Bp Variant Research Articles

Exploring the Sheep MAST4 Gene Variants and Their Associations with Litter Size.

The economic efficiency of sheep breeding can be improved by enhancing sheep productivity. A recent genome-wide association study (GWAS) unveiled the potential impact of the MAST4 gene on prolificacy traits in Australian White sheep (AUW)). Herein, whole-genome sequencing (WGS) data from 26 different sheep breeds worldwide (n = 1507), including diverse meat, wool, milk, or dual-purpose sheep breed types from China, Europe, and Africa, were used. Moreover, polymerase chain reaction (PCR) genotyping of the MAST4 gene polymorphisms in (n = 566) Australian white sheep (AUW) was performed. The 3 identified polymorphisms were not homogeneously distributed across the 26 examined sheep breeds. Findings revealed prevalent polymorphisms (P3-ins-29 bp and P6-del-21 bp) with varying frequencies (0.02 to 0.97) across 26 breeds, while P5-del-24 bp was presented in 24 out of 26 breeds. Interestingly, the frequency of the P3-ins-29 bp variant was markedly higher in Chinese meat or dual-purpose sheep breeds, while the other two variants also showed moderate frequencies in meat breeds. Notably, association analysis indicated that all InDels were associated with AUW sheep litter size (p < 0.05). These results suggest that these InDels within the MAST4 gene could be useful in marker-assisted selection in sheep breeding.

Identification of Various InDel-II Variants of the White Spot Syndrome Virus Isolated from Frozen Shrimp and Bivalves Obtained in the Korean Commercial Market.

White spot syndrome virus (WSSV) poses a significant threat to the global shrimp industry. We investigated the presence of WSSV in frozen shrimp (n = 86) and shellfish (n = 185) from the Korean market (2010-2018). The detection rate of first-step polymerase chain reaction (PCR) in domestic shrimp was 36.8% (7/19), whereas that in imported shrimp was 0.01% (1/67). Furthermore, the WSSV genome was amplified from domestic bivalve mollusks by first- and second-step PCR with accuracies of 3.4% (5/147) and 15.6% (23/147), respectively. The genetic relatedness of InDel-II regions among WSSVs detected in domestic shrimp groups revealed four variants (777, 5649, 11,070 and 13,046 bp insertion or deletion), and imported shrimp groups had four variants (10,778, 11,086, 11,500 and 13,210 bp) compared with the putative ancestor WSSV strain. The 5649 bp variant was the dominant type among the WSSV variants detected in domestic shrimp (54.5%, 6/11). Notably, bivalve mollusks exhibited six variants (777, 5649, 5783, 5876, 11,070 and 13,046 bp), including four variants detected in shrimp, indicating that bivalve mollusks could facilitate WSSV tracking. In a challenge test, whiteleg shrimp (Litopenaeus vannamei) exhibited varying mortality rates, indicating a link between InDel-II deletion and viral replication. These findings highlight the complexity of WSSV transmission.

Screen of small fragment mutations within the sheep thyroid stimulating hormone receptor gene associated with litter size

The thyroid stimulating hormone receptor (TSHR), a glycoprotein hormone receptor, plays an important role in metabolic regulation and photoperiod control in the time of reproduction in birds and mammals. Previous genome-wide association studies revealed that the TSHR gene was related to reproduction and its function was identified in female reproduction, but rare studies reported the polymorphism of TSHR gene. However, the molecular mutations of the TSHR gene in sheep have not been reported so far. Herein, we explored potential polymorphisms of the sheep TSHR gene, and a 29 bp nucleotide sequence variant (rs1089565492) was identified in the AUW sheep. There were three genotypes of the 29 bp variant locus detected which named ‘II’ ‘DD’ and ‘ID’ been identified. Association analysis results showed the 29 bp variant was significantly associated with the litter size of the AUW sheep (p < 0.05). This finding suggests that the 29 bp nucleotide sequence variant within TSHR gene could be a candidate marker of reproduction traits for sheep breeding improving through the marker-assisted selection (MAS).

