Gene (v.499, #1)

Editorial Board (iv-v).

Cell cycle progression is negatively regulated by the retinoblastoma family of pocket proteins and CDK inhibitors (CKIs). In contrast, CDKs promote progression through multiple phases of the cell cycle. One prominent way by which CDKs promote cell cycle progression is by inactivation of pocket proteins via hyperphosphorylation. Reactivation of pocket proteins to halt cell cycle progression requires dephosphorylation of multiple CDK-phosphorylated sites and is accomplished by PP2A and PP1 serine/threonine protein phosphatases. The same phosphatases are also implicated in dephosphorylation of multiple CDK substrates as cells exit mitosis and reenter the G1 phase of the cell cycle. This review is primarily focused on the role of PP2A and PP1 in the activation of pocket proteins during the cell cycle and in response to signaling cues that trigger cell cycle exit. Other functions of PP2A during the cell cycle will be discussed in brief, as comprehensive reviews on this topic have been published recently (De Wulf et al., 2009; Wurzenberger and Gerlich, 2011).► Cell cycle progression is negatively regulated by the pRB pocket protein family. ► Pocket proteins also promote differentiation in a context dependent manner. ► Reactivation of pocket proteins requires dephosphorylation of multiple CDK sites. ► PP2A/PP1 phosphatases dephosphorylate pocket proteins and mitotic CDK substrates. ► This review focuses on PP2A's role in pocket protein activation by cellular cues.
Keywords: p107; pRB; Retinoblastoma; p130; B55alpha; E2F;

Complexity of a complex trait locus: HP, HPR, haemoglobin and cholesterol by Philip A.I. Guthrie; Santiago Rodriguez; Tom R. Gaunt; Debbie A. Lawlor; George Davey Smith; Ian N.M. Day (8-13).
HP and HPR are related and contiguous genes in strong linkage disequilibrium (LD), encoding haptoglobin and haptoglobin-related protein. These bind and chaperone free Hb for recycling, protecting against oxidation. A copy number variation (CNV) within HP (Hp1/Hp2) results in different possible haptoglobin complexes which have differing properties. HPR rs2000999 (G/A), identified in meta-GWAS, influences total cholesterol (TC) and LDL-cholesterol (LDL-C). We examined the relationship between HP CNV, HPR rs2000999, Hb, red cell count (RCC), LDL-C and TC in the British Women's Heart and Health Study (n = 2779 for samples having CNV, rs2000999, and phenotypes). Analysing single markers by linear regression, rs2000999 was associated with LDL-C (β = 0.040 mmol/L, p = 0.023), TC (β = − 0.040 mmol/L, p = 0.019), Hb (β = − 0.044 g/dL, p = 0.028) and borderline with RCC (β = − 0.032 × 1012/L, p = 0.066). Analysis of CNV by linear regression revealed an association with Hb (Hp1 vs Hp2, β = 0.057 g/dL, p = 0.004), RCC (β = 0.045 × 1012/L, p = 0.014), and showed a trend with LDL-C and TC. There were 3 principal haplotypes (Hp1-G 36%; Hp2-G 45%; Hp2-A 18%). Haplotype comparisons showed that LDL-C and TC associations were from rs2000999; Hb and RCC associations derived largely from the CNV. Distinct genotype–phenotype effects are evident at the genetic epidemiological level once LD has been analysed, perhaps reflecting HPHPR functional biology and evolutionary history. The derived Hp2 allele of the HP gene has apparently been subject to malaria-driven positive selection. Haptoglobin-related protein binds Hb and apolipoprotein-L, i.e. linking HPR to the cholesterol system; and the HPR/apo-L complex is specifically trypanolytic. Our analysis illustrates the complex interplay between functions and haplotypes of adjacent genes, environmental context and natural selection, and offers insights into potential use of haptoglobin or haptoglobin-related protein as therapeutic agents.► HP CNV/HPR SNP haplotype analysis shows association of HP CNV with Hb levels/RCC. ► HP CNV/HPR SNP haplotype analysis shows association of HPR SNP with LDL-C/TC. ► The HP CNV/Hb-related association may be via Hp2 allele advantage in malaria zones. ► The HPR SNP/cholesterol association is likely via apolipoproteins in TLF-1 and -2. ► We infer that HP CNV duplication preceded HPR SNP mutation.
Keywords: HP; HPR; Haemoglobin; Cholesterol; Malaria; Trypanosome;

Genomic organization, phylogenetic comparison and expression profiles of annexin gene family in tomato (Solanum lycopersicum) by Yongen Lu; Bo Ouyang; Junhong Zhang; Taotao Wang; Chen Lu; Qinqin Han; Shengnan Zhao; Zhibiao Ye; Hanxia Li (14-24).
Annexins have been suggested to play pivotal roles in stress resistance and plant development. However, related studies on fruit-bearing plants, especially on fruit development, are very limited. In the present study, we provide a comprehensive overview of the annexin family in tomato, describing the gene structure, promoter cis-regulatory elements, organ expression profile, and gene expression patterns under hormone and stress treatments.Bioinformatic analysis revealed that the nine tomato annexins were structurally different from their animal counterparts, but highly conserved annexin domains were still found in most of them. Cis-regulatory element prediction showed that there were important elements in the 2 kb upstream promoter regions, including stress- and hormone-responsive-related elements. The expression patterns of these genes were investigated, and the results revealed that they were regulated under developmental processes and environmental stimuli. Among them, AnnSl1.1 and AnnSl2 were highly expressed in most of the tested organs. Genes preferentially or specifically expressed in organs, such as stigma or ovary (AnnSl6), stamen (AnnSl8), and fruit pericarp (AnnSl1.2 and AnnSl9), were identified. Some annexin genes were induced by plant hormones including abscisic acid (AnnSl3, AnnSl6, AnnSl8, and AnnSl9) and gibberellic acid (AnnSl1.1, AnnSl1.2, AnnSl4, and AnnSl7). Most of these annexin genes were induced by salt, drought, wounding, and heat or cold stresses. The present study provides significant information for understanding the diverse roles of annexins in tomato growth and development.► Conserved annexin domains were found in most tomato annexins. ► Putative stress- and hormone-responsive-related elements were identified. ► Tomato annexins showed tissue and development preferential expression patterns. ► Most tomato annexin genes were induced by abiotic stresses.
Keywords: Annexin; Tomato; Abiotic stress; Plant hormone; Gene expression;

Molecular cloning, bacterial expression and promoter analysis of squalene synthase from Withania somnifera (L.) Dunal by Wajid Waheed Bhat; Surrinder K. Lattoo; Sumeer Razdan; Niha Dhar; Satiander Rana; Rekha S. Dhar; Shabnam Khan; Ram A. Vishwakarma (25-36).
Withania somnifera (ashwagandha) is a rich repository of large number of pharmacologically active secondary metabolites known as withanolides. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, but there is sparse information about the genes responsible for biosynthesis of these compounds. In this study, we have cloned and characterized a gene encoding squalene synthase (EC from a withaferin A rich variety of W. somnifera, a key enzyme in the biosynthesis of isoprenoids. Squalene synthase catalyses dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for sterols and triterpenes. A full-length cDNA consisting of 1765 bp was isolated and contained a 1236 bp open reading frame (ORF) encoding a polypeptide of 411 amino acids. Recombinant C-terminus truncated squalene synthase (WsSQS) was expressed in BL21 cells (Escherichia coli) with optimum expression induced with 1 mM IPTG at 37 °C after 1 h. Quantitative RT-PCR analysis showed that squalene synthase (WsSQS) expressed in all tested tissues including roots, stem and leaves with the highest level of expression in leaves. The promoter region of WsSQS isolated by genome walking presented several cis-acting elements in the promoter region. Biosynthesis of withanolides was up-regulated by different signalling components including methyl-jasmonate, salicylic acid and 2, 4-D, which was consistent with the predicted results of WsSQS promoter region. This work is the first report of cloning and expression of squalene synthase from W. somnifera and will be useful to understand the regulatory role of squalene synthase in the biosynthesis of withanolides.► Squalene synthase gene was cloned from Withania somnifera (L) Dunal. ► Recombinant C-terminus truncated WsSQS was expressed in E. coli. ► qPCR analysis showed higher expression of WsSQS in leaves as compared to root. ► WsSQS promoter presented several cis elements involved in secondary metabolism. ► Biosynthesis of withanolides was upregulated by different signalling phytohormones.
Keywords: Withania somnifera; Squalene synthase; Withanolides; Withaferin A; Promoter; SDS-PAGE;

