The rs225017 Polymorphism in the 3′UTR of the Human DIO2 Gene Is Associated with Increased Insulin Resistance

The Thr92Ala (rs225014) polymorphism in the type 2 deiodinase (DIO2) gene has been associated with insulin resistance (IR) and decreased enzyme activity in human tissues but kinetic studies failed to detect changes in the mutant enzyme, suggesting that this variant might be a marker of abnormal DIO2 expression. Thus, we aimed to investigate whether other DIO2 polymorphisms, individually or in combination with the Thr92Ala, may contribute to IR. The entire coding-region of DIO2 gene was sequenced in 12 patients with type 2 diabetes mellitus (T2DM). Potentially informative variants were evaluated in 1077 T2DM patients and 516 nondiabetic subjects. IR was evaluated using the homeostasis model assessment (HOMA-IR) index. DIO2 gene sequencing revealed no new mutation but 5 previously described single nucleotide polymorphisms (SNPs). We observed that all T2DM patients displaying high HOMA-IR index (n = 6) were homozygous for the rs225017 (T/A) polymorphism. Further analysis showed that the median fasting plasma insulin and HOMA-IR of T2DM patients carrying the T/T genotype were higher than in patients carrying the A allele (P = 0.013 and P = 0.002, respectively). These associations were magnified in the presence of the Ala92Ala genotype of the Thr92Ala polymorphism. Moreover, the rs225017 and the Thr92Ala polymorphisms were in partial linkage disequilibrium (|D′| = 0.811; r ² = 0.365). In conclusion, the rs225017 polymorphism is associated with greater IR in T2DM and it seems to interact with the Thr92Ala polymorphism in the modulation of IR.     

Molecular Evolution of Daphnia Immunity Genes: Polymorphism in a Gram-Negative Binding Protein Gene and an α-2-Macroglobulin Gene

Studies of DNA polymorphism have shown that some immune system genes of mammals and plants are exceptionally diverse, indicating that coevolution between these taxa and their parasites mediates positive selective sweeps and/or balancing selection. The genes of the arthropod immune system remain comparatively unstudied. We isolated two putative immune system genes from the cladoceran crustacean Daphnia and examined DNA sequence diversity. For one gene, encoding a putative gram-negative binding protein, we found evidence of only purifying selection, indicating that this gene is under strong functional constraint and that selection acts to eliminate amino acid variation. For another gene, encoding a putative -2-macroglobulin, we found evidence of positive selection, indicating the possible involvement of this gene in a host–parasite arms race. We discuss the assumed function of these genes and offer speculation regarding which components of the arthropod immune system might experience diversifying adaptive evolution.[Reviewing Editor: Dr. Martin Kreitman]     

14-3-3ε Gene variants in a Japanese patient with left ventricular noncompaction and hypoplasia of the corpus callosum

Background

Left ventricular noncompaction (LVNC) is a cardiomyopathy characterized by a prominent trabecular meshwork and deep intertrabecular recesses, and is thought to be due to an arrest of normal endomyocardial morphogenesis. However, the genes contributing to this process remain poorly understood. 14-3-3ε, encoded by YWHAE, is an adapter protein belonging to the 14-3-3 protein family which plays important roles in neuronal development and is involved in Miller–Dieker syndrome. We recently showed that mice lacking this gene develop LVNC. Therefore, we hypothesized that variants in YWHAE may contribute to the pathophysiology of LVNC in humans.

Methods and results

In 77 Japanese patients with LVNC, including the probands of 29 families, mutation analysis of YWHAE by direct DNA sequencing identified 7 novel variants. One of them, c.− 458G > T, in the YWHAE promoter, was identified in a familial patient with LVNC and hypoplasia of the corpus callosum. The − 458G > T variant is located within a regulatory CCAAT/enhancer binding protein (C/EBP) response element of the YWHAE promoter, and it reduced promoter activity by approximately 50%. Increased binding of an inhibitory C/EBPβ isoform was implicated in decreasing YWHAE promoter activity. Interestingly, we had previously shown that C/EBPβ is a key regulator of YWHAE.

Conclusions

These data suggest that the − 458G > T YWHAE variant contributes to the abnormal myocardial morphogenesis characteristic of LVNC as well as abnormal brain development, and implicate YWHAE as a novel candidate gene in pediatric cardiomyopathies.     

The Family of Peanut Fatty Acid Desaturase Genes and a Functional Analysis of Four ω-3 AhFAD3 Members

Plant Molecular Biology Reporter - The synthesis of α-linolenic acid (ALA) requires the activity of ω-3 fatty acid desaturases (ω-3 FADs). The quality of peanut oil would be much...     

