The new sequencer on the block: comparison of Life Technology’s Proton sequencer to an Illumina HiSeq for whole-exome sequencing

We assessed the performance of the new Life Technologies Proton sequencer by comparing whole-exome sequence data in a Centre d’Etude du Polymorphisme Humain trio (family 1463) to the Illumina HiSeq instrument. To simulate a typical user’s results, we utilized the standard capture, alignment and variant calling methods specific to each platform. We restricted data analysis to include the capture region common to both methods. The Proton produced high quality data at a comparable average depth and read length, and the Ion Reporter variant caller identified 96 % of single nucleotide polymorphisms (SNPs) detected by the HiSeq and GATK pipeline. However, only 40 % of small insertion and deletion variants (indels) were identified by both methods. Usage of the trio structure and segregation of platform-specific alleles supported this result. Further comparison of the trio data with Complete Genomics sequence data and Illumina SNP microarray genotypes documented high concordance and accurate SNP genotyping of both Proton and Illumina platforms. However, our study underscored the problem of accurate detection of indels for both the Proton and HiSeq platforms.     

Apobec-1 Increases Cyclooxygenase-2 and Aggravates Injury in Oxygen-Deprived Neurogenic Cells and Middle Cerebral Artery Occlusion Rats

Given that cyclooxygenase-2 (COX-2) plays a crucial role during cerebral ischemia and Apobec-1 is a critical regulator of COX-2 mRNA stabilization in gastrointestinal settings, the correlation of COX-2 and Apobec-1 was investigated in neurogenic cells and rat model of cerebral ischemia. After neurogenic SH-SY5Y, NG108-15 and PC12 cells were exposed to oxygen-glucose deprivation, cell viability, LDH leakage and Apobec-1 expression were determined. The effect of Apobec-1 overexpression on injury severity of oxygen-glucose deprivation, COX-2 expression, C-to-U editing of COX-2 mRNA were measured in vitro. Then the correlation of Apobec-1 level and injury severity was analyzed in cells with oxygen-glucose deprivation and in rats with middle cerebral artery occlusion. Apobec-1 expression was elevated along with upregulation of COX-2 and injury severity of oxygen-glucose deprivation in the three cell lines. Apobec-1 overexpression aggravated injury of oxygen-glucose deprivation in vitro and could be correlated to injury severity in vivo. Meanwhile, Apobec-1 increased COX-2 expression and COX-2 mRNA stabilization in neurogenic cells, and failed to catalyze C-to-U editing of COX-2 mRNA. Apobec-1 could upregulate COX-2 expression in neurogenic cells by stabilizing COX-2 mRNA, and might aggravate injury of oxygen-glucose deprivation in neurogenic cells as well as in rats with cerebral ischemia.     

The Expression Pattern of Classical MHC Class I Molecules in the Development of Mouse Central Nervous System

Classical major histocompatibility complex (MHC) class I, first identified in the immune system, is also expressed in the developing and adult central nervous system (CNS). Although the MHC class I molecules have been found to be expressed in the CNS of different species, a necessary step to elucidate the temporal and spatial expression patterns of MHC class I molecules in the brain development has never been taken. Frozen sections were made from the brains of embryonic and postnatal C57BL/6 J mice, and the expression of H-2D^b mRNA was examined by in situ hybridization. Immunofluorescence was also performed to define the cell types that express H2-D^b in P15 mice. At E10.5, the earliest stage we examined, H2-D^b was expressed in neuroepithelium of the brain vesicles. From E12.5 to P0, H2-D^b expression was mainly located at cerebral cortex, neuroepithelium of the lateral ventricle, neuroepithelium of aquaeductus and developing cerebellum. From P4 to adult, H2-D^b mRNA was detected at olfactory bulb, hippocampus, cerebellum and some nerve nuclei. The major cell types expressing H-2D^b in P15 hippocampus, cerebral cortex and olfactory bulb were neuron. H2-K^b signal paralleled that of H2-D^b and the expression levels of the two molecules were comparable throughout the brain. The investigation of the expression pattern of H-2D^b at both embryonic and postnatal stages is important for further understanding the physiological and pathological roles of H2-D^b in the developing CNS.     

Cloning and expression analysis of the cytochrome P450c17s enzymes during the reproductive cycle in ovoviviparous Korean rockfish (Sebastes schlegeli)

Cytochrome P450c17 (CYP17, 17α-hydroxylase/17, 20-lyase) plays a critical role in the production of androgens and estrogens in vertebrates. We isolated the full length cDNAs of P450c17-I and P450c17-II from Sebastes schlegeli. The cDNA sequences of P450c17-I and P450c17-II encoded 515 and 533 amino acid residues respectively. The putative P450c17-I and P450c17-II enzymes of Korean rockfish share high sequence identity with that of Japanese flounder (92% and 81%) respectively. Our current study describes that P450c17s of Korean rockfish are mainly expressed in gonads, head kidney and kidney by RT-PCR. Quantitative real-time PCR showed that the expression patterns of Korean rockfish P450c17s were developmental stage-dependency. In addition, the testosterone (T) and gonadosomatic index (GSI) levels further support the important role of P450c17-I during shift in steroidogenesis. Taken together, this study provides information about the Korean rockfish P450c17s characterization and mRNA expression as such helps in further understanding of its function in gonadal development.     