High GC content of simple sequence repeats in Herpes simplex virus type 1 genome by Qingjian Ouyang; Xiangyan Zhao; Haiping Feng; You Tian; Dan Li; Mingfu Li; Zhongyang Tan (37-40).
The presence, locations and composition of simple sequence repeats (SSRs) in Herpes simplex virus type 1 (HSV-1) genome were extracted and analyzed by using the software Imperfect Microsatellite Extractor (IMEx). There were 663 mon-, 502 di-, 184 tri-, 20 tetra-, 4 penta- and 4 hexanucleotide SSRs that were observed in different distribution between coding and noncoding regions in the HSV-1 genome. G/C, GC/CG, and (GGC)n were predominant in mononucleotide, dinucletide, trinucleotide repeats respectively. Indeed, the results showed that GC content in simple sequence repeats was notably higher than that in entire HSV-1 genome. Our data might be helpful for studying the pathogenesis, genome structure and evolution of HSV-1.► We analyzed the distribution of SSRs in coding, noncoding regions of HSV-1 genome. ► G/C, GC/CG, (GGC)n were excessive in mono-, di-, trinucleotide repeats respectively. ► In all SSRs the GC content was higher than that in entire genome. ► High GC content SSRs might be related to genome structure and pathogenesis of HSV-1.
Keywords: Simple sequence repeat; Microsatellite; Herpes simplex virus type1;

Low dependency of retrotransposition on the ORF1 protein of the zebrafish LINE, ZfL2-1 by Masaki Kajikawa; Tomohiro Sugano; Ryosuke Sakurai; Norihiro Okada (41-47).
The zebrafish long interspersed element (LINE), ZfL2-1, which belongs to the L2 clade, contains two open reading frames, ORF1 and ORF2. ORF1 encodes a protein containing a coiled-coil motif and an esterase domain, whereas ORF2 encodes a protein containing an endonuclease and a reverse transcriptase domain. To elucidate the functional significance of ORF1 in retrotransposition, we constructed many variants of ZfL2-1 and examined their retrotransposition ability. We concluded: 1) the ORF1 protein is not essential for ZfL2-1 retrotransposition in cultured cells; 2) the translation of ORF1 is required for the translation of ORF2; and 3) ORF2 translation probably occurs via suppression of the ORF1 stop codon, the efficiency of which is influenced by the context of the sequence juxtaposed to the 3′ side of the stop codon. These results offer a new perspective on the evolution of the L2 clade LINEs.► The ORF1 protein has no apparent essential function in retrotransposition of ZfL2-1. ► An upstream coding sequence, however, is required for retrotransposition of ZfL2-1. ► We propose a model for the translation of ORF2 through the ORF1 in ZfL2-1.
Keywords: Non-LTR retrotransposon; Transposable element;

Eight-alanine duplication in homeobox D13 in a Chinese family with synpolydactyly by Qian Xin; Lin Li; Jiangxia Li; Rongfang Qiu; Chenhong Guo; Yaoqin Gong; Qiji Liu (48-51).
Human synpolydactyly (SPD), belonging to syndactyly (SD) II, is an inherited autosomal-dominant limb malformation characterized by SD of finger 3 or 4 or toe 4 or 5, usually with digit duplication. Previous studies have demonstrated that homeobox protein D13 (HOXD13) is responsible for this Mendelian disorder. In this paper, we report on a family with SPD — 7 members show typical SPD malformations. We used PCR and Sanger sequencing of DNA from peripheral blood samples and found an 8-Ala expansion in exon 1 of HOXD13 by mutation detection; this variant was absent in unaffected members and in 50 unaffected non-related subjects. This study further confirmed the correlation between SPD and alanine expansion in HOXD13.► Confirmed HOXD13 mutation and synpolydactyly. ► Found an eight-alanine expansion in HOXD13. ► This mutation leads to obviously variable expressivities.
Keywords: Synpolydactyly; HOXD13; Polyalanine expansion mutation;

Identification of cis-regulatory elements specific for different types of reactive oxygen species in Arabidopsis thaliana by Veselin Petrov; Vanessa Vermeirssen; Inge De Clercq; Frank Van Breusegem; Ivan Minkov; Klaas Vandepoele; Tsanko S. Gechev (52-60).
The type of reactive oxygen species (ROS) is a major factor that determines the specificity of biological responses. These responses may be elicited by activation of transcription factors that recognize ROS-specific cis-regulatory elements in target genes. In search for Arabidopsis promoter motifs specific for particular types of ROS, genome-wide microarray expression profiles for 283 abiotic stress-related conditions were subjected to cluster analysis to identify gene groups induced by singlet oxygen, superoxide radicals, and H2O2. Promoters of these gene groups were analyzed to identify cis-regulatory elements that are associated with specific types of ROS. Eleven ROS-specific de novo identified elements, seven known promoter motifs and several sequences enriched in ROS-responsive clusters but lacking in specificity are reported. The conservation of the identified motifs was determined in orthologous genes in C. papaya, V. vinifera and P. trichocarpa. Finally, biological functions were attributed to the motifs by calculation of GO-term enrichment for genes with conserved ROS-responsive elements.► Clusters enriched in genes responsive to a single type of ROS were obtained. ► ROS-specific clusters were subjected to 3 motif finding algorithms. ► Cis-regulatory elements enriched in the ROS-specific clusters were reported. ► The conservation of the reported motifs was studied by phylogenetic footprinting. ► Genes with conserved ROS-responsive elements were analyzed for GO-term enrichment.
Keywords: Reactive oxygen species; Promoter motifs; Cluster analysis; Gene expression profiles;

Polymorphisms of interleukin-1 and interleukin-6 genes on the risk of ischemic stroke in a meta-analysis by Fei Ye; Xiao-Qing Jin; Guang-Hui Chen; Xiao-Ling Den; Yong-Qiang Zheng; Cheng-Yan Li (61-69).
Many epidemiological studies have investigated the associations between polymorphisms of interleukin-1 (IL1) and interleukin-6 (IL6) genes and risk of ischemic stroke (IS), but no conclusions are available because of conflicting results. The aim of this study was to assess the relationships by meta-analysis. The databases of Pubmed, Embase and Wangfang, updated to August 1st, 2011, were retrieved. Odds ratio (OR) and corresponding 95% confidence interval (95% CI) as effect size were calculated by a fixed- or random-effect model. In total, three case–control studies for IL1α-889C/T, eight studies for IL1β-511C/T, eight studies for IL1-Ra and seven studies for IL6-147G/C were included in this meta-analysis. Combined analysis indicated that IL1β-511C/T polymorphism was not overall associated with risk of IS [OR (95% CI) = 1.22 (0.85–1.87) for TT vs. CC]. However, when subgroup analyses for countries were conducted, the results indicated that T allele was associated with increased risk of IS for Polish and associated with a trend of increased risk of IS for Chinese although it did not reach statistical significance [TT vs. CC: OR (95% CI) = 1.97 (1.22–3.17) for Polish and 1.40 (0.99–1.99) for Chinese]. In addition, overall and subgroup analyses indicated that IL1α-889C/T, IL1-Ra and IL6-147G/C polymorphisms were also not associated with risk of IS [OR (95% CI) = 1.21 (0.86–1.70) for TT vs. CC of IL1α-889C/T, 1.22 (0.85–1.75) for RN2/RN2 vs. RN1/RN1 for IL1-Ra and 1.09 (0.84–1.40) for G carriers vs. C carriers for IL6-147G/C]. This study inferred that IL1β-511C/T polymorphism might be moderately associated with increased risk of IS, but no sufficient evidence was available to support any associations between IL1-Ra and IL6-147G/C polymorphisms and IS. We could not draw a conclusion between IL1α-889C/T polymorphism and risk of IS based on the limited data, and further large sample-sized studies were required.
Keywords: Inflammation; Interleukin-1; Interleukin-6; Ischemic stroke; Risk; Meta-analysis;

