5' UTR CGG repeat expansion in GIPC1 is associated with oculopharyngodistal myopathy.

Oculopharyngodistal myopathy is a late-onset degenerative muscle disorder characterized by ptosis and weakness of the facial, pharyngeal, and distal limb muscles. A recent report suggested a non-coding trinucleotide repeat expansion in LRP12 to be associated with the disease. Here we report a genetic study in a Chinese cohort of 41 patients with the clinical diagnosis of oculopharyngodistal myopathy (21 cases from seven families and 20 sporadic cases). In a large family with 12 affected individuals, combined haplotype and linkage analysis revealed a maximum two-point logarithm of the odds (LOD) score of 3.3 in chromosomal region chr19p13.11-p13.2 and narrowed the candidate region to an interval of 4.5 Mb. Using a comprehensive strategy combining whole-exome sequencing, long-read sequencing, repeat-primed polymerase chain reaction and GC-rich polymerase chain reaction, we identified an abnormal CGG repeat expansion in the 5' UTR of the GIPC1 gene that co-segregated with disease. Overall, the repeat expansion in GIPC1 was identified in 51.9% independent pedigrees (4/7 families and 10/20 sporadic cases), while the repeat expansion in LRP12 was only identified in one sporadic case (3.7%) in our cohort. The number of CGG repeats was <30 in controls but >60 in affected individuals. There was a slight correlation between repeat size and the age at onset. Both repeat expansion and retraction were observed during transmission but somatic instability was not evident. These results further support that non-coding CGG repeat expansion plays an essential role in the pathogenesis of oculopharyngodistal myopathy.

Identification of gene mutations in patients with primary periodic paralysis using targeted next-generation sequencing.

BACKGROUND: Primary periodic paralysis is characterized by recurrent quadriplegia typically associated with abnormal serum potassium levels. The molecular diagnosis of primary PP previously based on Sanger sequencing of hot spots or exon-by-exon screening of the reported genes. METHODS: We developed a gene panel that includes 10 ion channel-related genes and 245 muscular dystrophy- and myopathy-related genes and used this panel to diagnose 60 patients with primary periodic paralysis and identify the disease-causing or risk-associated gene mutations. RESULTS: Mutations of 5 genes were discovered in 39 patients (65.0%). SCN4A, KCNJ2 and CACNA1S variants accounted for 92.5% of the patients with a genetic diagnosis. CONCLUSIONS: Targeted next-generation sequencing offers a cost-effective approach to expand the genotypes of primary periodic paralysis. A clearer genetic profile enables the prevention of paralysis attacks, avoidance of triggers and the monitoring of complications.

Comparing Mycobacterium tuberculosis genomes using genome topology networks.

BACKGROUND: Over the last decade, emerging research methods, such as comparative genomic analysis and phylogenetic study, have yielded new insights into genotypes and phenotypes of closely related bacterial strains. Several findings have revealed that genomic structural variations (SVs), including gene gain/loss, gene duplication and genome rearrangement, can lead to different phenotypes among strains, and an investigation of genes affected by SVs may extend our knowledge of the relationships between SVs and phenotypes in microbes, especially in pathogenic bacteria. RESULTS: In this work, we introduce a 'Genome Topology Network' (GTN) method based on gene homology and gene locations to analyze genomic SVs and perform phylogenetic analysis. Furthermore, the concept of 'unfixed ortholog' has been proposed, whose members are affected by SVs in genome topology among close species. To improve the precision of 'unfixed ortholog' recognition, a strategy to detect annotation differences and complete gene annotation was applied. To assess the GTN method, a set of thirteen complete M. tuberculosis genomes was analyzed as a case study. GTNs with two different gene homology-assigning methods were built, the Clusters of Orthologous Groups (COG) method and the orthoMCL clustering method, and two phylogenetic trees were constructed accordingly, which may provide additional insights into whole genome-based phylogenetic analysis. We obtained 24 unfixable COG groups, of which most members were related to immunogenicity and drug resistance, such as PPE-repeat proteins (COG5651) and transcriptional regulator TetR gene family members (COG1309). CONCLUSIONS: The GTN method has been implemented in PERL and released on our website. The tool can be downloaded from http://homepage.fudan.edu.cn/zhouyan/gtn/ , and allows re-annotating the 'lost' genes among closely related genomes, analyzing genes affected by SVs, and performing phylogenetic analysis. With this tool, many immunogenic-related and drug resistance-related genes were found to be affected by SVs in M. tuberculosis genomes. We believe that the GTN method will be suitable for the exploration of genomic SVs in connection with biological features of bacterial strains, and that GTN-based phylogenetic analysis will provide additional insights into whole genome-based phylogenetic analysis.

Evidence of positive selection at signal peptide region of interferon gamma.