Complete Chloroplast Genome Variants Reveal Discrete Long-Distance Dispersal Routes of Rhizophora in the Western Indian Ocean

Historical processes of long-distance migration and ocean-wide expansion feature the global biogeographic pattern ofRhizophoraspecies. Throughout the Indian Ocean,Rhizophora stylosaandRhizophora mucronataseem to be a young phylogenetic group with an expansion ofR. mucronatatoward the Western Indian Ocean (WIO) driven by the South Equatorial Current (SEC). Nuclear microsatellites revealed genetic patterns and breaks; however, the estimation of propagule dispersal routes requires maternally inherited cytoplasmic markers. Here, we examine the phylogeography of 21R. mucronataprovenances across a &amp;gt;4,200 km coastal stretch in the WIO usingR. stylosaas an outgroup. Full-length chloroplast genome (164,474 bp) and nuclear ribosomal RNA cistron (8,033 bp) sequences were assembled. The boundaries, junction point, sequence orientation, and stretch between LSC/IRb/SSC/IRa/LSC showed no differences withR. stylosachloroplast genome. A total of 58 mutations inR. mucronataencompassing transitions/transversions, insertions-deletions, and mononucleotide repeats revealed three major haplogroups. Haplonetwork, Bayesian maximum likelihood (ML), and approximate Bayesian computation (ABC) analyses supported discrete historical migration events. An ancient haplogroup A in the Seychelles and eastern Madagascar was as different from other haplogroups as fromR. stylosa. A star-like haplonetwork referred as the recent range expansion of haplogroup B from northern Madagascar toward the African mainland coastline, including a single variant spanning &amp;gt;1,800 km across the Mozambique Channel area (MCA). Populations in the south of Delagoa Bight contained haplogroup C and was originated from a unique bottleneck dispersal event. Divergence estimates of pre- and post-Last Glacial Maximum (LGM) illustrated the recent emergence ofRhizophoramangroves in the WIO compared to other oceans. Connectivity patterns could be aligned with the directionality of major ocean currents. Madagascar and the Seychelles each harbored haplogroups A and B, albeit among spatially separated populations, explained from a different migration era. Likewise, the Aldabra Atoll harbored spatially distinct haplotypes. Nuclear ribosomal cistron (8,033 bp) variants corresponded to haplogroups and confirmed admixtures in the Seychelles and Aldabra. These findings shed new light on the origins and dispersal routes ofR. mucronatalineages that have shaped their contemporary populations in large regions of the WIO, which may be the important information for defining marine conservation units both at ocean scale and at the level of small islands.

Helicobacter Pylori Variants with ABC-Type Tyrosine Phosphorylation Motif in Gastric Biopsies of Ghanaian Patients.

Background Helicobacter pylori pathogenicity and disease severity are determined by the tyrosine phosphorylation motifs of CagA protein. This study is aimed at detecting the presence of H. pylori and identifying the CagA tyrosine phosphorylation motifs in Ghanaian patients. Material and Methods. A total of 94 archival genomic DNA samples from gastric biopsies were used for the study, and H. pylori was detected by amplifying the 16S rRNA gene. The 3′-end variable region of the cagA gene was amplified, and the entire 3′-end was sequenced and translated into amino acids. Results H. pylori was detected in 53.2% (50/94) of the samples, and all the detected bacteria harboured the cagA gene. Two variants of the bacteria were identified based on the size of the amplified cagA gene: 207 bp and 285 bp. The 207 bp and 285 bp variants accounted for 74% and 22%, respectively, and 4% showed both fragments. Translated amino acid sequence of the cagA gene showed EPIYA-A, EPIYA-B, and EPIYA-C (ABC type) motifs, indicating the Western variant. The CagA protein C-terminal showed insertion of amino acids in the sequence flanking the EPIYA-A motif at the N-terminal and a complete deletion of the EPIYA-CC and EPIYA-CCC motifs together with the flanking sequences. Conclusions H. pylori identified were Western variant (ABC type) with unique amino acid insertions, suggesting unique variants in Ghanaian patients. Further investigation is however required to understand the role of the molecular diversity of the variant in gastric disease outcome.

