hsp70 PCR-RFLP as an alternative tool to identify Sauroleishmania species.

Sauroleishmania spp. comprises one of the four Leishmania subgenera, which has been historically considered a non-pathogenic protozoan of reptiles. However, some strains appear to be transiently infective to mammals, and recent findings have detected these parasites in dogs and humans in areas where leishmaniasis is endemic. Herein, the digestion pattern of PCR-RFLP of the 234 bp-hsp70 fragment was evaluated as a simpler and cheaper tool to distinguish the Sauroleishmania species from the other Leishmania subgenera. As a result, the digestion of the 234 bp-hsp70 fragments with HaeIII produced a banding pattern specific to the four Sauroleishmania strains assessed. This technique could contribute to the identification of Leishmania parasites isolated from sandflies, reptiles, or even mammals in fieldworks as an alternative to the use of laborious and expensive methodologies.

[Identification of Cervi Cornu (Cervus elaphus) and its formula granules based on PCR-RFLP].

Cervi Cornu is the ossified antler, or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C. nippon, as a precious traditional Chinese medicine, has been recognized for its medicinal value and widely used in clinical practice. However, the origins of Cervi Cornu are miscellaneous, and Cervi Cornu is even mixed with adulterants in the market. Currently, there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cornu. So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants. In this study, the differences in the mitochondrial barcode cytochrome b(Cytb) gene sequences of C. elaphus, C. nippon and their related species were compared and the specific single nucleotide polymorphism(SNP) sites on the Cytb sequences of Cervi Cornu were screened out. According to the screened SNPs, Cervi Cornu-specific primers dishmy-F and dishmy-R were designed. The PCR system was established and optimized, and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated. The amplification products of C. elaphus and C. nippon were digested using the restriction enzyme MseⅠ. The results showed that after electrophoresis of the product from PCR with the annealing temperature of 56 ℃ and 35 cycles, a single specific band at about 100 bp was observed for C. elaphus samples, and the product of C. elaphus samples was 60 bp shorter than that of C. nippon samples. There was no band for adulterants from other similar species such as Alces alces, Rangifer tarandus, Odocoileus virginianus, O. hemionus, Cap-reolus pygargus, Przewalskium albirostis and negative controls. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method established in this study can quickly and accurately identify Cervi Cornu originated from C. elaphus in crude drugs, standard decoctions, and formula granules, and distinguish the origins of Cervi Cornu products, i.e., C. nippon and similar species. This study can be a reference for other studies on the quality standard of other formula granules of traditional Chinese medicines.

Prevalence and Clinical Characteristics of Mitochondrial DNA Mutations in Korean Patients With Sensorineural Hearing Loss.

BACKGROUND: Mutations in mitochondrial DNA (mtDNA) are associated with several genetic disorders, including sensorineural hearing loss. However, the prevalence of mtDNA mutations in a large cohort of Korean patients with hearing loss has not yet been investigated. Thus, this study aimed to investigate the frequency of mtDNA mutations in a cohort of with pre- or post-lingual hearing loss of varying severity. METHODS: A total of 711 Korean families involving 1,099 individuals were evaluated. Six mitochondrial variants associated with deafness (MTRNR1 m.1555A>G, MTTL1 m.3243A>G, MTCO1 m.7444G>A and m.7445A>G, and MTTS1 m.7471dupC and m.7511T>C) were screened using restriction fragment length polymorphism. The prevalence of the six variants was also analyzed in a large control dataset using whole-genome sequencing data from 4,534 Korean individuals with unknown hearing phenotype. RESULTS: Overall, 12 of the 711 (1.7%) patients with hearing loss had mtDNA variants, with 10 patients from independent families positive for the MTRNR1 m.1555A>G mutation and 2 patients positive for the MTCO1 m.7444G>A mutation. The clinical characteristics of patients with the mtDNA variants were characterized by post-lingual progressive hearing loss due to the m.1555A>G variant (9 of 472; 1.9%). In addition, 18/4,534 (0.4%) of the Korean population have mitochondrial variants associated with hearing loss, predominantly the m.1555A>G variant. CONCLUSION: A significant proportion of Korean patients with hearing loss is affected by the mtDNA variants, with the m.1555A>G variant being the most prevalent. These results clarify the genetic basis of hearing loss in the Korean population and emphasize the need for genetic testing for mtDNA variants.