Quantification of mRNA of genes related to metabolism, immunity and cellular stress was examined in relation to a massive mortality event during the culture of American oyster larvae, Crassostrea virginica which was probably, in regard to previous microbiological analysis, induced by Vibrio infection. To document molecular changes associated with the mortality event, mRNA levels were compared to biochemical and physiological data, previously described in a companion paper. Among the 18 genes studied, comparatively to the antibiotic control, 10 showed a lower relative gene expression when the massive mortality occurred. Six of them are presumed to be related to metabolism, corroborating the metabolic depression associated with the mortality event suggested by biochemical and physiological analyses. Relationships between the regulation of antioxidant enzyme activities, lipid peroxidation, and the mRNA abundance of genes linked to oxidative stress, cytoprotection, and immune response are also discussed. Finally, we observed an increase in the transcript abundance of two genes involved in apoptosis and cell regulation simultaneously with mortality, suggesting that these processes might be linked.► Gene expression associated to massive mortality emergence during larval development. ► Gene expression in relation to physiological and biochemical parameters. ► Genes related to metabolism, immunity and cellular stress. ► Integrative approach of mortality event.
Keywords: Massive mortality; Relative gene expression; Metabolism; Immunity; Cellular stress; Larvae;

Applying DNA barcodes for identification of plant species in the family Araliaceae by Zhihua Liu; Xu Zeng; Dan Yang; Guiyan Chu; Zhengrong Yuan; Shilin Chen (76-80).
An effective DNA marker in authentication of the family Araliaceae was screened out of the five DNA regions (matK, rbcL, ITS2, psbA-trnH and ycf5). In the present study, 1113 sequences of 276 species from 23 genera (Araliaceae) were collected from DNA sequencing and GenBank, in which 16 specimens were from 5 provinces in China and Japan. All of the sequences were assessed in the success rates of PCR amplifications, intra- and inter-specific divergence, DNA barcoding gaps and efficiency of identification. Compared with other markers, ITS2 showed superiority in species discrimination with an accurate identification of 85.23% and 97.29% at the species and genus levels, respectively, in plant samples from the 589 sequences derived from Araliaceae. Consequently, as one of the most popular phylogenetic markers, our study indicated that ITS2 was a powerful barcode for Araliaceae identification.Display Omitted►ITS2 had a high authentication success rate of Araliaceae species. ►ITS2 provided a great efficiency of PCR amplification. ►ITS2 exhibited significantly high levels of inter-specific discriminatory ability. ►ITS2 was more suitable than other barcodes in Araliaceae species discrimination.
Keywords: Araliaceae; DNA barcoding; ITS2;

Different mutation profiles associated to P53 accumulation in colorectal cancer by Ignacio López; Ligia P. Oliveira; Paula Tucci; Fernando Álvarez-Valín; Renata A. Coudry; Mónica Marín (81-87).
The tumor suppressor TP53 gene is one of the most frequently mutated in different types of human cancer. Particularly in colorectal cancer (CRC), it is believed that TP53 mutations play a role in the adenoma–carcinoma transition of tumors during pathological process. In order to analyze TP53 expressed alleles in CRC, we examined TP53 mRNA in tumor samples from 101 patients with sporadic CRC. Samples were divided in two groups defined according to whether they exhibit positive or negative P53 protein expression as detected by immunohistochemistry (IHC). The presence of TP53 mutation was a common event in tumors with an overall frequency of 54.5%. By direct sequencing, we report 42 different TP53 sequence changes in 55 CRC patients, being two of them validated polymorphisms. TP53 mutations were more frequent in positive than in negative P53 detection group (p < 0.0001), being the precise figures 79.6% and 30.8%, respectively. In addition, the mutation profiles were also different between the two groups of samples; while most of the mutations detected in P53 positive group were missense (38 out of 39), changes in P53 negative detection group include 7 insertions/deletions, 6 missense, 2 nonsense and 1 silent mutation. As previously observed, most mutations were concentrated in regions encoding P53 DNA binding domain (DBD). Codons 175, 248 and 273 together account for 36.7% of point mutations, in agreement with previous observations provided that these codons are considered mutation hotspots. Interestingly, we detected two new deletions and two new insertions. In addition, in three samples we detected two deletions and one insertion that could be explained as putative splicing variants or splicing errors.► We analyzed TP53 mutation pattern and P53 level in colorectal cancer in Brazil. ► There was no association between mutations & protein accumulation with clinical data. ► TP53 mutations were more frequent in positive than in negative P53 detection group. ► We detected two new deletions and two new insertions in cancer. ► We also detected three changes that could be explained as splicing variants or errors.
Keywords: Colorectal cancer; TP53 mutation pattern; TP53 mRNA;

Widespread occurrence of power-law distributions in inter-repeat distances shaped by genome dynamics by Alexandros Klimopoulos; Diamantis Sellis; Yannis Almirantis (88-98).
Repetitive DNA sequences derived from transposable elements (TE) are distributed in a non-random way, co-clustering with other classes of repeat elements, genes and other genomic components. In a previous work we reported power-law-like size distributions (linearity in log–log scale) in the spatial arrangement of Alu and LINE1 elements in the human genome. Here we investigate the large-scale features of the spatial arrangement of all principal classes of TEs in 14 genomes from phylogenetically distant organisms by studying the size distribution of inter-repeat distances. Power-law-like size distributions are found to be widespread, extending up to several orders of magnitude. In order to understand the emergence of this distributional pattern, we introduce an evolutionary scenario, which includes (i) Insertions of DNA segments (e.g., more recent repeats) into the considered sequence and (ii) Eliminations of members of the studied TE family. In the proposed model we also incorporate the potential for transposition events (characteristic of the DNA transposons' life-cycle) and segmental duplications. Simulations reproduce the main features of the observed size distributions. Furthermore, we investigate the effects of various genomic features on the presence and extent of power-law size distributions including TE class and age, mode of parental TE transmission, GC content, deletion and recombination rates in the studied genomic region, etc. Our observations corroborate the hypothesis that insertions of genomic material and eliminations of repeats are at the basis of power-laws in inter-repeat distances. The existence of these power-laws could facilitate the formation of the recently proposed “fractal globule” for the confined chromatin organization.► We study the genomic distribution of transposable elements (TEs). ► We find the distribution to be a power-law across multiple species and TE classes. ► We implement an evolutionary model to explain the observed distribution ► The model includes genomic length increase and random TE deletions. ► We suggest that TE power-laws contribute to the “fractal globule” genome structure.
Keywords: transposable elements; power-law distribution; genome evolution; fractal globule;

Bioinformatic selection of putative epigenetically regulated loci associated with obesity using gene expression data by Valérie Turcot; Alexandra Groom; James C. McConnell; Mark S. Pearce; Catherine Potter; Nicholas D. Embleton; Daniel C. Swan; Caroline L. Relton (99-107).
There is considerable interest in defining the relationship between epigenetic variation and the risk of common complex diseases. Strategies which assist in the prioritisation of target loci that have the potential to be epigenetically regulated might provide a useful approach in identifying concrete examples of epigenotype–phenotype associations. Focusing on the postulated role of epigenetic factors in the aetiopathogenesis of obesity this report outlines an approach utilising gene expression data and a suite of bioinformatic tools to prioritise a list of target candidate genes for more detailed experimental scrutiny. Gene expression microarrays were performed using peripheral blood RNA from children aged 11–13 years selected from the Newcastle Preterm Birth Growth Study which were grouped by body mass index (BMI). Genes showing ≥ 2.0 fold differential expression between low and high BMI groups were selected for in silico analysis. Several bioinformatic tools were used for each following step; 1) a literature search was carried out to identify whether the differentially expressed genes were associated with adiposity phenotypes. Of those obesity-candidate genes, putative epigenetically regulated promoters were identified by 2) defining the promoter regions, 3) then by selecting promoters with a CpG island (CGI), 4) and then by identifying any transcription factor binding modules covering CpG sites within the CGI. This bioinformatic processing culminated in the identification of a short list of target obesity-candidate genes putatively regulated by DNA methylation which can be taken forward for experimental analysis. The proposed workflow provides a flexible, versatile and low cost methodology for target gene prioritisation that is applicable to multiple species and disease contexts.► We proposed in silico tools to target obesity candidate loci putatively regulated epigenetically. ► Microarrays revealed differentially expressed genes between variable adiposity index. ► Further selection of target loci was achieved using a gene-adiposity literature search. ► Promoters of target loci were analysed for potential methylation regulation.
Keywords: DNA methylation; CpG island; Promoter in silico analysis; Body mass index; Adiposity;