Cloning and truncation modification of trehalose-6-phosphate synthase gene from Selaginella pulvinata

A homologous sequence was amplified from resurrection plant Selaginella pulvinta by RACE technique, proved to be the full-length cDNA of trehalose-6-phosphate synthase gene by homologous alignment and yeast complementation assay, and nominated as SpTPS1 gene. The open reading frame of this gene was truncated 225 bp at the 5′-end, resulting the N-terminal truncation modification of 75 amino acids for its encoding protein. The TPS1 deletion mutant strain YSH290 of the brewer's yeast transformed by the truncated gene SpTPS1Δ and its original full-length version restored growth on the medium with glucose as a sole carbon source and displayed growth curves with no significant difference, indicating their encoding proteins functioning as TPS enzyme. The TPS activity of the mutant strain transformed by the truncated gene SpTPS1Δ was about six fold higher than that transformed by its original version, reasoning that the extra N-terminal extension of the full-length amino acid sequence acts as an inhibitory domain to trehalose synthesis. However, the trehalose accumulation of the mutant strain transformed by the truncated gene SpTPS1Δ was only 8% higher than that transformed by its original version. This result is explained by the feedback balance of trehalose content coordinated by the comparative activities between trehalose synthase and trehalase. The truncated gene SpTPS1Δ is suggested to be used in transgenic operation, together with the inhibition of trehalase activity by the application of validamycin A or genetic deficiency of the endogenous trehalase gene, for the enhancement of trehalose accumulation and improvement of abiotic tolerance in transgenic plants.     

Association between the XRCC3 T241M polymorphism and risk of cancer: Evidence from 157 case–control studies

The T241M polymorphism in the X-ray cross-complementing group 3 (XRCC3) had been implicated in cancer susceptibility. The previous published data on the association between XRCC3 T241M polymorphism and cancer risk remained controversial. Hence, we performed a meta-analysis to investigate the association between cancer susceptibility and XRCC3 T241M (61,861 cases and 84,584 controls from 157 studies) polymorphism in different inheritance models. We used odds ratios with 95% confidence intervals to assess the strength of the association. Overall, significantly increased cancer risk was observed in any genetic model (dominant model: odds ration [OR] = 1.07, 95% confidence interval [CI] = 1.00–1.13; recessive model: OR = 1.15, 95% CI = 1.08–1.23; additive model: OR = 1.17, 95% CI = 1.08–1.28) when all eligible studies were pooled into the meta-analysis. In further stratified and sensitivity analyses, the elevated risk remained for subgroups of bladder cancer and breast cancer, especially in Caucasians. In addition, significantly decreased lung cancer risk was also observed. In summary, this meta-analysis suggests the participation of XRCC3 T241M in the susceptibility for bladder cancer and breast cancer, especially in Caucasians, and XRCC3 T241M polymorphism is associated with decreased lung cancer risk. Moreover, our work also points out the importance of new studies for T241M association in some cancer types, such as gastric cancer, colorectal cancer, and melanoma skin cancer, where at least some of the covariates responsible for heterogeneity could be controlled, to obtain a more conclusive understanding about the function of the XRCC3 polymorphism in cancer development.     

Angiotensin-converting enzyme insertion/deletion polymorphism and risk of myocardial infarction in an updated meta-analysis based on 34 993 participants

The association between angiotensin-converting enzyme insertion/deletion (ACE I/D) polymorphism and risk of myocardial infarction (MI) has been extensively studied. However, the results were in controversy. This study aimed to explore the association between ACE I/D polymorphism and risk of MI by using a meta-analysis. We retrieved the following databases to indentify eligible studies: Medline, Embase, ISI, VIP, CBM and Wan Fang database. The latest update was 10th May, 2012. Odds ratio and 95% confidence interval (95% CI) were used to present the strength of the association. A total of 40 case–control studies with 34 993 participants were included. Overall, D allele of ACE I/D polymorphism was significantly associated with an increased risk of MI in genetic comparison models (OR (95% CI): 1.41 (1.22–1.64) for DD vs. II; 1.11 (1.01–1.21) for ID vs. II; 1.23 (1.10–1.37) for D carriers vs. II; 1.28 (1.15–1.43) for DD vs. I carriers and 1.06 (1.02–1.10) for D carriers vs. I carriers). Subgroup analyses, according to ethnicities and countries of participants also indicated that D allele was significantly associated with an increased risk of MI in Asians (especially for Chinese) and Caucasians (especially for English, French, Germans and Italians) (OR (95% CI) of DD vs. ID + II: 2.11 (1.65–2.70) for Asians and 1.15 (1.05–1.27) for Caucasians). In conclusion, this meta-analysis indicated that D allele of ACE I/D polymorphism was a possible risk factor for MI incidence for both Asians and Caucasians.     