Development of a Molecular Tool for Identification of a New Neopestalotiopsis sp. Associated with Disease Outbreaks on Strawberry.

A new Neopestalotiopsis sp. was recently reported causing outbreaks of leaf spot and fruit rot on strawberry in Florida, Georgia, and South Carolina. In contrast to other Pestalotiopsis pathogens, the new species appears more aggressive and destructive on strawberry. Current chemical options for management are disease suppressive at best, and affected growers have been experiencing major yield losses. In this study, we developed a molecular method based on polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) for identification of the new Neopestalotiopsis sp. from strawberry. Isolates of the new Neopestalotiopsis sp. collected in Florida; isolates of N. rosae, N. honoluluana, N. ellipsopora, N. saprophytica, N. samarangensis, and P. rhododendri; and isolates from South Carolina suspected to be the new Neopestalotiopsis sp. were included in this study. This method is based on PCR amplification of a ß-tubulin gene fragment using a previously published set of primers (Bt2a and Bt2b), followed by use of the restriction enzyme BsaWI. The enzyme cuts the PCR product from the new Neopestalotiopsis sp. twice, yielding fragments of 290 base pairs (bp) and 130 and 20 bp in size, whereas fragments from other species are only cut once, yielding fragments of 420 and 20 bp. This method will aid research labs and diagnostic clinics in the accurate and fast identification of the aggressive Neopestalotiopsis sp. variant from strawberry.

Detection and Genotypic Analysis of Anaplasma bovis and A. phagocytophilum in Horse Blood and Lung Tissue.

A clinical case of Anaplasma bovis was reported for the first time in our previous study (2019) in a horse, a nondefinitive host. Although A. bovis is a ruminant and not a zoonotic pathogen, it is responsible for persistent infections in horses. In this follow-up study, the prevalence of Anaplasma spp., including A. bovis, was assessed in horse blood and lung tissue samples to fully understand Anaplasma spp. pathogen distribution and the potential risk factors of infection. Among 1696 samples, including 1433 blood samples from farms nationwide and 263 lung tissue samples from horse abattoirs on Jeju Island, a total of 29 samples (1.7%) tested positive for A. bovis and 31 (1.8%) samples tested positive for A. phagocytophilum, as determined by 16S rRNA nucleotide sequencing and restriction fragment length polymorphism. This study is the first to detect A. bovis infection in horse lung tissue samples. Further studies are needed to clarify the comparison of sample types within cohorts. Although the clinical significance of Anaplasma infection was not evaluated in this study, our results emphasize the need to clarify the host tropism and genetic divergence of Anaplasma to enable the development of effective prevention and control measures through broad epidemiological studies.

Changes in the Endophytic Bacterial Community of Brassica rapa after Application of Systemic Insecticides.

Insecticides not only control target pests but also adversely affect non-target communities including humans, animals, and microbial communities in host plants and soils. The effect of insecticides on non-target communities, especially endophytic bacterial communities, remains poorly understood. Two phases of treatments were conducted to compare the trends in endophytic bacterial response after insecticide application. Endophytic bacteria were isolated at 2 and 4 weeks after germination. Most insecticide treatments showed a declining trend in bacterial diversity and abundance, whereas an increasing trend was observed in the control. Therefore, insecticide use negatively affected non-target endophytic bacterial communities. Bacillus spp. was mostly dominant in the early stage in both insecticide treatment and control groups. Nevertheless, in the matured stage, mostly bacteria including Pseudomonas spp., Priestia spp. were dominant in groups treated with high insecticide concentrations. Therefore, plants can regulate and moderate their microbiome during their lifecycle depending on surrounding environmental conditions.

Molecular Characterization of Haemoglobin E.