Genome-wide analysis of the mitogen-activated protein kinase gene family in Solanum lycopersicum by Fuling Kong; Jie Wang; Lin Cheng; Songyu Liu; Jian Wu; Zhen Peng; Gang Lu (108-120).
Mitogen-activated protein kinases (MAPKs), a family of Ser/Thr protein kinases, play an essential role in mediating biotic and abiotic stress responses in plants. In this study, we investigated 16 putative SlMAPK genes from tomato genome and compared them with those from Arabidopsis. The full cDNA sequences of 13 novel SlMAPKs in tomato were obtained through PCR ampilification. A comprehensive genome-wide analysis of SlMAPKs in tomato is presented, including their gene structure, phylogeny, genome localization, and expression profiles. Phylogenic analysis of the 16 SlMAPKs and 20 AtMAPKs from Arabidopsis indicated that the SlMAPK genes were clustered into four major groups, and genes within the same groups had similar exon–intron structures. All SlMAPK proteins in groups A, B and C had a Thr-Glu-Tyr (TEY) activation domain, whereas those in group D contained a Thr-Asp-Tyr (TDY) activation domain. The analysis of 5′-upstream region of SlMAPKs revealed a group of putative cis-acting elements related to stress responses. Expression analysis of SlMAPK genes using RT-PCR and real-time quantitative PCR demonstrated that all SlMAPK transcripts were able to be detected in at least one investigated tissue, and some of them exhibited tissue-specific expression patterns. The transcript abundance of nearly all SlMAPK genes was increased in response to heat stress treatment. Our data provided an insight into the evolution of the gene family and a useful reference for further functional analysis of MAPK family genes in tomato.► A comprehensive genome-wide analysis of SlMAPK gene family in tomato is presented. ► The full cDNA sequences of 13 novel SlMAPKs were identified with PCR methods. ► Some stress-responsive cis-acting elements were found in promoter regions. ► SlMAPKs show different expression pattern in various organs and stages. ► Heat stress markedly affects the expression levels of SlMAPKs.
Keywords: Solanum lycopersicum; Mitogen-activated protein kinase; Phylogenetic analysis; Expression analysis; QRT-PCR;

Non-association of Crohn's disease with NOD2 gene variants in Moroccan patients by I. Hama; I. Ratbi; S. Reggoug; F. Elkerch; G. Kharrasse; I. Errabih; H. Ouazzani; A. Sefiani (121-123).
Crohn's disease is a chronic inflammatory bowel disease, with multifactorial traits, that can involve any part of the gastrointestinal tract. In recent years, a dozen genome-wide association scan and meta-analysis were published bringing the number of susceptibility alleles to more than 30 variations. However, the major susceptibility gene for Crohn's disease is NOD2, located on proximal 16q, which is involved in the innate immune response. Three main variants of this gene: two single nucleotide polymorphisms p.Arg702Trp and p.Gly908Arg substitutions and frameshift polymorphism p.Leu1007fsinsC are involved in susceptibility to Crohn's disease.There is no data about the frequency of these allelic variants in Moroccan patients with Crohn's disease. The aim of our study is to genotype the NOD2 gene to assess the involvement of these three variants in susceptibility to Crohn's disease for Moroccans.We carried out genotyping for the three variants p.Arg702Trp, p.Gly908Arg and p.Leu1007fsinsC of NOD2 gene using PCR-sequencing among 101 Moroccan patients with Crohn's disease and 107 healthy controls.The three main variants of NOD2 gene were present in Moroccan patients with no significant difference compared to controls.This preliminary study shows no evidence association of NOD2 gene with Crohn's disease in the Moroccan population.► We recruited a group of Moroccan patients with Crohn’s disease, and a control group. ► We genotyped three major variants of the NOD2 gene in the two groups. ► We compared the genotype frequencies between the two groups by statistical test. ► The three variants are not involved in Crohn’s disease susceptibility in Moroccans.
Keywords: Crohn's disease; NOD2/CARD15; Variant; Moroccan population;

Molecular characterization, tissue distribution and expression analysis of Interleukin-12 receptor β2 chain in sheep by Xuelian Meng; Aijiang Guo; Wei Gong; Wanzhong Jia; Xuenong Luo; Junjun Zhai; Yongxi Dou; Xuepeng Cai (124-129).
Ovine β2 subunit of the interleukin (IL)-12 receptor (IL-12Rβ2) was cloned from mRNA preparation of mitogen-activated peripheral blood mononuclear cells (PBMCs). The complete coding sequence for ovine IL-12 Rβ2 was found to be 2586 nucleotides in length encoding 862-amino-acid residue protein. It showed 96.4% homology at the nucleotide level and 94.1% homology at the amino acid level with bovine IL-12 Rβ2. The ovine IL-12 Rβ2 subunit shares common structural and functional elements with their counterparts from the other species. Phylogenetic tree showed that ovine IL-12Rβ2 was clustered into the Artiodactyla group, together with those of cattle and pig, which was distinct from the other groups. Real-time RT-PCR was used to investigate expression of the IL-12Rβ2 in different tissues of sheep in order to determine the characterization of this receptor in tissue. Expression analysis showed that IL-12Rβ2 mRNA expression was detected at all the detected tissues with the exception of thymus.
Keywords: IL-12Rβ2; Sheep; Molecular characterization; Expression; Tissue distribution;

A novel silk-like shell matrix gene is expressed in the mantle edge of the Pacific oyster prior to shell regeneration by Jun Takahashi; Masaya Takagi; Yumiko Okihana; Kei Takeo; Takahisa Ueda; Ken Touhata; Shingo Maegawa; Haruhiko Toyohara (130-134).
During shell formation, little is known about the functions of organic matrices, especially about the biomineralization of shell prismatic layer. We identified a novel gene, shelk2, from the Pacific oyster presumed to be involved in the shell biosynthesis. The Pacific oyster has multiple copies of shelk2. Shelk2 mRNA is specifically expressed on the mantle edge and is induced during shell regeneration, thereby suggesting that Shelk2 is involved in shell biosynthesis. To our surprise, the database search revealed that it encodes a spider silk-like alanine-rich protein. Interestingly, most of the Shelk2 primary structure is composed of two kinds of poly-alanine motifs—GXNAn(S) and GSAn(S)—where X denotes Gln, Arg or no amino acid. Occurrence of common motifs of Shelk2 and spider silk led us to the assumption that shell and silk are constructed under similar strategies despite of their living environments.► We identified a novel gene, shelk2, in the Pacific oyster. ► Database search revealed that Shelk2 carries a spider silk-like protein. ► Shelk2 is expressed specifically in the mantle edge prior to shell biosynthesis. ► Shelk2 is assumed to be involved in the biosynthesis of shell prismatic layer. ► Our findings suggest oyster shell is constructed by similar strategy to spider silk.
Keywords: Biomineralization; Calcite; Pacific oyster; Poly-alanine; Spider-silk protein;