Evaluating individual genome similarity with a topic model.

Evaluating genome similarity among individuals is an essential step in data analysis. Advanced sequencing technology detects more and rarer variants for massive individual genomes, thus enabling individual-level genome similarity evaluation. However, the current methodologies, such as the principal component analysis (PCA), lack the capability to fully leverage rare variants and are also difficult to interpret in terms of population genetics. Here, we introduce a probabilistic topic model, latent Dirichlet allocation, to evaluate individual genome similarity. A total of 2535 individuals from the 1000 Genomes Project (KGP) were used to demonstrate our method. Various aspects of variant choice and model parameter selection were studied. We found that relatively rare (0.001<allele frequency < 0.175) and sparse (average interval > 20000 bp) variants are more efficient for genome similarity evaluation. At least 100000 such variants are necessary. In our results, the populations show significantly less mixed and more cohesive visualization than the PCA results. The global similarities among the KGP genomes are consistent with known geographical, historical and cultural factors. The source code and data access are available at: https://github.com/lrjuan/LDA_genome. Supplementary data are available at Bioinformatics online.

A clinical study of the safe use of pneumatic tourniquet in orthopaedic surgery

Specific objective: A study was undertaken to evaluate the superiority of Lowest occlusion pressure method of using the pneumatic tourniquet verus the other routine ways of usage of Pneumatic Tourniquets in Orthopaedics surgery which include the Fixed and Systolic Blood Pressure variants. Materials and methods: This study included 120 patients of both upper and lower limbs trauma. These patients were randomly included any of the three categories of study after fullfilling the inclusion and exclusion criteria. Three modalities of application of toruniquet were included in this study. They were Fixed, Systolic Blood pressure (SBP) and Lowest Occlusion Pressure methods (LOP). Each category included 40 patients and the results were analysed according to final outcome using Ishii et al. grading, VAS Score criteria. Results: In our study 120 cases managed by LOP had better results compared to fixed variant and Systolic BP variant in terms of less pressure needed to keep tourniquet, good operative field, pain score (VAS) at tourniquet application region and skin injury (reddening). But no complications like compartment syndrome, deep vein disorder, paresis, nerve injury has occurred before and after surgery Conclusion: LOP method of using the pneuamtic tourniquet is superior to the other methods of using Tourniquet in terms of less pressure needed for elevating tourniquet, Good operative (blood less) field, no skin abrasions.

Genetic Polymorphisms of One-carbon Enzymes Interactively Modify Metabolic Folate Stress and Risks of Hepatocellular Carcinoma Development

Few studies have comprehensively evaluated how genetic modifiers of metabolic one-carbon stress as folate deficiency and hyperhomocysteinemia interact to modify the host-susceptibility of human heaptocellular carcinoma (HCC) development. Genetic polymorphisms of 3 key one-carbon enzymes (methylenetetrahydrofolate reductase: MTHFR, methionine synthase: MS, and thymidylate synthase: TS) at 5 loci were characterized in a case-control and hospital-based Asian population (n=398). The monopolymorphic analysis revealed that the T variant allele at MTHFR 677 loci in relative to the other genotyped variant alleles (MTHFR 1298C, MS 2756G, TSER 2R, TS3’UTR 1494+6bp insertion) was associated with a significant 40% reduction of HCC risks in the dominant model (adjusted ORs: 0.6, 95% CI: 0.4-0.9, P=0.03). Among individuals with low metabolic folate stress (serum folate>6 ng/mL), the MTHFR CC wild-type interacted with the TSER 2R variant alleles to increase HCC risks (OR: 0.14 vs. 0.3; 95% CI: 0.1-0.8), whereas 2-fold reduced HCC were associated with the compound MTHFR T and TSER 2R variant alleles (OR: 0.14 vs. 0.07; 95% CI: 0.02-0.2) (P for interaction: 0.044) after adjustment for serum homocysteine (Hcy) levels. The TSER 2R or TS3’UTR+6bp variant alleles interacted with MTHFR T variant allele to reverse its lowering serum folate and elevating Hcy effects (P for trend=0.009 and 0.001, respectively). Taken together, our data demonstrated that MTHFR 677 T and TSER 2R variant allele interacted to alleviate metabolic one-carbon folate stress, which folate-genetic interactions may be the important elements in favor of reduced HCC risks.