The Mammalian 2′-5′ Oligoadenylate Synthetase Gene Family: Evidence for Concerted Evolution of Paralogous Oas1 Genes in Rodentia and Artiodactyla

Multiple 2′-5′ oligoadenylate (2-5A) synthetases are important components of innate immunity in mammals. Gene families encoding these proteins have previously been studied mainly in humans and mice. To reconstruct the evolution of this gene family in mammals, a search for additional 2-5A synthetase genes was performed in rat, cattle, pig, and dog. Twelve 2′-5′ oligoadenylate synthetase (Oas) genes were identified in the rat genome, including eight Oas1 genes, two Oas1 pseudogenes, single copies of Oas2 and Oas3, and two Oas-like genes, Oasl1 and Oasl2. Four OAS genes were detected in the pig genome and five OAS genes were found in both the cattle and dog genomes. An OAS3 gene was not found in either the cattle or the pig genome. While two tandemly duplicated OAS-like (OASL) genes were identified in the dog genome, only a single OASL orthologue was found in both the cattle and the pig genomes. The bovine and porcine OASL genes contain premature stop codons and encode truncated proteins, which lack the typical C-terminal double ubiquitin domains. The cDNA sequences of the rat, cattle, pig, and dog OAS genes were amplified, sequenced and compared with each other and with those in the human, mouse, horse, and chicken genomes. Evidence of concerted evolution of paralogous 2′-5′ oligoadenylate synthetase 1 genes was obtained in rodents (Rodentia) and even-toed ungulates (Artiodactyla). Calculations using the nonparametric Kolmogorov-Smirnov test suggested that the homogenization of paralogous OAS1 sequences was due to gene conversion rather than stabilizing selection. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Reviewing Editor: Dr. Martin Kreitman     

Functional Promoter -308G>A Variant in Tumor Necrosis Factor α Gene Is Associated with Risk and Progression of Gastric Cancer in a Chinese Population

Background

Tumor necrosis factor-α (TNF-α) plays a crucial role in the development and progression of gastric cancer. A functional polymorphism, -308 G>A (rs1800629), which is located in the promoter of TNFA gene, has been suggested to alter the production of TNF-α and influence cancer risk. In the present study, we sought to investigate whether this polymorphism has effects on the risk and progression of gastric cancer in a Chinese population.

Methods

We genotyped the TNFA -308 G>A polymorphism using the TaqMan method in a two-stage case-control study comprising a total of 1686 gastric cancer patients and 1895 cancer-free subjects. The logistic regression was used to assess the genetic associations with occurrence and progression of gastric cancer.

Results

We found a significant association between the variant genotypes and increased risk of gastric cancer [P = 0.034, odds ratio (OR) = 1.39, 95% confidence interval (CI) = 1.01–1.67, GA/AA vs. GG]. Similar results were observed in the follow-up replication study. When combined the data from the two studies, we found a more significant association (P = 0.001, OR = 1.34, 95%CI = 1.13–1.59), especially for older subjects (>65 years). Furthermore, the patients carrying the variant genotypes had a significantly greater prevalence of T4 stage of disease (P = 0.001, OR = 2.19, 95%CI = 1.39–3.47) and distant metastasis (P = 0.013, OR = 1.61, 95%CI = 1.10–2.35).

Conclusions

Our results suggest that the functional promoter -308 G>A polymorphism in TNFA influence the susceptibility and progression of gastric cancer in the Chinese population.     

Molecular Cytogenetic Characterization of a Wheat - Leymus mollis 3D（3Ns） Substitution Line with Resistance to Leaf Rust

Leymus mollis （Trin.） Pilger （NsNsXmXm, 2n = 28）, a wild relative of common wheat, possesses many potentially valuable traits that could be transferred to common wheat during breeding programs. In this study, the karyotypic constitution of a wheat - L. mollis 3D（3Ns#1） disomic substitution line isolated from the F5 progeny of octoploid Tritileymus M842-16 x Triticum durum cv. D4286, which was designated as 10DM57, was determined using genomic in situ hybridization （GISH）, fluorescent in situ hybridization （FISH）, SSR markers, and EST- STS markers. Screening of mitosis and meiosis showed that 10DM57 had a chromosome karyotype of 2n = 42 =21Ⅱ. GISH indicated that 10DM57 was a line with 40 chromosomes from wheat and two of the Ns chromosomes from L. mollis, which formed a ring bivalent in pollen mother cells at metaphase I. FISH analysis showed that the chromosome 3D may be replaced by 3Ns#1 in 10DM57. DNA markers, including SSR and EST-STS primers, showed that the pair of wheat chromosome 3D in 10DM57 was substituted by the pair of chromosome 3Ns#t from L. mollis. Evaluation of the agronomic traits showed that, compared with its common wheat relative 7182, 10DM57 was resistant to leaf rust while the spike length and number of spikes per plant were improved significantly, which correlated with a higher wheat yield. The new germplasm, 10DM57, could be exploited as an intermediate material in wheat genetic and breeding programs.     