The chitinase-like 1 protein, YKL-40, is involved in inflammation and tissue remodeling. Patients with coronary heart disease (CHD) and acute myocardial infarction have elevated levels of serum YKL-40. The goal of the present study was to investigate whether the chitinase-like 1 gene-329G/A variant (rs10399931) confers susceptibility to CHD, and whether it is associated with the clinical phenotype and severity of disease.We performed a case-control study of 410 unrelated CHD patients (coronary stenosis ≥ 50% or documented myocardial infarction) and 442 controls from China. A ligase detection reaction was used to determine a single-nucleotide polymorphism in rs10399931. The genotypic and allelic associations of this single-nucleotide polymorphism with CHD, phenotypes and severity were also evaluated. Plasma levels of YKL-40 were measured using ELISA assays.Three genotypes, CC, CT, and TT, existed in rs10399931 and there were no significant differences found in either the genotypic or allelic frequencies between the CHD cases and controls. Patients with CHD had higher YKL-40 levels compared to controls and those with acute myocardial infarction had the highest levels of YKL-40 compared to patients with either stable or unstable angina pectoris (all p  < 0.01). Rs10399931 affected neither the main anthropometric or metabolic characteristics, nor did there exists any association between rs10399931 and the severity of coronary lesions assessed by Gensini scores (all p  > 0.05).Our results do not support that rs10399931 is associated with clinical phenotypes of CHD and the extent of coronary lesions; however, YKL-40 levels are higher in CHD patients and associated with its clinical phenotypes.► Chitinase 3-like 1 gene polymorphism is not associated with clinical phenotypes of coronary heart disease. ► YKL-40 levels are higher in patients with coronary heart disease than in controls. ► YKL-40 levels are associated with clinical phenotypes of coronary heart disease.
Keywords: Atherosclerosis; Coronary heart disease; Gene; Phenotypes; Single-nucleotide polymorphism;

Detection of truncated dystrophin lacking the C-terminal domain in a Chinese pedigree by next-generation sequencing by Shuqi Xie; Zhangzhang Lan; Ning Qu; Xiaoming Wei; Ping Yu; Qian Zhu; Guanghui Yang; Jinming Wang; Quan Shi; Wei Wang; Ling Yang; Xin Yi (139-142).
► Next-generation sequencing platform and bioinformatics are applied in this study. ► A nonsense mutation is identified by next-generation sequencing in a pedigree. ► The nonsense mutation, c.10141C>T (p.Arg3381X), is located at the exon 70. ► This is a first report of truncated dystrophin lacking C-terminal domain in China.
Keywords: Duchenne Muscular Dystrophy (DMD); Dystrophin (DMD) gene; Next-generation sequencing; Bioinformatics;

Promoter contribution to the testis-specific expression of Stellate gene family in Drosophila melanogaster by Oxana M. Olenkina; Ksenia S. Egorova; Mikhail V. Kibanov; Yuri V. Gervaziev; Vladimir A. Gvozdev; Ludmila V. Olenina (143-153).
Testis-specific tandemly repeated Stellate genes are part of the SteSu(Ste) genetic system required for male fertility in Drosophila melanogaster. Stellate genes encode a functional homolog of the β-subunit of protein kinase CK2. Derepression of Stellate results in their over-expression, meiotic disturbances and male sterility. Stellate genes are represented by clustered copies in the X chromosome and carry promoters shared with another X-chromosome cluster, βNACtes genes, encoding putative β-subunits of the nascent polypeptide-associated complex. Using Electrophoretic Mobility Shift Assay, we revealed in the Stellate promoter three cis-acting elements, E-boxes, the loss of which greatly diminished the reporter gene expression in Drosophila testes. We identified that these E-boxes were recognized by helix–loop–helix protein, dUSF (Drosophila ortholog of mammalian USF) in testis nuclear extract. All three E-boxes were preserved in the promoters of both euchromatic and heterochromatic Stellate clusters. Two analogous E-boxes were detected in the promoters of 5′-copies of the duplicated βNACtes gene pairs, whereas the 3′-copies lacked these sites but possessed a new binding site for a testis protein distinct from dUSF. Here we characterized a new type of testis-specific core promoter and identified dUSF as its interacting transcription factor.► Regulation of SteSu(Ste) system in Drosophila spermatogenesis. ► Cis-regulatory elements, E-boxes, were found in Stellate promoter. ► E-box-binding transcription factor dUSF is expressed in the testes. ► SteSu(Ste) and βNACtes genes employ common regulatory elements in shared promoters. ► A new type of testis-specific promoter was described.
Keywords: Spermatogenesis; Promoter; E-box; Stellate; dUSF;

The St genome, which is present in nearly half of all Triticeae species, originates from the genus Pseudoroegneria. However, very little is known about the high molecular weight (HMW) subunits of glutenin which are encoded by the St genome. In this paper, we report the isolation from Pd. libanotica of four sequences encoding HMW subunits of glutenin. The four genes were all small compared to standard glutenin genes. All four sequences resemble y-type glutenins rather than x-types. However, their N-terminal domains contain a glutamine residue which is present in all x-type, but very few y-type subunits, and their central repetitive domains included some irregular motifs. The indication is therefore that the Glu-1St genes evolved earlier than other modern day homoeologues, so that they represent an intermediate state in the divergence between x- and y-type subunits. No x-type Glu-1St subunit genes were identified.► We cloned four novel HMW-GS genes from Pseudoroegneria libanotica. ► All four genes were small; two of them were the smallest HMW-GS genes until now. ► They were evolutionarily older than y-type subunits didn't possess an extra Q residue. ► They represent an intermediate state in the divergence of x- and y-type subunits. ► The St genome differentiated earlier from other Triticeae genomes
Keywords: Evolution; Glu-1; HMW-GS; Pseudoroegneria; St genome;

A previous genome-wide association study (GWAS) failed to discover any nucleotide sequence variant associated with susceptibility to vascular dementia (VaD) and remained a problem of false negatives produced by a low statistical power. The current study was conducted to identify such potential false negatives and to provide comprehensive evidence for the most plausible predisposing genetic factor using large-scale Korean cohorts. We identified the gene encoding retinitis pigmentosa GTPase regulator-interacting protein 1-like (RPGRIP1L) with multiple nucleotide variants associated with susceptibility to VaD by a modest significant threshold (P < 10− 4). Genetic associations were intensively examined with its sequence variants using 207 VaD patients and 207 age- and gender-matched control subjects. Genetic association analysis with dense variants in the region associated with VaD revealed 3 variants (P < 0.0017) in strong linkage. Further analysis with VaD-related phenotypes using Korean Association REsource (KARE) cohort data showed that the region of the gene was associated with alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood pressure (BP) (P < 7.6 × 10− 4). The current study provided the first evidence of the association between RPGRIP1L gene and susceptibility of VaD. Functional studies are needed to understand underlying biological mechanism of the genetic association.► We examine genetic factors for vascular dementia. ► RPGRIP1L gene is associated with susceptibility to vascular dementia. ► Further associations are identified with vascular dementia-related phenotypes.
Keywords: Dementia; Genetic association; RPGRIP1L; Single nucleotide polymorphism; Vascular cognitive impairment;

MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs about 18 to 26 nucleotides long. Their conserved nature among the various organisms makes them a good source of new miRNAs discovery by comparative genomics approach. The study resulted in novel 75 precursor miRNAs containing 102 mature sequences belonging to 46 families in an important aquatic environmental monitoring fish (Salmo salar). All the miRNA families (let-7, mir-1, 7, 9, 21, 22, 92, 96, 122, 126, 128, 129, 132, 133, 142, 144, 147, 148, 196, 202, 212, 223, 375, 429, 430, 449, 451, 457, 466, 682, 700, 1388, 1594, 1600, 1607, 1616, 1642, 1681, 1701, 1720, 1772, 1782, 1787, 1814, 2189 and 3540) are found for the first time in S. salar. All 75 miRNA precursors form stable minimum free energy stem loop and the mature miRNAs reside in the stem portion of the stem loop structure. Their target proteins are involved in transcription factors (28%), metabolism (23%), signaling (18%), transportation (9%), immunity (8%), stress related activity (5%), cancer and tumor related activity (5%), growth and development (3%), and cell division (1%).► 102 novel S. salar MicroRNAs are reported. ► 258 targets for these miRNAs are identified. ► 21 pre-miRNA clusters were also identified.
Keywords: Blast; Comparative genomics; MicroRNAs; Salmo salar;