Novel SLC20A2 mutations identified in southern Chinese patients with idiopathic basal ganglia calcification

Idiopathic basal ganglia calcification (IBGC) is a rare neuropsychiatric disorder characterized by bilateral and symmetric cerebral calcifications. Recently, SLC20A2 was identified as a causative gene for familial IBGC, and three mutations were reported in a northern Chinese population. Here, we aimed to explore the mutation spectrum of SLC20A2 in a southern Chinese population. Sanger sequencing was employed to screen mutations within SLC20A2 in two IBGC families and 14 sporadic IBGC cases from a southern Han Chinese population. Four novel mutations (c.82G > A p.D28N, c.185T > C p.L62P, c.1470_1478delGCAGGTCCT p.Q491_L493del and c.935-1G > A) were identified in two families and two sporadic cases, respectively; none were detected in 200 unrelated controls. No mutation was found in the remaining 12 patients. Different mutations may result in varied phenotypes, including brain calcification and clinical manifestations. Our study supports the hypothesis that SLC20A2 is a causative gene of IBGC and expands the mutation spectrum of SLC20A2, which facilitates the understanding of the genotype–phenotype correlation of IBGC.     

SNV and haplotype analysis reveals new CSRP1 variants associated with growth and carcass traits

The cysteine and glycine-rich protein 1 and 2 genes (CSRP1 and CSRP2) are an effective growth factor in promoting skeletal muscle growth in vitro and vivo. However, in cattle, the information on the CSRP1 and CSRP2 genes is very limited. The aim of this study was to examine the association of the CSRP1 and CSRP2 variants with growth and carcass traits in cattle breeds. Three single nucleotide variants (SNVs) were identified within the bovine CSRP1 gene, whereas CSRP2 gene has not detected any SNVs, using DNA pooled sequencing, PCR-RFLP, and forced PCR-RFLP methods. These SNVs include g. 801T>C (Intron 2), g. 46T>C (Exon 3) and g. 99C>G (Intron 3). Besides, we also investigated haplotype frequencies and linkage disequilibrium (LD) coefficients for three SNVs in all study populations. LD and haplotype structure of CSRP1 were different between breeds. The result of haplotype analysis demonstrated eight haplotype present in QC (Qinchuan) and one haplotype in CH (Chinese Holstein). Only haplotype 1 (TTC), shared by all two populations, comprised 10.74% and 100.00%, of all haplotypes observed in QC and CH, respectively. Haplotype 5 (CTC) had the highest haplotype frequencies in QC (30.98%) and haplotype 1 had the highest haplotype frequencies in CH (100.00%). The statistical analyses indicated that one single SNV and 19 combined haplotypes were significantly or highly significantly associated with growth and carcass traits in the QC cattle population (P < 0.05 or P < 0.01). Quantitative real-time PCR (qRT-PCR) analyses showed that the bovine CSRP1 and CSRP2 genes were widely expressed in many tissues. The results of this study suggest that the CSRP1 gene possibly is a strong candidate gene that affects growth and carcass traits in the Chinese beef cattle breeding.     

Comparison and Effects of Acute Lamotrigine Treatment on Extracellular Excitatory Amino Acids in the Hippocampus of PTZ-Kindled Epileptic and PTZ-Induced Status Epilepticus Rats

In this communication, the effect of acute treatment with lamotrigine (LTG) was investigated on release of main excitatory amino acids (EAA) such as glutamate (Glu) and aspartate (Asp) in the hippocampus of pentylenetetrazol (PTZ)-induced and PTZ-kindled freely moving rats using micro dialysis. The results show that, levels of Glu and Asp significantly increased in the rat hippocampus during the seizure/interical periods for PTZ-status epilepticus (SE) and PTZ-kindled epileptic (EP) rats. The levels of Glu and Asp increased more in EP rat hippocampus than in SE rat hippocampus. After administration of 20 mg/kg LTG, the levels of Glu and Asp significantly decreased in the SE and EP rat hippocampus. The results indicate that: (a) excitability of the PTZ-kindled epileptogenic model is higher than that of the status epilepticus model; (b) the modulation of LTG on the EAA neurotransmitters certainly plays an important role in antiepileptic efficacy, especially in PTZ-kindled epileptic model where the release of EAA was influenced more markedly by acute application of 20 mg/kg LTG.