OBJECTIVE: To detect mutation in cases having haemoglobin A2 level >7% on high performance liquid chromatography. METHODS: The cross-sectional, descriptive study was conducted from July 2017 to December 2018 at the Department of Haematology and Human Genetics and Molecular Biology, University of Health Sciences, Lahore, Pakistan, and comprised patients of either gender with haemoglobin A2 ≥7%. The samples were collected from different cities of Punjab in collaboration with the Punjab Thalassemia Prevention Programme, Lahore. The samples were subjected to complete blood count and high performance liquid chromatography using automated haematology analysers and variant-II beta thalassemia short programme, respectively. To analyse haemoglobin E mutations at the molecular level, polymerase chain reaction-restriction fragment length polymorphism (PCR_RFLP) was performed using a type IIS restriction endonuclease known as Mnl1 (derived from Moraxella nonliquefaciens) to cleave DNA at specific sites and the results were further confirmed on randomly selected samples using Sanger sequencing. Data was analysed using SPSS 25. RESULTS: Of the 39 patients, 15(38.5%) were males and 24(61.5%) were females. The overall median age was 14 (23) years. There were 29 (74.4%) patients with thalassemia family history, and 22(56.4%) had a positive family history of transfusion related to thalassemia, while no patient had a family history of iron therapy. The median haemoglobin A, haemoglobin A2 and haemoglobin F levels were 72.2 (65.2-79.1) %, 26.6 (19.1-34.0) % and 0.9 (-0.8-2.6) %, respectively. After molecular investigation, HbAE mutation was found in 23(59%) patients, while wild type HbAA genotype was found in 16(41%). The heterozygous HbE mutation was present in 23(59%) patients. CONCLUSIONS: Frequently missed/undiagnosed cases of haemoglobin E that co-elute with haemoglobin A2 in the same high performance liquid chromatography window were detected among those with haemoglobin A2 ≥7%.

Responses of potential ammonia oxidation and ammonia oxidizers community to arsenic stress in seven types of soil.

Soil arsenic contamination is of great concern because of its toxicity to human, crops, and soil microorganisms. However, the impacts of arsenic on soil ammonia oxidizers communities remain unclear. Seven types of soil spiked with 0 or 100 mg arsenic per kg soil were incubated for 180 days and sampled at days 1, 15, 30, 90 and 180. The changes in the community composition and abundance of ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) were analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis, clone library sequencing, and quantitative PCR (qPCR) targeting amoA gene. Results revealed considerable variations in the potential ammonia oxidation (PAO) rates in different soils, but soil PAO was not consistently significantly inhibited by arsenic, probably due to the low bioavailable arsenic contents or the existence of functional redundancy between AOB and AOA. The variations in AOB and AOA communities were closely associated with the changes in arsenic fractionations. The amoA gene abundances of AOA increased after arsenic addition, whereas AOB decreased, which corroborated the notion that AOA and AOB might occupy different niches in arsenic-contaminated soils. Phylogenetic analysis of amoA gene-encoded proteins revealed that all AOB clone sequences belonged to the genus Nitrosospira, among which those belonging to Nitrosospira cluster 3a were dominant. The main AOA sequence detected belonged to Thaumarchaeal Group 1.1b, which was considered to have a high ability to adapt to environmental changes. Our results provide new insights into the impacts of arsenic on the soil nitrogen cycling.

Association of Single Nucleotide Polymorphisms from Angiogenesis-Related Genes, ANGPT2, TLR2 and TLR9, with Spontaneous Preterm Labor.

In this study, we hypothesized that the changes localized at angiopoietin-2 (ANGPT2), granulocyte-macrophage colony-stimulating factor (CSF2), fms-related tyrosine kinase 1 (FLT1) and toll-like receptor (TLR) 2, TLR6 and TLR9 genes were associated with spontaneous preterm labor (PTL), as well as with possible genetic alterations on PTL-related coagulation. This case-control genetic association study aimed to identify single nucleotide polymorphisms (SNPs) for the aforementioned genes, which are correlated with genetic risk or protection against PTL in Polish women. The study was conducted in 320 patients treated between 2016 and 2020, including 160 women with PTL and 160 term controls in labor. We found that ANGPT2 rs3020221 AA homozygotes were significantly less common in PTL cases than in controls, especially after adjusting for activated partial thromboplastin time (APTT) and platelet (PLT) parameters. TC heterozygotes for TLR2 rs3804099 were associated with PTL after correcting for anemia, vaginal bleeding, and history of threatened miscarriage or PTL. TC and CC genotypes in TLR9 rs187084 were significantly less common in women with PTL, compared to the controls, after adjusting for bleeding and gestational diabetes. For the first time, it was shown that three polymorphisms-ANGPT2 rs3020221, TLR2 rs3804099 and TLR9 rs187084 -were significantly associated with PTL, adjusted by pregnancy development influencing factors.