Association of the FADS2 Gene with ω-6 and ω-3 PUFA Concentration in the Egg Yolk of Japanese Quail

This study focused on the association of polymorphisms of the FADS2 gene with fatty acid profiles in egg yolk of eight Japanese quail lines selected for high and low ω-6:ω-3 PUFA ratio (h2 = 0.36–0.38). For the identification of polymorphisms within the FADS2 gene 1350 bp of cDNA sequence were obtained encoding 404 amino acids. Five synonymous SNPs were found by comparative sequencing of animals of the high and low lines. These SNPs were genotyped by single base extension on 160 Japanese quail. The association analysis, comprising analysis of variance and family based association test (FBAT), revealed significant effects of SNP3 and SNP4 genotypes on the egg yolk fatty acid profiles, especially the ω-6 and ω-3 PUFAs (P < 0.05). No effects of the other SNPs were found—indicating that these are not in linkage disequilibrium with the causal polymorphism. The results of this study promote FADS2 as a functional candidate gene for traits related to ω-6 and ω-3 PUFA concentration in the egg yolk.     

Polymorphic Sites at the 3’ Untranslated Region of the HLA-G Gene Are Associated with Differential hla-g Soluble Levels in the Brazilian and French Population

HLA-G molecule has well-recognized tolerogenic properties, and the encoding gene shows lower frequency of polymorphism at the coding region but higher variability at regulatory 5’ and 3’ untranslated (3’UTR) regions. At least three 3’UTR polymorphic sites have been associated with HLA-G mRNA regulation, including the 14 base pair (14bp) Insertion/Deletion, +3142C-G and +3187A-G. We studied the association of polymorphic sites at 3’UTR (sequencing analysis, encompassing the 14bp Ins-Del/+3003T-C/+3010C-G/+3027C-A/+3035C-T/+3142C-G/+3187A-G/+3196C-G polymorphic sites) with plasma soluble HLA-G levels (sHLA-G, detected by ELISA) in 187 French and 153 Brazilian healthy individuals. Allele and genotype frequencies were closely similar in both populations; however, Brazilians showed a higher HLA-G 3’UTR haplotype diversity. Considering sHLA-G levels in both populations altogether, individuals presenting 14bp Del/Del showed higher levels compared to 14bpIns/Ins genotype (P <0.05); those presenting +3010C/G showed higher levels compared to the +3010C-C genotype (P< 0.05); those presenting +3027C-C showed higher levels than the +3027A-A genotype (P< 0.05); and those bearing +3035C-C showed higher levels compared to the +3035C-T (P < 0.01) and +3035T-T (P < 0.05) genotypes. The analyses of 3’UTR haplotypes showed that UTR-1 (DelTGCCCGC) was associated with higher expression of sHLA-G, whereas UTR-5 (InsTCCTGAC) and UTR-7 (InsTCATGAC) with lower expression and other UTRs (UTR-2/3/4/6) exhibited intermediate levels. Since the differential expression of HLA-G may be beneficial or harmful depending on the underlying condition, the identification of individuals genetically programmed to differentially express HLA-G may help on defining novel strategies to control the immune response against the underlying disorder.     

Novel Polymorphisms in Porcine 3′UTR of the Leptin Gene,Including a Rare Variant within Target Sequence for MIR-9 Gene in Duroc Breed,not Associated with Production Traits

The leptin gene (LEP) is considered as a functional candidate for production traits in livestock due to its crucial role in energy homeostasis. Because polymorphisms in regulatory sequences may affect gene expression, we searched for them in the 3′UTR of LEP and analyzed their association with production traits. Four breeds and a composite line were studied. In the Polish Landrace and Polish Large White breeds, 8 SNPs and 1 indel were observed; whereas, in the Duroc breed, 9 specific SNPs were found. Pietrain and Line 990 were monomorphic. One SNP (g.+168C>T), observed in the Duroc breed only, was located within a target site for microRNA (miR-9). Association studies showed a weak association between one SNP (c.+846C>T) and abdominal fat weight in the Polish Landrace only. Thus, we concluded that contribution of polymorphisms in the 3′UTR to phenotypic variability of pig production traits is marginal. Moreover, we presented an overview of known polymorphisms (128) in the pig leptin gene.     