Interferon gamma (IFNG) is a major cytokine and plays crucial roles in pathogen clearance. About the course of evolution of IFNG, it has been reported that IFNG is being subjected to adaptive selection, which is proved at the level of gene. Neighbor-joining method was used to reconstruct the phylogenetic tree of all IFNG protein-coding sequences. The pair-wise computation of Ka/Ks between every exon homologs, branch-specific model, and site-specific model of the likelihood method were performed to detect positive selection of IFNG. We reported, for the first time, that the signal peptide region of IFNG is under significant positive selection, evolving faster than other parts. We provide evidence at the level of individual exon and individual amino acid site that IFNG is under adaptive evolution, which establishes the basis for further researches about IFNG.

Evidence of positive selection at codon sites localized in the C-terminal peptide of ORC6.

Origin recognition complex 6 (Orc6) plays a central role in the initiation of DNA replication in all eukaryotic systems. The exact contribution of Orc6 to replication initiation has yet to be elucidated. Here, we analyzed the evolutionary dynamics of Orc6 in 15 vertebrates. Positive selection was detected in the region of exon 6 of the Orc6 gene. Site tests revealed a proportion of codon sites that displayed evidence of positive selection (ω > 1) within the coding sequences of the vertebrate Orc6 gene. Seven positively selected amino acid sites were identified and three were located in exon6. These results suggest that amino acid residues present in the middle region of the protein are more selectively constrained, whereas amino acid residues in the C-terminal peptide of the protein evolve at a faster rate, possibly because of heightened selective pressure during the course of evolution.

Hox genes from the parasitic flatworm Schistosoma japonicum.

Hox genes are characterized by a highly conserved peptide domain and contribute to antero-posterior axis patterning during embryogenesis. These genes have been widely studied in a variety of animal species due to their central role in evolutionary developmental biology. Based on the published genome assembly and unpublished re-sequencing project data, we present the first genome-wide characterization and comparative genomic analysis of the Hox gene family within Schistosoma japonicum. Eight Hox genes were identified and validated in our investigation. Phylogenetic analysis revealed that these genes are distributed among seven orthology groups of the Hox gene family. Our study further suggested that differences in the Lox5 gene copy number existed between the two closely related species, S. japonicum and Schistosoma mansoni. Semi-quantitative real-time polymerase chain reaction experiments revealed that Lox5 and Hox4 gene expression was high in the schistosomulum stage, and all four genes investigated showed highest expression within the eggs.

An intronic polymorphism rs2237062 in the CXCL14 gene influences HBV-related HCC progression in Chinese population.

CXCL14 (C-X-C motif chemokine ligand 14) is a conserved member of chemokine family and functions as a chemoattractant for multiplicate immunocytes. CXCL14 expression is constitutive in normal tissues, but absent in wide range of epithelial tumors. Many reports have claimed its important role in tumorigenesis and vascularization. An association between rs2237062 polymorphism and hepatocellular carcinoma (HCC) susceptibility was found in patients with chronic HCV infection in Japanese population. Here we analyzed, by using a polymerase chain reaction-ligation detection reaction (PCR-LDR), the polymorphism in 202 non-HCC patients with HBV infection, 361 HBV-related HCC patients and 407 healthy controls. The aim was to detect the possible association of this single-nucleotide polymorphism (SNP) with HBV-related HCC susceptibility and progression. However, no association was found between rs2237062 polymorphism and susceptibility to HBV infection or HBV-related HCC. Intriguingly, our stratification analysis revealed that HBV-related HCC patients in advanced phase (TNM-II-IV stage) had significantly higher C allele frequency at this polymorphism than patients at early stage (TNM-I stage) (33.5% vs. 25.7%), and its odds ratio reached 1.47 (95% CI 1.06-2.04, P = 0.021). These results suggest that the rs2237062 polymorphism in the CXCL14 gene might influence HBV-related HCC progression in Chinese population.

CTLA4 A49G polymorphism shows significant association with glioma risk in a Chinese population.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA4) A49G is a polymorphism that is extensively studied in various cancers. To investigate whether it is associated with the occurrence of glioma in Chinese patients, we performed a case-control research study with 670 patients and 680 controls. In this group, we found that the genotype at this locus is significantly associated with glioma risk (GG vs. AA: P = 0.045; GG + AG vs. AA: P = 0.013). In some subgroups, G allele carriers are significantly less represented. We also observed significant correlations between the polymorphism genotype and glioma risk in patients with WHO histologic stages. We conclude that CTLA4 A49G might be a potential clinical biomarker for distinguishing persons with a high risk for developing gliomas.

An intronic polymorphism in the corticotropin-releasing hormone receptor 2 gene increases susceptibility to HBV-related hepatocellular carcinoma in Chinese population.