Molecular assays for determining sulphadoxine-pyrimethamine drug resistance in India: a systematic review.

A plethora of studies analyse the molecular markers of drug resistance and hence help in guiding the evidence-based malaria treatment policies in India. For reporting mutations, a number of techniques including DNA sequencing, restriction-fragment length polymorphism and mutation-specific polymerase chain reaction have been employed across numerous studies, including variations in the methodology used. However, there is no sufficient data from India comparing these methods as well as report the prevalence of polymorphisms in SP drug resistance molecular markers independently using such methods. Therefore, all data from Indian studies available for molecular marker studies of Plasmodium falciparum drug resistance to sulphadoxine-pyrimethamine was gathered, and a systematic review was performed. This systematic review identifies the molecular methods in use in India and compares each method for detecting sulphadoxine-pyrimethamine drug resistance marker. To delay the spread of drug-resistant parasite strains, a simplified and standardized molecular method is much needed which can be obtained by analysing the performance of each method in use and answering the necessity of newer methodological approaches.

Rapid Detection of Red Rot Disease Pathogens (Pythium chondricola and P. porphyrae) in Pyropia yezoensis (Rhodophyta) with PCR-RFLP.

Red rot disease is one of the best-known algal diseases infecting red algae Pyropia species. This disease decreases the quality and quantity of Pyropia aquaculture products in Korea, Japan, and China. Recently we found that Pythium chondricola (Oomycetes) infects blades of Pyropia yezoensis. Therefore, two Pythium species (P. chondricola and P. porphyrae) have been reported as red rot disease pathogens. In this study, we developed a species-specific molecular marker for distinguishing between the two red rot disease pathogens. Using a polymerase chain reaction restriction fragment length polymorphism method based on the mitochondrial cytochrome c oxidase subunit 2 (cox2) and nuclear ribosomal RNA large subunit regions, we classified these two Pythium species without a sequencing step. This new method had high specificity and efficiency for detecting red rot disease pathogens at the species level for both of the cultured and field samples. Therefore, the molecular markers developed in this study are effective for long-term monitoring of the infection and distribution pattern of each Pythium species in Pyropia aquaculture farms. Moreover, molecular monitoring can provide useful information for predicting infection and preventing mass mortality of Pyropia species by red rot disease.

Association of PFKM gene polymorphisms and susceptibility to cryptorchidism in a Chinese Han population.

BACKGROUND: Cryptorchidism is one of the most common congenital anomalies in newborn boys. There are various risk factors that have been verified to have relationship with cryptorchidism, including exogenous and genetic, but the pathogenesis of cryptorchidism remains unclear. PFKM gene is a critical gene encodes for a regulatory enzyme, which limits the rate in the pathway of glycolysis. We assumed that cryptorchidism risk may associated with PFKM gene single-nucleotide polymorphisms (SNPs). Thus we selected three tag SNPs in the PFKM gene and aimed to investigate the possible association between PFKM gene polymorphisms and cryptorchidism risk. METHODS: The SNPs were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. 140 cases and 227 controls were enrolled in this study, including 105 unilateral cryptorchidism and 35 bilateral cases. The testis position was decided by the higher one in bilateral cases. RESULTS: The frequency of allele G of SNP rs2228500 is increased in cryptorchidism patients compared to that in controls (p < 0.05). Genotypic frequencies of rs2228500 are associated with the susceptibility of cryptorchidism in the codominant model (p < 0.05). And compared with G/G genotype in the dominant model, notable decreased frequencies of A carriers (A/G-A/A genotypes) were observed in cryptorchidism patients (p = 0.0069, OR = 1.80, 95% CI 1.17-2.75). CONCLUSIONS: This research first revealed that PFKM gene polymorphisms were associated with cryptorchidism in a Chinese Han population. We have offered primary evidence that the G allele and the G/G genotype of rs2228500 SNP in the PFKM gene are more frequent in patients with cryptorchidism than healthy controls.