Resistance Profiles of (+) 2′-Deoxy-3′-oxa-4′-thiocytidine and (-) 2′-Deoxy-3′-oxa-4′-thio-5-fluorocytidine

Abstract

Resistant variants were selected in vitro against two novel nucleoside analogues, (+) dOTC and (-) dOTFC using the HIV-1 molecular clone HXB2D. The variants obtained displayed 6.5-fold and 10-fold resistance to these compounds, respectively. Cloning and sequencing of the RT genes of the selected viruses identified two mutations, M184I for (+) dOTC and M184V for (-) dOTFC. Results with mutated recombinant clones of HXB2D confirmed the importance of these mutations in MT-4 cells. The resistance profiles of clinical samples with wild-type or 3TC-resistant phenotypes were also studied; low to moderate levels of cross-resistance were observed against the novel compounds.     

Resistance to Insecticides in Insect Vectors of Disease:estα3, a Novel Amplified Esterase Associated with Amplifiedestβ1from Insecticide Resistant Strains of the MosquitoCulex quinquesfasciatus

Vector control programmes in many countries face the dual problems of parasite drug resistance and insecticide resistance in the insect vectors of the disease. Here we report for the first time a new esterase-based insecticide resistance mechanism in the filariasis vectorCulex quinquefasciatus.The field collected COL strain ofC. quinquefasciatusfrom Columbia was heterogeneous for organophosphorus insecticide resistance. On native polyacrylamide gels it had an elevated β-naphthyl acetate specific esterase with the same R_fas that for the Estβ1s involved in insecticide resistance in other strains of this mosquito species. After five generations of temephos insecticide selection, both the esterase specific activity withp-nitrophenyl acetate and the temephos LC₅₀values were increased, suggesting that elevation of esterase activity was the underlying mechanism of resistance. Western blots with antisera raised to Estα2¹and Estβ2¹fromC. quinquefasciatusindicated that the COL strain had an elevated Estα3 enzyme which co-migrated on native gels with Estβ1. Southern blots indicated that anestα3gene was amplified in the COL strain and a Cuban mosquito strain (MRes), although the restriction digest patterns of theestβ1 genes in these two strains are different. In contrast, the Californian TEMR strain, with the amplifiedestβ1¹gene, had no associated elevated Estα.Restriction digest patterns for COL and TEMR DNA suggest that they contain an identicalestβ1¹gene, but our data suggest that theestα3gene occurs on the same amplicon as anestβ1 gene although the genes are probably >10 kb apart. Hence, either the COL strain has twoestβ1genes or theestβ1¹amplicon in TEMR has been disrupted at some stage during the long colonisation of this strain and the amplifiedestα has been lost.     

Glycogen Synthase Kinase-3β Regulates Equilibrium Between Neurogenesis and Gliogenesis in Rat Model of Parkinson’s Disease: a Crosstalk with Wnt and Notch Signaling

Neurogenesis involves generation of functional newborn neurons from neural stem cells (NSCs). Insufficient formation or accelerated degeneration of newborn neurons may contribute to the severity of motor/nonmotor symptoms of Parkinson’s disease (PD). However, the functional role of adult neurogenesis in PD is yet not explored and whether glycogen synthase kinase-3β (GSK-3β) affects multiple steps of adult neurogenesis in PD is still unknown. We investigated the possible underlying molecular mechanism of impaired adult neurogenesis associated with PD. Herein, we show that single intra-medial forebrain bundle (MFB) injection of 6-hydroxydopamine (6-OHDA) efficiently induced long-term activation of GSK-3β and reduced NSC self-renewal, proliferation, neuronal migration, and neuronal differentiation accompanied with increased astrogenesis in subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Indeed, 6-OHDA also delayed maturation of neuroblasts in the DG as witnessed by their reduced dendritic length and arborization. Using a pharmacological approach to inhibit GSK-3β activation by specific inhibitor SB216763, we show that GSK-3β inhibition enhances radial glial cells, NSC proliferation, self-renewal in the SVZ, and the subgranular zone (SGZ) in the rat PD model. Pharmacological inhibition of GSK-3β activity enhances neuroblast population in SVZ and SGZ and promotes migration of neuroblasts towards the rostral migratory stream and lesioned striatum from dorsal SVZ and lateral SVZ, respectively, in PD model. GSK-3β inhibition enhances dendritic arborization and survival of granular neurons and stimulates NSC differentiation towards the neuronal phenotype in DG of PD model. The aforementioned effects of GSK-3β involve a crosstalk between Wnt/β-catenin and Notch signaling pathways that are known to regulate NSC dynamics.