Delivery of AP-2α siRNA into cultured bovine trophoblast cells by electroporation repressed key placenta-specific gene expression by Xuan Zhou; Zhenyun Wang; Zhen Zhang; Qunwei Cui; Yachun Wang; Genlin Wang (169-175).
Binucleate trophoblast giant cells (BNC) characteristically appear early in gestation in the bovine placenta. They secret pivotal hormones and cytokines for feto-maternal communication, for example, expression of placental lactogens (CSH1), prolactin-related protein 1 (PRP1) and pregnancy-associated glycoprotein 1 (PAG1) are necessary for pregnancy establishment in bovine. These genes transcription are regulated in a temporal and spatial manner, however, molecular mechanisms by which these gene transcriptions are regulated in this manner have not been firmly elucidated. In this study, a cell culture model for bovine trophoblast cells was initially established, small interfering RNA duplexes against Activator Protein-2α (TFAP2A) was transfected into the cells by electroporation, and transcripts of CSH1, PRP1 and PAG1 were measured by qPCR. The results showed that trophoblast giant cells were confluent for 90% after cultured for 10 days, and BNC constituted of a population of more than 45% of the total cells. Using a fluorescein-labeled non-silencing siRNA duplex, an electroporation protocol yielding routinely > 93% positive cells could be established, and siRNA duplex transfection demonstrated an efficient knockdown of cellular AP-2α mRNA level by 72.30 ± 3.28% in electroporated cells. Finally, CSH1, PRP1 and PAG1 genes expression were effectively down-regulated by 65.45 ± 6.38% (P  < 0.01), 40.73 ± 11.72% (P  < 0.01) and 11.59 ± 1.88% (P  < 0.05), respectively. It was therefore suggested that electroporating siRNA into bovine trophoblast cells could be an efficient method to manipulate BNC function and to study the regulation mechanism of specific gene transcription without the use of chemical transfection reagents. It was suggested that AP-2α could be at least involved in the regulation of expression CSH1 and PRP1 transcripts.► We established a cell culture model for bovine trophoblast cells. ► siRNA duplexes against TFAP2A was effectively transfected into the cells. ► Electroporating siRNA into bovine trophoblast cells could be an efficient method. ► AP-2α could be involved in regulation of expression CSH1 and PRP1 transcripts.
Keywords: Bovine binucleate trophoblast cell; AP-2α siRNA; Electroporation; BNC-specific genes;

Euchromatic and heterochromatic compositional properties emerging from the analysis of Solanum lycopersicum BAC sequences by Miriam Di Filippo; Alessandra Traini; Nunzio D'Agostino; Luigi Frusciante; Maria Luisa Chiusano (176-181).
The consortium responsible for the sequencing of the tomato (Solanum lycopersicum) genome initially focused on the sequencing of the euchromatic regions using a BAC-by-BAC strategy. We analyzed the compositional features of the whole collection of BAC sequences publically available. This analysis highlights specific peculiarities of heterochromatic and euchromatic BACs, in particular: the whole BAC collection has i) a large variability in repeat and gene content, ii) a positive and significant correlation of LTR retrotransposons of the Gypsy class with the repeat content and iii) the preferential location of the SINEs (short interspersed nuclear elements) in BAC sequences showing a low repeat content. Our results point out a typical design of the tomato chromosomes and pave the way for further investigations on the relationship between DNA primary structure and chromatin organization in Solanaceae genomes.► LTR retrotransposons and SINEs significantly correlate with the repeat content. ► Compositional properties of euchromatic and heterochromatic regions emerge from BAC sequences.
Keywords: Tomato genome; Heterochromatin; Euchromatin; Repetitive elements; Gene content; Repeat content;

A submicroscopic deletion involving part of the CREBBP gene detected by array-CGH in a patient with Rubinstein–Taybi syndrome by Angeline H.M. Lai; Maggie S. Brett; Wai-Hoe Chin; Eileen C.P. Lim; Jasmine S.H. Ng; Ene-Choo Tan (182-185).
We report a girl with Rubinstein–Taybi syndrome (RSTS) who was found to have copy number loss on 16p13.3 by array-CGH. She has developmental delay and other features of RSTS including downslanting palpebral fissures, a prominent nose with the nasal septum extending below the alae nasi, broad thumbs and big toes, postaxial polydactyly of the right foot and constipation from birth. We report the junction sequence across the breakpoint region for a microdeletion in RSTS. The sequencing results also showed that the deletion was 81.4 kb involving three genes DNASE 1, TRAP 1, and CREBBP.► We report a Chinese patient with features of Rubinstein–Taybi syndrome (RSTS). ► She also has postaxial polydactyly which is not common for RSTS. ► Array-CGH showed copy number loss for 16p13.3. ► Sequence at the breakpoint junction showed the structure of a DNASE 1CREBBP fusion gene. ► Report is the first on the sequence at the breakpoint of a microdeletion for this syndrome.
Keywords: CREBBP; DNASE 1; Microdeletion; Rubinstein–Taybi syndrome; TRAP 1;

The enzymes 3β-hydroxysteroid dehydrogenase (3βHSD) and 17β-hydroxysteroid dehydrogenase (17βHSD) regulate the steroid metabolism in mammals. In this study, we aimed to characterize the steroid related transcription factors at the 5′ flanking region of these two genes. A series of 5′ deletions of approximately 1 kb of 5′-flanking region on both genes were fused to a pGL3 basic vector containing firefly luciferase cDNA, and then transfected to human hepatocellular liver carcinoma cell line (HepG2). Luciferase activity assay indicated the region from − 574 to − 617 bp of the 3βHSD1 promoter, and from − 850 to − 868 bp of 17βHSD7 promoter induced the highest luciferase activity. A putative transcription factor, i.e. the proline and acidic amino acid-rich basic leucine zipper (PAR/bZIP) family of 3βHSD1 gene, and three-amino acid loop extension (TALE) homeodomain class of 17βHSD7 were identified respectively by sequence homology. Gel shift assay further confirmed the binding capacity of the putative elements to nuclear extract. Our study gives new insights to the transcriptional regulation of 3βHSD1 and 17βHSD7 and further hints to their involvement in steroid metabolism.► We studied steroid related transcription factor of porcine 3βHSD1 and 17βHSD7 genes. ► Core regulation regions of both genes were identified by transfection analysis. ► EMSA further confirmed the binding capacity of putative elements to nuclear extract.
Keywords: Pig; 3β-Hydroxysteroid dehydrogenase; 17β-Hydroxysteroid dehydrogenase; Transcription factor;

Analysis of JAG1 gene variant in Chinese patients with Alagille syndrome by Honglian Wang; Xiaohong Wang; Qiaoli Li; Shiting Chen; Liyan Liu; Zhiyun Wei; Lei Wang; Yun Liu; Xinzhi Zhao; Lin He; Jianshe Wang; Qinghe Xing (191-193).
Alagille syndrome (AGS) is an autosomal dominant disorder characterized by bile duct paucity. It can be caused by variations in the JAG1 gene encoding a protein of Notch ligand and by variations in the NOTCH2 gene encoding a Notch receptor. In this study we identified 15 different JAG1 gene variations in 17 Chinese patients, nine of which were novel alterations including c.766G > T, c.819delC, c.826delT, c.3099_3100delCA, c.1323_1326delCTGG, c.1771_1775delGTGCGinsT, c.1868delG, c. 2791_2792insA and c.866delG. These alterations were located in the extracellular domain of JAG1, in particular in the DSL and EGF-like repeat domain. All the specific variations in five inheritance cases investigated were de novo. Furthermore, no sequence variation of NOTCH2 was detected in JAG1 alteration negative patients.► We identified 15 different JAG1 gene variations in 17 Chinese AGS patients. ► Nine of the JAG1 gene variations we identified were novel. ► All the specific variations in the five inheritance cases were de novo. ► No mutation of NOTCH2 was detected in JAG1 variation negative patients. ► No apparent correlation between genotype and phenotype was observed in our study.
Keywords: Alagille syndrome; JAG1; NOTCH2; Chinese;