Association of C-262 polymorphism in catalase gene with rheumatoid arthritis.

OBJECTIVE: To evaluate the association of C-262 polymorphism in Catalase gene (CAT) with Rheumatoid Arthritis. METHODS: The comparative cross-sectional study was conducted at the Department of Biochemistry and Molecular Biology, Army Medical College, Rawalpindi, in collaboration with the Rheumatology Department, Pak Emirates Military Hospital, Rawalpindi, Pakistan, from January to December 2020, and comprised Deoxyribonucleic acid extraction of samples. Samples in group I belonged to diagnosed rheumatoid arthritis patients of either gender aged 30-60 years who were on disease-modifying anti-rheumatic drugs. Group II had an equal number of healthy controls. The promoter region of the CAT gene having the polymorphic segment was amplified through polymerase chain reaction, and its products were then subjected to restriction fragment length polymorphism for the analysis of polymorphic region of the CAT gene. Genotypic frequency equilibrium and the association of polymorphism with rheumatoid arthritis was checked. Also, association between fasting lipid profile and haemoglobin was assessed. Data was analysed using SPSSS 22. RESULTS: Of the 60 samples, 30(50%) belonged to each of the two groups. The mean age was 44.90±10.50 years (range: 30-60 years). Overall, there were 34(56.7%) males and 26(43.3%) were females. Two alleles and three genotypes of the polymorphism was detected. The frequency of CC genotype was higher in group I 23(76.6%), but no association of any of the genotype of polymorphism was found significant (p <0.05). Haemoglobin and lipid profile levels were significantly different in the two groups (p≤0.05). CONCLUSIONS: There was no significant association found between C-262 polymorphism in CAT gene and rheumatoid arthritis.

Microbiological quality of tempeh with different wraps: banana leaf versus plastic.

Tempeh is a traditional Indonesian fermented food widely consumed and became staple food in some Indonesian diet. Commercially, tempeh is available in banana leaf or plastic wraps. The wraps are not only important for fermentation and giving final form of tempeh,  but also potential source of food microbiome. This study aimed to investigate the effect of different packaging materials on the lactic acid bacteria (LAB) quantity and community composition of tempeh from three manufacturers with different production environments. Each tempeh wrapped with banana leaf or plastic during the fermentation process and then LAB from every tempeh sample were quantified using qPCR and plate count method. Terminal Restriction Fragment Length Polymorphism analysis was carried out to assign bacterial community composition from these samples. The LAB population quantity of fresh tempeh from three manufacturers were essentially similar around 10 log CFU/g. However, there is a difference in the proportion of cultured and uncultured bacteria. Principal Coordinate Analyses based on Bray-Curtis similarity matrices showed an apparent clustering pattern for tempeh samples according to tempeh manufacturers. Meanwhile, packaging materials did not significantly influence changes in  bacterial community composition. The result of this work could be used for determination, authentication, and improvement of tempeh quality.

The new Internal Transcribed Spacer 2 diagnostic tool clarifies the taxonomic position and geographic distribution of the North American malaria vector Anopheles punctipennis.

BACKGROUND: The malaria mosquito Anopheles punctipennis, a widely distributed species in North America, is capable of transmitting human malaria and is actively involved in the transmission of the ungulate malaria parasite Plasmodium odocoilei. However, molecular diagnostic tools based on Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA are lacking for this species. Anopheles punctipennis is a former member of the Anopheles maculipennis complex but its systematic position remains unclear. METHODS: In this study, ITS2 sequences were obtained from 276 An. punctipennis specimens collected in the eastern and midwestern United States and a simple and robust Restriction Fragment Length Polymorphism approach for species identification was developed. The maximum-likelihood phylogenetic tree was constructed based on ITS2 sequences available through this study and from GenBank for 20 species of Anopheles. RESULTS: The analysis demonstrated a consistent ITS2 sequence length and showed no indications of intragenomic variation among the samples based on ITS2, suggesting that An. punctipennis represents a single species in the studied geographic locations. In this study, An. punctipennis was found in urban, rural, and forest settings, suggesting its potential broad role in pathogen transmission. Phylogeny based on ITS2 sequence comparison demonstrated the close relationship of this species with other members of the Maculipennis group. CONCLUSIONS: This study developed molecular tools based on ITS2 sequences for the malaria vector An. punctipennis and clarified the phylogenetic position of the species within the Maculipennis group.

