by using SPSS version 21. groups, respectively (p=0.201). The mean serum C3 levels were 1120.9±265 µg/ml and 2241.0±293 µg/ml in the control and endometriosis patient groups, respectively (p <0.001). RESULTS: The mean serum albumin levels were 4.62±0.88 g/dl and 4.42±0.52 g/dl in the control and endometriosis Conclusion: Serum C3 complement were significantly increased in endometriosis patients but not in controls; whereas, serum albumin levels were not significantly different in endometriosis cases as compared to the controls.