Evolutionary rates of commonly used nuclear and organelle markers of Arabidopsis relatives (Brassicaceae) by Chi-Chun Huang; Kuo-Hsiang Hung; Wei-Kuang Wang; Chuan-Wen Ho; Chao-Li Huang; Tsai-Wen Hsu; Naoki Osada; Chi-Chuan Hwang; Tzen-Yuh Chiang (194-201).
Recovering the genetic divergence between species is one of the major interests in the evolutionary biology. It requires accurate estimation of the neutral substitution rates. Arabidopsis thaliana, the first whole-genome sequenced plant, and its out-crossing relatives provide an ideal model for examining the split between sister species. In the study, rates of molecular evolution at markers frequently used for systematics and population genetics, including 14 nuclear genes spanning most chromosomes, three noncoding regions of chloroplast genome, and one intron of mitochondrial genome, between A. thaliana and four relatives were estimated. No deviation from neutrality was detected in the genes examined. Based on the known divergence between A. thaliana and its sisters about 8.0–17.6 MYA, evolutionary rates of the eighteen genes were estimated. Accordingly, the ratio of rates of synonymous substitutions among mitochondrial, chloroplast and nuclear genes was calculated with an average and 95% confidence interval of 1 (0.25–1.75): 15.77 (7.48–114.09): 74.79 (36.27–534.61). Molecular evolutionary rates of nuclear genes varied, with a range of 0.383–0.856 × 10− 8 for synonymous substitutions per site per year and 0.036–0.081 × 10− 9 for nonsynonymous substitutions per site per year. Compared with orthologs in Populus, a long life-span tree, genes in Arabidopsis evolved faster in an order of magnitude at the gene level, agreeing with a generation time hypothesis. The estimated substitution rates of these genes can be used as a reference for molecular dating.► We examine evolutionary rates in 18 genomic regions of Arabidopsis. ► We detect a clock-like evolution in most sequenced regions. ► Nuclear genes exhibit high synonymous substitution rates. ► Synonymous substitution rates are much higher than nonsynonymous rates, as expected. ► Herb Arabidopsis evolves faster than tree Populus in an order of magnitude.
Keywords: Arabidopsis; Evolutionary rates; Generation time; Nonsynonymous substitutions; Populus; Synonoymous substitutions;

Genome wide computational analysis of Brugia malayi helicases: A comparison with human host by Renu Tuteja; Abulaish Ansari; Anita; Manish Kumar Suthar; Jitendra Kumar Saxena (202-208).
The availability of Brugia malayi genome sequence has paved ways for the search of homologues for a variety of genes. Helicases are ubiquitous enzymes involved in all the nucleic acid metabolic pathways and are essential for the development and growth. The genome wide analysis of B. malayi for different helicases showed the presence of a number of DEAD box helicases, 7 DEAH box helicases, RecQ helicases, repair helicases, super killer helicases, MCM2-7 complex, Rad54 and two subunits of Ku helicase. The comparison of protein sequence of each helicase with its human counterpart indicated characteristic differences in filarial helicases. There are noticeable differences in some of the filarial helicases such as DHX35, RecQL1 and Ku. Further characterization of these helicases will help in understanding physiological significance of these helicases in filarial parasites, which in future can be utilized for chemotherapy of parasitic infection.Genome wide analysis of helicases from Brugia malayi shows that there are some differences in domain structure of some of the helicases of B. malayi.Display Omitted► Comprehensive analysis of filarial parasite Brugia malayi helicases is reported. ► Our results show that genome contains a number of DEAD box and 7 DEAH box helicases. ► B. malayi genome also contains Ku, MCM, Rad54, Rec Q and super killer helicases. ► The comparison of all B. malayi helicases with human homologues is also presented.
Keywords: DEAD box; DEAH box; Helicase; Parasite; Brugia malayi; Unwinding;

POLG mutation in a patient with cataracts, early-onset distal muscle weakness and atrophy, ovarian dysgenesis and 3-methylglutaconic aciduria by Mir Reza Bekheirnia; Wei Zhang; Tanya Eble; Alecia Willis; Aziz Shaibani; Lee-Jun C. Wong; Fernando Scaglia; Shweta U. Dhar (209-212).
Mutations in POLG account for one of the most frequent nuclear encoded causes of mitochondrial disorders to date. Individuals harboring POLG mutations exhibit fairly heterogeneous clinical presentations leading to increasing difficulties in classifying these patients into defined clinical phenotypes. This study aims to investigate the molecular basis of a mitochondrial cytopathy in a patient with 3-methylglutaconic aciduria and to expand the clinical phenotype associated with POLG mutations.Clinical, molecular and genetic analyses as well as neurophysiological examinations were carried out for a 23-year-old woman of mixed Caucasian and Latin American ancestry with a history of cataracts diagnosed at age 1year, she had onset of distal muscle weakness at age 2years progressing to atrophy and ovarian dysgenesis at puberty. The patient was found to have 3-methylglutaconic acid with normal 3 hydroxyisovaleric acid on urine organic acid analysis. POLG sequencing was done and a heterozygous variant, c.2851T>A (p.Y951N) was found which is predicted to be deleterious. There are limited reports of POLG mutations in individuals with 3-methylglutaconic aciduria. This case report of a young woman with a heterozygous mutation in POLG, presenting with muscle weakness and atrophy at a young age aims to aid clinicians in similar challenging diagnostic situations as well as enhances our understanding of POLG-related disease phenotypes.► We tested a 23-year-old female with suspected mitochondrial disease for POLG mutation. ► Patient was affected with distal muscle atrophy, cataract and ovarian dysgenesis. ► We also detected 3-methylglutaconic acid on urine organic acid analysis. ► We identified a heterozygous p.Y951N variant in POLG, predicted to be deleterious. ► These findings enhance our understanding of POLG-related disease phenotypes.
Keywords: POLG; 3-Methylglutaconic aciduria; Muscle weakness; Cataracts; Ovarian dysgenesis;

Association between rs4673 (C/T) and rs13306294 (A/G) haplotypes of NAD(P)H oxidase p22phox gene and severity of stenosis in coronary arteries by Mohammad Najafi; Behnam Alipoor; Mohammad Shabani; Abdollah Amirfarhangi; Hassan Ghasemi (213-217).
Phagocytic NADH/NADPH oxidase is an important enzyme producing reactive oxygen species within subendothelial space of vessels. Findings have shown that p22phox subunit is an essential element related to the enzyme activity. Since some p22phox polymorphisms are thought to have functional roles in the enzyme thus, we studied the association between rs4673 (C242T) and rs13306294 (A/G) haplotypes and the severity of stenosis in coronary arteries. One hundred eighty-two subjects undergoing coronary angiography were recruited on the base of study design. Patients (n = 114) had at least a stenosed coronary artery (> 50% stenosis) and subdivided into three subgroups; SVD (n = 28), 2VD (n = 31) and 3VD (n = 55) while controls (n = 68) had the normal coronary arteries (< 5% stenosis). The direct haplotyping technique of SNPs was performed using ARMS–RFLP–PCR method. Furthermore, alphabet-based tools predicted the changes of secondary structure at the rs4673 position. All haplotypes being proposed theoretically were found in the study population. The distribution of two-allele haplotypes had no significant difference between patients and controls (P = 0.1). Although the rs4673 allele frequency was not significant between the groups (P > 0.5), chi square test and multinomial regression analysis showed an observed high risk for rs13306294 A allele among patients. The bioinformatics tools predicted that the p22phox secondary structure is not changed due to the substitution of Tyr → His at the rs4673 position. We concluded that the polymorphisms have no allele linkage on the chromosome. In addition, the rs13306294 A allele is a potential factor of stenosis of coronary arteries that increases susceptibility for the extent of disease.► The first study on direct haplotyping of SNPs. ► The first study in evaluation of rs13306294 in CAD patients. ► Use of bioinformatics tools for study of rs4673 substitution in secondary structure.
Keywords: P22phox; rs4673; rs13306294; Haplotype; Polymorphism; Coronary artery;