DNA identification of species of the Anopheles maculipennis complex and first record of An. daciae in Belgium.

The present study aimed at identifying the members of the Anopheles maculipennis complex (Diptera: Culicidae) occurring in Belgium. Therefore, the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI) loci were sequenced in 175 and 111 specimens, respectively, collected between 2007 and 2019. In parallel, the suitability of two species-diagnostic PCR-RFLP assays was tested. The identified specimens included: An. maculipennis s.s. (N = 105), An. daciae (N = 62), An. atroparvus (N = 6) and An. messeae (N = 2). Each species was characterized by unique ITS2 haplotypes, whereas COI only supported the monophyly of An. atroparvus, a historical malaria vector in Belgium. Species identification results were further supported by unique PCR-RFLP banding patterns. We report for the first time An. daciae in Belgium, where it was found to co-occur with An. maculipennis s.s. The latter was the most prevalent in the collection studied (60%) and appears to have the widest distribution in Belgium. As in other studies, An. daciae and An. messeae appeared the most closely related species, up to the point that their species status remains debatable, while their ecological differences, including vector competences, need further study.

Correlation of -160C > A and -347GA > G polymorphisms in E-cadherin gene and gastric cancer in north of Iran.

BACKGROUND: E-cadherin (CDH1 gene) is a protein involved in cell-cell adhesion. There are reports on the association of -160C > A (rs16260) and -347GA > G (rs5030625) polymorphisms in the 5'-promoter region of the CDH1 gene with tumor development and progression of gastric cancer. This study aimed to examine the potential relationship between these two polymorphisms and gastric cancer in patients from Mazandaran province, Northern Iran. MATERIALS AND METHODS: A case-control study was conducted to test 97 patients and 95 healthy controls. Genomic DNA was extracted from peripheral blood followed by polymerase chain reaction amplification. Genotyping analysis was carried out using restriction fragment length polymorphism analysis for two potentially functional polymorphisms. RESULTS: Heterozygous genotype GA/G versus GA/GA of rs5030625 (-347 GA > G) was found to be associated with increased risk of gastric cancer in the people studied (odds ratio = 5.73, 95% confidence interval = 2.11-15.56, P = 0.001). Furthermore, AA or CA genotype in -160C > A polymorphism did not show any increased risk of gastric cancer (P = 0.559). CONCLUSION: The present study revealed that GA/G genotype of rs5030625 (-347 GA > G) polymorphism is associated with gastric cancer in Northern Iran.

A significant association of the CTLA4 gene variants with the risk of autoimmune Graves' disease in ethnic Kashmiri population.

Graves' disease (GD) is the commonest cause of hyperthyroidism in populations with adequate iodine intake. It results from an abnormality in the immune system, which produces unique antibodies causing over production of thyroid hormones and glandular hyperplasia in individuals with genetic susceptibility. The Cytotoxic Lymphocyte Associated Antigen-4 (CTLA4) gene product serves the important function of immunomodulation, thereby helping in maintenance of peripheral self-tolerance. Studies on the association of the CTLA4 SNPs with GD have shown variations in the results from different populations. Since no such study has been carried out in ethnic Kashmiri population, we aimed to study a possible association of the CTLA4 SNPs (+49 A/G, -318C/T, CT 60 A/G and -1661 A/G) with GD. A total of 285 individuals (135 patients with GD and 150 healthy individuals) were genotyped using PCR-RFLP method and the results showed statistically significant differences in genotypic and allelic frequencies of cases and controls for + 49 A/G SNP (p=<0.001; OR = 5.14; CI = 2.17-12.19) and CT 60 A/G SNP (p = < 0.001; OR = 6.9; CI = 2.8-16.6), while -318C/T and -1661 A/G SNPs showed no significant association. We also studied the mRNA expression of the CTLA4 in patients with GD and healthy individuals by Real-Time PCR and found a decreased expression of the CTLA4 mRNA in PBMCs of patients with GD as compared to healthy controls with a -3.71-fold change. We conclude that the CTLA4 + 49 A/G and CT 60 A/G SNPs have a significant association with the risk of GD development in Kashmiri population and CTLA4 mRNA expression is significantly decreased in GD.