Differential genome evolution and speciation of Coix lacryma-jobi L. and Coix aquatica Roxb. hybrid guangxi revealed by repetitive sequence analysis and fine karyotyping.

BackgroundCoix, Sorghum and Zea are closely related plant genera in the subtribe Maydeae. Coix comprises 9–11 species with different ploidy levels (2n = 10, 20, 30, and 40). The exclusively cultivated C. lacryma-jobi L. (2n = 20) is widely used in East and Southeast Asia for food and medicinal applications. Three fertile cytotypes (2n = 10, 20, and 40) have been reported for C. aquatica Roxb. One sterile cytotype (2n = 30) closely related to C. aquatica has been recently found in Guangxi of China. This putative hybrid has been named C. aquatica HG (Hybrid Guangxi). The genome composition and the evolutionary history of C. lacryma-jobi and C. aquatica HG are largely unclear.ResultsAbout 76% of the genome of C. lacryma-jobi and 73% of the genome of C. aquatica HG are repetitive DNA sequences as shown by low coverage genome sequencing followed by similarity-based cluster analysis. In addition, long terminal repeat (LTR) retrotransposable elements are dominant repetitive sequences in these two genomes, and the proportions of many repetitive sequences in whole genome varied greatly between the two species, indicating evolutionary divergence of them. We also found that a novel 102 bp variant of centromeric satellite repeat CentX and two other satellites only appeared in C. aquatica HG. The results from FISH analysis with repeat probe cocktails and the data from chromosomes pairing in meiosis metaphase showed that C. lacryma-jobi is likely a diploidized paleotetraploid species and C. aquatica HG is possibly a recently formed hybrid. Furthermore, C. lacryma-jobi and C. aquatica HG shared more co-existing repeat families and higher sequence similarity with Sorghum than with Zea.ConclusionsThe composition and abundance of repetitive sequences are divergent between the genomes of C. lacryma-jobi and C. aquatica HG. The results from fine karyotyping analysis and chromosome pairing suggested diploidization of C. lacryma-jobi during evolution and C. aquatica HG is a recently formed hybrid. The genome-wide comparison of repetitive sequences indicated that the repeats in Coix were more similar to those in Sorghum than to those in Zea, which is consistent with the phylogenetic relationship reported by previous work.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1025) contains supplementary material, which is available to authorized users.

Genetic variant rs16430 6bp &gt; 0bp at the microRNA‐binding site in TYMS and risk of sporadic breast cancer risk in non‐hispanic white women aged ≤55 years

Thymidylate synthase (TYMS) is involved in the folate metabolism and provision of nucleotides needed for DNA synthesis and repair. Thus, functional genetic variants in TYMS may alter cancer risk. In the study, we evaluated associations of three germline variants (rs2790 A > G, rs16430 6 bp > 0 bp, and rs1059394 C > T) in the predicted miRNA-binding sites of TYMS with risk of sporadic breast cancer in non-Hispanic white women aged ≤ 55. We found that carriers of the rs16430 0 bp variant allele had an increased risk of breast cancer [adjusted odd ratio (OR) = 1.37, 95% confidence interval (CI): 1.08-1.73; P = 0.010], compared with carriers of the 6 bp/6 bp genotype. This increased risk was more evident in older subjects (OR = 1.47, 95% CI = 1.06-2.03, P = 0.022), never smokers (OR = 1.67, 95% CI = 1.23-2.25, P < 0.001), never drinkers (OR = 1.44, 95% CI = 1.01-2.05, P = 0.043), and estrogen receptor-positive patients (OR = 1.46, 95% CI = 1.11-1.92, P = 0.006), regardless of tumor stages. The results are consistent with the functional analyses of rs16430 as previously reported, which showed that the 0 bp allele had a decrease in both luciferase activity by ∼ 70% and mRNA levels by ∼ 50% compared with the 6bp allele. Additionally, the rs16430 variant was predicted to influence the binding activity of miR-561. Taken together, these findings indicate that the TYMS rs16430 may contribute to the etiology of sporadic breast cancer in non-Hispanic white women aged ≤ 55 yr. Further validation in large population-based or cohort studies is needed.