The impact of severe LDL receptor mutations on SREBP-pathway regulation in homozygous familial hypercholesterolemia (FH) by Muhidien Soufi; Volker Ruppert; Bilgen Kurt; Juergen R. Schaefer (218-222).
Familial hypercholesterolemia (FH), Niemann–Pick disease type C (NPC) and Tangier disease (TD) are genetic inherited disorders with impaired processing of cholesterol, caused by mutations in genes that regulate cellular uptake, intracellular movement and transport of cholesterol. Various studies have shown a crucial regulatory role of the SREBP-pathway for cellular cholesterol homeostasis in these diseases. Since cholesterol is an essential structural component of cells, we assessed the impact of a severe FH causing LDLR mutation (FH p.W556R) on the SREBP pathway in primary FH fibroblasts. Primary FH fibroblasts derived from patients with the LDL receptor mutation p.W556R were used for gene expression experiments. Gene expression studies revealed increased expressions of SREBP regulated genes HMGCR, LDLR, SREBP-2, SREBP-1, SR-BI, INSIG-1, but interestingly not SCAP. In contrast expression of ABCA1, was strongly decreased in homozygous, but not in heterozygous p.W556R fibroblasts. Gene expression experiments with LDL receptor lacking primary FH fibroblasts, revealed that SR-BI and ABCA1 are important regulators for cholesterol acquisition in FH cells, consistent with findings in cells from NPC and TD patients.► We examined SREPB pathway regulation in FH-Fibroblasts. ► SREBP-2 activates SR-BI expression in FH-Fibroblasts. ► SREBP-2 mediated ABCA1 expression is decreased in homozygous FH-Fibroblasts. ► Cholesterol pools regulate SREBP-2 function for ABCA1 transcription in FH.
Keywords: Familial hypercholesterolemia; LDL receptor; SREBP pathway; SR-BI; ABCA1;

Ellis-van Creveld syndrome in a fetus with rhizomelia and polydactyly. Report of a case diagnosed by genetic analysis, and correlation with pathological andradiologic findings by Milena Peraita-Ezcurra; Mónica Martínez-García; Víctor L. Ruiz-Pérez; María Eugenia Sánchez-Gutiérrez; María Fenollar-Cortés; Camilo Vélez-Monsalve; Carmen Ramos-Corrales; Ignacio Pastor; Carlos Santonja; María José Trujillo-Tiebas (223-225).
Ellis-van Creveld syndrome is an autosomal recessive disorder mainly characterized by a disproportionate limb dwarfism, chondroectodermal dysplasia, congenital heart disease, postaxial polydactyly, and dysplastic fingernails and teeth. Only 300 cases have been published worldwide. We report a 21-week fetus with rhizomelia and polydactyly detected. Gross photographs, radiologic studies and pathological study were performed leading to the clinico-pathological suspicion of EvC. DNA from fresh fetal tissue was extracted for sequencing the EVC and EVC2 genes. p.W215X and p.R677X mutations were identified in the EVC2 gene in the fetal sample. Parental sample analysis showed the p.W215X mutation to be inherited from the mother and the p.R677X mutation from the father. The clinical information is essential not only to arrive at a correct diagnosis in fetuses with pathologic ultrasound findings, but also to offer a proper genetic counseling to the parents and their relatives.► EvC syndrome: characterized by chondroectodermal dysplasia and postaxial polydactyly. ► Only 300 cases of EvC syndrome have been reported worldwide. ► This condition has been found to be associated with EVC and EVC2 mutations. ► All the clinical information is essential to arrive at a correct diagnosis in fetuses.
Keywords: Ellis-van Creveld syndrome; Polydactyly; EVC2 gene; Fetus; Clinico-pathological approach;

Contribution of complement factor H Y402H polymorphism to sudden sensorineural hearing loss risk and possible interaction with diabetes by Naoki Nishio; Masaaki Teranishi; Yasue Uchida; Saiko Sugiura; Fujiko Ando; Hiroshi Shimokata; Michihiko Sone; Hironao Otake; Ken Kato; Tadao Yoshida; Mitsuhiko Tagaya; Tatsuya Hibi; Tsutomu Nakashima (226-230).
Sudden sensorineural hearing loss (SSNHL) is one of the most common diseases encountered by otolaryngologists; however, the etiology is unclear. The aim of this study was to assess the association between SSNHL and polymorphism of complement factor H (CFH) Y402H, which is implicated in age-related macular degeneration. We conducted a case–control study, in which the cases were 72 SSNHL patients and the controls were 2161 residents selected randomly from the resident register. The odds ratio (OR) for SSNHL risk was determined using the additive-genetic model of CFH Y402H polymorphism. The OR for SSNHL risk was 1.788 (95% confidence interval [CI]: 1.008–3.172) with no adjustments and 1.820 (CI: 1.025–3.232) after adjusting for age and sex. Of the three lifestyle-related diseases hypertension, dyslipidemia, and diabetes, only diabetes was significantly associated with SSNHL risk. We classified both the controls and SSNHL patients into those with or without diabetes, and the OR for SSNHL risk was 6.326 (CI: 1.885–21.225) in diabetic subjects and 1.214 (CI: 0.581–2.538) in nondiabetic subjects. We conclude that CFH Y402H polymorphism and SSNHL risk are significantly related, and that diabetic CFH Y402H minor allele carriers may be susceptible to SSNHL.► We studied the association between SSNHL and CFH Y402H polymorphism. ► We conducted a case–control study. ► The cases were 72 SSNHL patients and the controls were 2161 residents. ► CFH Y402H polymorphism and SSNHL risk are significantly related. ► Diabetic CFH Y402H minor allele carriers may be susceptible to SSNHL.
Keywords: Complement factor H; Sudden sensorineural hearing loss; CFH Y402H; Diabetes; Polymorphism;

The families of TGF-β proteins are the most important growth factors in the ovary for growth and differentiation of early ovarian follicles. Three related oocyte-derived members of the transforming growth factor-β superfamily, namely GDF9, BMP15 and BMPR-IB have been shown to be essential for follicular growth and ovulation. The objective of the present study was to detect the incidence of mutation in intronic portion of BMP 15 gene in Corriedale and local Kashmir valley sheep breeds. Blood samples were collected from 85 ewes and genomic DNA was extracted using the modified phenol chloroform method. The quantity and quality of extracted DNA was examined using spectrophotometry and gel electrophoresis, respectively. A fragment with the size of 356 bp was amplified using polymerase chain reaction (PCR) with a pair of specific primers. The amplified PCR products were digested with Mph11031 restriction enzyme. In the presence of mutation at this locus, the Mph11031 enzyme cannot recognize the restriction site. However, here in the absence of mutations, the enzyme recognizes one restriction site and divides the amplified fragment into two fragments of 152 and 204 bp. The 356 bp fragment was also analyzed for polymorphism by SSCP technique. The results indicated two different banding patterns AA and AB for this fragment. Later on two different allelic forms A and B were confirmed by nucleotide sequencing. The 356 bp nucleotide sequence was subjected to alignment analysis and it was observed that sequence similarity of this fragment with that of other sheep and Jining grey goat was more than 97.8%. Phylogenetic analysis revealed that both designated A and B alleles as well as published sequence of sheep form a common cluster indicating their evolutionary closeness. The origin of Jining grey goat was located some distance away from the sheep. The overall frequencies of AA and AB genotypes were 0.79 and 0.21. The breed wise frequencies were 0.78 and 0.22 in Corriedale sheep and the frequencies in Kashmir valley sheep were 0.80 and 0.20 for AA and AB genotypes, respectively. The overall allelic frequencies of A and B alleles were 0.89 and 0.11 whereas allelic frequencies Corriedale sheep was 0.89 and 0.11 and that of Kashmir valley sheep were 0.90 and 0.10.► Molecular characterization of BMP15 gene in local Kashmir Valley Sheep. ► PCR-SSCP analysis to explore the polymorphism. ► Sequencing to confirm the two allelic variants of BMP15 gene. ► Phylogenetic analysis done on the basis of nucleotide sequencing. ► A new SNP in intronic region of BMP15 gene stands identified.
Keywords: BMP 15 gene; PCR-RFLP; PCR-SSCP; Polymorphism; Sheep;