Variants of SMAD1 gene increase the risk of colorectal cancer in the Bangladeshi population.

Colorectal cancer is the fourth most common type of malignancy worldwide that may develop due to the accumulation of several genetic variations. Different single nucleotide polymorphisms of SMAD1 gene are assumed to be linked with increased colorectal cancer risk. The current case-control study was conducted to verify the association of genetic polymorphisms of SMAD1 (rs11100883 and rs7661162) with colorectal cancer in the Bangladeshi population. This study was performed on 275 colorectal cancer patients and 300 healthy volunteers using polymerase chain reaction-restriction fragment length polymorphism method. The odds ratios were adjusted for age and sex with logistic regression analysis. In case of SMAD1 rs11100883 polymorphism, GA heterozygous genotype, GA + AA (dominant model), and minor allele "A" were significantly associated with colorectal cancer (adjusted odds ratio = 1.55, 95% confidence interval = 1.09-2.20, p = 0.014; adjusted odds ratio = 1.59, 95% confidence interval = 1.13-2.23, p = 0.008; and odds ratio = 1.35, 95% confidence interval = 1.06-1.73, p = 0.015, respectively) and the significance exists after the Bonferroni correction. Again, single nucleotide polymorphism rs7661162 showed significant association with an elevated colorectal cancer risk for AG heterozygous genotype, AG + GG (dominant model), AG versus AA + GG (overdominant model), and minor allele "G" (adjusted odds ratio = 1.78, 95% confidence interval = 1.24-2.56, p = 0.002; adjusted odds ratio = 1.68, 95% confidence interval = 1.18-2.39, p = 0.004; adjusted odds ratio = 1.76, 95% confidence interval = 1.23-2.53, p = 0.002; and odds ratio = 1.47, 95% confidence interval = 1.08-2.00, p = 0.014, respectively) and significance withstands after the Bonferroni correction. No significant age and gender differences between cases and controls were observed. In silico, gene expression analysis showed that the SMAD1 mRNA level was downregulated in the colon and rectal cancer tissues compared to healthy tissues. In conclusion, our findings indicate that SMAD1 rs11100883 and rs7661162 polymorphisms are responsible for increasing the susceptibility of colorectal cancer development in the Bangladeshi population.

Polymorphism rs1800255 from COL3A1 gene and the risk for pelvic organ prolapse.

INTRODUCTION AND HYPOTHESIS: Genetic variations of type III collagen may compromise the supportive structures of the female pelvic floor and consequently favor pelvic organ prolapse. The single nucleotide polymorphism G/A rs1800255 located in the coding region for type III collagen (COL3A1) was evaluated as a risk factor for pelvic organ prolapse. METHODS: A single-center prospective cohort study including women with clinical diagnosis of stage III and IV prolapse (POP group) and prolapse stage 0 or I (control group). Sociodemographic, clinical data and obstetric history were retrieved by physician interview. DNA including the rs1800255 polymorphism was amplified by polymerase chain reaction from blood genomic cells and digested with AluI restriction enzyme for distinction of G and A variants. Qualitative variables were compared using the chi-square and Fisher's exact tests and unpaired t-test for quantitative variables. After stratification of the groups, risk factors for POP were estimated using odds ratios (ORs) from the binary logistic regression model. RESULTS: A total of 292 women were included, 112 in the POP group and 180 in the control group. There was no significant difference between groups regarding rs1800255. Age and home birth were the only significant risk factors for pelvic organ prolapse. CONCLUSION: Polymorphism rs1800255 from COL3A1 gene was not a risk factor for pelvic organ prolapse.