Chromosome 18p11.2 harbors susceptibility marker: D18S452, for bipolar affective disorder

Aim:The aim of our study was to investigate whether the tandem repeat polymorphism in D18S452 microsatellite marker at locus 18p11.2 is a risk factor of bipolar affective disorder (BPAD) in Kashmiri population.Materials and Methods:The repeat polymorphism in D18S452 was evaluated by polymerase chain reaction (PCR) analysis of in 74 diagnosed BPAD patients and 74 controls subjects.Results:Tandem repeat (300 bp*) allele frequency was found to be 1.35% in controls and 8.108% in cases. The tandem repeat (250 bp*) allele frequency was found to be in 91.89% in cases and 98.65% in controls. The 252 bp/252 bp genotype was found to be present in 89.18% of cases and 98.64% of controls, the 300 bp/300 bp genotype in 5.40% of cases and 1.35% of controls and the 252 bp/300 bp variant in 5.40% of cases and none among the controls. Although the proportion of patients homozygous for tandem repeat (300 bp/300 bp) was higher in cases than in controls, the difference was not statistically significant when 252 bp/252 bp genotype was taken as reference (odds ratio [OR]=4.4242; 95% confidence interval [CI] 0.4822-40.5924); P=0.1529). However, when the frequency of heterozygous genotype (252 bp/300 bp) was compared with 252 bp/252 bp statistical significance was observed (OR=8.0603; 95% CI 1.1112-58.4646; P=0.0383).Conclusion:This is the first study reporting a significant association between D18S452 maker with tandem repeat polymorphism in heterozygous condition (252 bp/300 bp) and the development of BPAD in Kashmiri population.

A rare variant in the osteoarthritis-associated locus GDF5 is functional and reveals a site that can be manipulated to modulate GDF5 expression

Osteoarthritis (OA) is a polygenic disease characterized by cartilage loss, with the single-nucleotide polymorphism (SNP) rs143383 (C/T) influencing OA susceptibility across a range of ethnic groups. The SNP resides within the 5′-UTR of the growth and differentiation factor 5 gene (GDF5), with the OA-associated T-allele mediating reduced GDF5 expression. As GDF5 codes for a cartilage anabolic protein, this reduced expression may explain why the T-allele of rs143383 is an OA risk factor. Our deep sequencing of GDF5 identified a C/A transversion located −41 bp relative to the gene's transcription start site. This promoter variant is predicted to affect transcription factor binding and it may therefore highlight a regulatory site that could be exploited to manipulate GDF5 expression and alleviate the detrimental effect mediated by the T-allele of rs143383. Here, we describe our functional assessment of the −41 bp variant. Using reporter constructs we demonstrated that the transversion leads to increased gene expression to such a degree that the A-allele is able to compensate for the reduced expression mediated by the T-allele of rs143383. Using electrophoretic mobility shift assays we identified YY1 as a trans-acting factor that differentially binds to the alleles of the −41 bp variant, with more avid binding to allele A. Knockdown of YY1 led to a significant reduction in GDF5 expression, supporting YY1 as a GDF5 activator. In conclusion, we demonstrated that the −41 bp variant is functional and we have identified a regulatory region of GDF5 that can be exploited to overcome the OA genetic deficit mediated by the T-allele of rs143383.

Genome-Wide Association Studies and Beyond

In spite of recent successes for other complex diseases, essential hypertension has been remarkably reluctant to reveal its underlying susceptibility genes through genome-wide association (GWA) studies. Only after shifting attention to the underlying quantitative traits (ie, systolic [SBP] and diastolic blood pressure [DBP]) and application of “brute force” GWA meta-analyses (n>29 000) of multiple population cohorts in the GlobalBPGen and CHARGE consortia was a tiny tip of the iceberg revealed.1,2 Six loci for SBP and 9 for DBP were discovered, of which 2 overlapped, yielding 13 independent genome-wide significant signals. However, without exception, the most significant single nucleotide polymorphisms (SNPs) representing these loci only explained a very small part of the total BP variance (≤0.11%; Figure). Heritability is well established for BP and typically ranges between 30% and 60%, as estimated in twin or family studies.3 This means that the majority of the BP heritability is still “missing” and remains at large.4 Expansion of GWA sample size (eg, in the International Consortium on BP-GWAS, a merger of GlobalBPGen and CHARGE) will likely uncover additional BP variants.5 However, they will have even smaller effect sizes, and a substantial increase in explained heritability of all loci combined is not expected. GWA studies in general experience a number of other inherent limitations, including a predominant focus on common variants (minor allele frequency: >5%) and a tendency for signals to map to noncoding sequence. Finding suitable answers to these challenges will to a large extent determine continued progress in chipping away at the missing BP heritability. The article by Tomaszewski et al6 in the present issue of Hypertension provides some intriguing clues as to the best way forward. Figure. A, Percentage of variance explained in SBP by the top SNPs of 6 loci identified in 2 large GWA meta-analysis …

The Role of Endothelial Nitric Oxide Synthase Gene Polymorphism in Clinically Classified Patients with Coronary Artery Disease

ABSTRACTThe aim of the study was to investigate the relationship of eNOS (4. intron 27bp) polymorphism in clinically classified patients with coronary artery disease (CAD) in Turkish population.PCR and restriction fragment length polymorphism analysis were used to detect the variant of the eNOS gene in 74 patients with CAD and 20 healthy controls. The CAD group was separated into 3 clinical groups depending on angiography criteria and clinical form designation: 1st Group Myocardial infarction (MI) (n:20); 2nd Group Unstable Angina Pectoris (UAP) (n:18); 3rd Group Stable Angina Pectoris (SAP) (n:36).When a and b allele frequencies in the CAD and control groups were compared, no statistically significant difference was found. No significant difference was observed in the 4. intron 27 bp variants of the eNOS gene when CAD patients were compared without distinguishing them clinically from the control group. When we assessed CAD patients classified according to their clinical form, no significant difference was determined in allele frequencies and genotype distribution in the subgroups except for subgroup SAP. When we compared SAP patients with the other subgroups and with the control group, it was found that there was a significant increase in the ab genotype and the a allele frequency, and a decrease in the bb genotype (p<0.05).In conclusion, CAD seemed to develop without any alterations in eNOS (4. intron 27bp) genotype frequency. However, the 27 bp repeat polymorphism of the eNOS gene in patients with SAP can be considered as SAP which may have a hereditary origin. High eNOS gene polymorphism in patients with SAP can be related to the increased risk of possible coronary occurrence in future. It was concluded that further studies of the relationship between eNOS gene polymorphism and CAD should take account of the clinical forms of CAD.

Frequent expression of the high molecular, 673-bp CD44v3,v8-10 variant in colorectal adenomas and carcinomas

CD44 is a transmembrane glycoprotein involved in the interaction between cells and the extracellular matrix. A large variety of alternatively spliced CD44 variants are expressed by different tumors with possible implication for tumor progression, formation of metastasis and survival. In colon carcinomas, previous reports described higher molecular bands of CD44 transcripts in neoplastic colonic tissue, although a complete analysis of multiple combinations of CD44v transcripts were not performed. We therefore analyzed the pattern of CD44 standard and variant (v2-v10) transcripts in colorectal adenomas and carcinomas by exon-specific RT-PCR amplification and sought CD44v transcripts specific for colonic neoplasias. Our data indicate that CD44 standard transcripts, including the epithelial form (C-v8,v9,v10-C) corresponding to a 720 bp transcript, were detected in 2/38 (5.2%) samples of normal mucosa, 20/20 (100%) adenomas and in 21/33 (63%) colorectal carcinomas. High molecular CD44v3,v8-10 (673 bp) transcripts were found in 2/33 (6%) samples from normal mucosa, 19/20 (95%) from adenomas and in 29/31 from colorectal carcinomas (93%). Similar CD44v3,v8-10 transcripts were detected in five from seven colorectal liver metastases, while normal liver did not contain high molecular CD44v3 variants. The same CD44v3,v8-10 (673 bp) variant was detected in HT-29 human colon carcinoma cells. Direct sequencing of the CD44v3 (673 bp) transcript in samples from colorectal carcinomas and HT-29 cells confirmed the assumed CD44v (-C-v3-v8-v9-v10-C-) cDNA sequence. Analysis of other CD44 variant transcripts (v4-v10) using exon specific primers were less frequently associated with colorectal neoplasias. These data report for the first time frequent expression of neoplasia-associated CD44v3,v8-10 variants in colorectal adenomas and carcinomas supporting the role of increased CD44 variant expression as an early event in colorectal carcinogenesis. The described CD44v3,v8-10 (673 bp) variant might be relevant for diagnosis and treatment of colorectal cancer.

Molecular cloning and characterization of porcine sirtuin genes

The sirtuin family of class III histone deacetylases (HDACs) is named after their homology to yeast silent information regulator 2 (SIR2). SIR2 and its mammalian derivatives (SIRT1-7) play a central role in gene silencing, cell cycle, aging and metabolism. Here we reported cDNA cloning, chromosome mapping,expression and evolutional analysis of sirtuin genes in Sus scrofa (Tongcheng pig). Sequence analysis showed that porcine sirtuin genes contain 7 members designated SIRT1-7. Tissue distribution analysis indicated porcine sirtuin genes ubiquitously expressed but with the highest abundance in brain, spinal cord and genital tissue. In silico and radiation hybrid mapping analysis mapped porcine SIRT1-7 to the chromosomes 14q23,6q11-12, 2q29, 14q19, 7p12, 2q11, and 12p15, respectively. We also isolated and characterized genomic sequence of porcine SIRT1, which spaned a region of 31,834 bp comprising 9 exons ranging in size from 80 bpto 2121 bp. The 5' flanking genomic region preceding an open reading frame of SIRT1 has a TATA box, a small300 bp CpG island and several putative Sp1 and p53 transcription factor binding sites. Moreover, we isolated two novel splicing SIRT6 variants with 346 bp (variant 2) in-frame deletions from lung and 327 bp(variant 3) in-frame deletions from spleen and brain. This is the first systematic report of molecular cloning and characterization of sirtuin genes in pigs, which will be helpful for a better understanding of the physiological role of sirtuin proteins in pigs.

The ACAA-insertion/deletion polymorphism at the 3′ UTR of the IGF-II receptor gene is associated with type 2 diabetes and surrogate markers of insulin resistance

The IGF-II receptor gene (IGFIIR) is located at chromosome 6q26, a region that harbors a genetic marker linked to insulin-resistant traits in Mexican-Americans. In the present study conducted in Spaniards, we tested a common polymorphism in IGFIIR for association with type 2 diabetes and insulin-resistant traits. Case-control association study. One hundred and forty-five type 2 diabetic patients and 217 non-diabetic controls were genotyped for the ACAA-insertion/deletion polymorphism at the 3' UTR of IGFIIR. Phenotyping included anthropometrics and a metabolic profile, including serum lipid levels and surrogate indexes of insulin resistance whenever possible. Diabetic patients were more frequently homozygous for the wild type 144 bp allele of IGFIIR compared with controls (diabetic patients 77.2%, controls 51.6%, P<0.001) suggesting a potential protective role against type 2 diabetes for the IGFIIR 140 bp variant. Carrying 140 bp alleles was associated with an odds ratio of having diabetes of 0.290 (95% confidence interval 0.109-0.770), and controls homozygous for the wild type 144 bp allele presented with lower insulin and triglyceride levels, which are proxies for insulin resistance. The ACAA-insertion/deletion polymorphism at the 3' UTR of IGFIIR is associated with type 2 diabetes and influences surrogate markers of insulin resistance in non-diabetic subjects.

Promoter Polymorphisms Regulating Corticotrophin-releasing Hormone TranscriptionIn Vitro

To investigate whether polymorphisms in the corticotrophin-releasing hormone (CRH) promoter are associated with altered CRH gene regulation, we studied the reactivity of three recently described promoter variants in vitro. The 3625 bp variants A1B1, A2B1 and A2B2 of the human CRH promoter were cloned in the 5' region to a luciferase reporter gene and transiently transfected into both mouse anterior pituitary cells AtT-20D16vF2 and pheochromocytoma cells PC12. Incubation with 8-Br-cAMP alone or in combination with cytokines significantly enhanced the promoter activity in both cell lines studied by up to 22-fold. However, dexamethasone antagonised cAMP effects on CRH expression in AtT-20 cells while showing no effect on PC12 cells, indicating that tissue-specific factors play a crucial role. Among the haplotypes studied, A1B1 exhibited the greatest reactivity on various stimuli. Electric mobility shift assay (EMSA) was performed to study whether the described polymorphic nucleotide sequences in the 5' region of the hCRH gene interfere with binding of nuclear proteins. A specific DNA protein complex was detected at position -2353 bp for the wild type sequence only, possibly interfering with a binding site for the activating transcription factor 6 (ATF6). Taken together, this is the first study to demonstrate that CRH promoter reactivity varies between the compound promoter alleles.

Polymorphisms of thymidylate synthase in the 5′- and 3′-untranslated regions associated with risk of gastric cancer in South China: a case–control analysis

Fruits and certain vegetables have a protective effect on gastric cancer (GC) and folate is one of the nutrients in fruits and vegetables. We hypothesized that the polymorphisms of thymidylate synthase (TYMS) gene involved in folate metabolism are associated with GC risk. In a population-based case-control study of 337 GC cases and 326 controls, frequency-matched by age, sex and residential areas in a southern Chinese population, we genotyped the 28 bp tandem repeat in the TYMS 5'-untranslated enhanced region (TSER) and the 6 bp deletion/insertion at bp 1494 in the TYMS 3'-untranslated region (TS3'UTR). We found that although the TSER polymorphism had no main effect on GC risk, the TS3'UTR 6 bp/6 bp genotype was associated with a significantly increased risk of GC [adjusted odds ratio (OR) = 1.96, 95% confidence interval (CI) = 1.18-3.25], especially the non-cardiac gastric cancer (2.16, 1.22-3.82), compared with the 0 bp/0 bp genotype. However, when we evaluated these two polymorphisms together and used the combined genotype with zero variant allele (TSER 2R and TS3'UTR 6 bp variant alleles) as the reference, we found that the combined genotype with three or four variant alleles was associated with a significantly increased risk of GC (2.06, 1.12-3.79), especially the non-cardiac gastric cancer (2.33, 1.19-4.59), and this significant association was more pronounced among older women (>60 years old), non-smokers, and never tea drinkers. In conclusion, the TYMS polymorphisms, especially the TS3'UTR polymorphism, are associated with GC risk, especially the non-cardiac gastric cancer, and the TSER 2R and TS3'UTR 6 bp alleles may jointly play a role in the etiology of GC in the southern Chinese population. Larger studies are warranted to verify these findings.

Intriguing diversity of Bacillus anthracis in eastern Poland – the molecular echoes of the past outbreaks

The multiple locus VNTRs analysis (MLVA) revealed the presence of five genotypes in a group of 10 Bacillus anthracis isolates from epidemiologically unrelated cases of bovine-anthrax in eastern Poland. Eight tested isolates possessed the pagA and capB genes indicating the presence of both virulence plasmids, while two isolates revealed only pagA and lacked pXO2. The MLVA and DNA sequence analysis indicated that seven tested isolates represent four novel genotypes. Five tested strains revealed a unique 144 bp vrrB2 variant as well as 220 bp variant of vrrB1, implying the relatedness to the lineage B2. Consequently, we propose establishing of novel B2 strains sub-lineage. Multiple anthrax outbreaks, which took place in Poland several decades ago were proposed as a cause of intriguing diversity of B. anthracis observed in this study.