Corticotropin-releasing hormone receptor 2 (CRHR2) plays a role in both the central nervous system (CNS) and the peripheral nervous system. CRHR2 together with its ligands, urocortins (Ucns) and corticotropin-releasing hormone (CRH), functions as a mediator of inflammatory response and inhibitor of angiogenesis. Recently, it has been reported to be expressed in many human cancers. An association between rs2267716 polymorphism in the CRHR2 gene and susceptibility to hepatocellular carcinoma (HCC) was found in patients with chronic hepatitis C virus (HCV) infection. In the present study we analyzed, using a polymerase chain reaction-ligation detection reaction (PCR-LDR), the rs2267716 polymorphism in 364 hepatitis B virus (HBV)-related HCC patients, 196 non-HCC patients with HBV infection, and 404 healthy controls. The aim was to detect the possible association of this single-nucleotide polymorphism (SNP) with susceptibility to HBV-related HCC. Significant differences of rs2267716 allele were detected between HBV-related HCC patients and healthy controls (OR = 1.55, 95% CI 1.13-2.15, P = 0.007) or non-HCC patients with HBV infection (OR = 1.61, 95% CI 1.13-2.31, P = 0.009). These results suggest that the rs2267716 polymorphism in the CRHR2 gene might influence the risk of developing HCC in patients with HBV infection in Chinese population.

CTLA-4 +49A>G polymorphism is associated with the risk but not with the progression of colorectal cancer in Chinese.

PURPOSE: Colorectal cancer (CRC) is one of the most common malignancies in the world and a multipathway disease. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a potent immunoregulatory molecule that suppresses antitumor response by down-regulating T-cell activation. The most studied +49A>G polymorphism of CTLA-4 gene has been associated with several autoimmune or cancer diseases. Our aim was to investigate the association between this genetic variant and the risk as well as progression of colorectal cancer in Chinese. METHODS: We conducted a case-control study of 124 colorectal cancer cases and 407 healthy controls. DNA was extracted from blood specimens, and +49A>G polymorphism in the CTLA-4 gene was genotyped by polymerase chain reaction-ligation detection reaction (PCR-LDR). RESULTS: In our study group, the frequency of AG or GG or carrying at least one G allele at position +49 was significantly different in colorectal cancer patients and the control group, indicating that the risk of CRC was significantly higher among subjects with the AG or GG genotype or carrying at least one G allele at position +49 than among the subjects with the AA genotype. However, we observed no association between CTLA-4 +49A>G polymorphism and the progression of CRC. Interestingly, the CTLA-4 +49A allele was in non-significantly higher numbers in CRC patients with distant metastasis. CONCLUSIONS: Our results suggested that CTLA-4 +49A>G polymorphism was associated with an increased risk of colorectal cancer, but this polymorphism did not play an important role in the progression of CRC in Chinese.

Electromyographic Features in a Chinese Cohort With Hereditary Skeletal Muscle Channelopathies.

PURPOSE: Hereditary skeletal muscle channelopathies are characterized by muscle stiffness and/or periodic muscle weakness because of different gene mutations. The objective of this study was to investigate the clinical and electromyographic phenotypes in Chinese patients with different skeletal ion channel mutations. METHODS: The electromyographic results of 61 Chinese patients with skeletal muscle channelopathies were retrospectively reviewed and the differential features were characterized. RESULTS: Myotonic discharges were in patients with chloride voltage-gated channel 1 and sodium voltage-gated channel alpha subunit 4 mutations. Subclinical myotonia was identified in four patients with hypokalemic periodic paralysis because of sodium voltage-gated channel alpha subunit 4 mutations. Patients with potassium voltage-gated channel subfamily J member 2 mutations had an early decline after exercise (5.7 ± 4.9 minutes) and patients with calcium voltage-gated channel subunit alpha 1S mutations have a relatively lower baseline amplitude (4.6 ± 2 mV). Specific patterns were characterized in patients with Becker disease and paramyotonia congenital after short exercise. CONCLUSIONS: Myotonic discharges help to discriminate chloride and sodium from other channelopathies. Early decline and low baseline compound motor action potential amplitude in long exercise test are significant in patients with potassium voltage-gated channel subfamily J member 2 and calcium voltage-gated channel subunit alpha 1S mutations, respectively. Electromyographic patterns in the electromyography study and exercise test may help in better providing the comprehensive picture for patients with primary skeletal muscle channelopathies.

Normoxic induction of cerebral HIF-1alpha by acetazolamide in rats: role of acidosis.

Acetazolamide has been recognized as an effective treatment for acute mountain sickness. The efficacy of acetazolamide is related to metabolic acidosis, which promotes chemoreceptors to respond to hypoxic stimuli at altitude. In this study, adult male Sprague-Dawley rats were treated with acetazolamide (100mg/kg or 50mg/kg, I.P.) for 3 days. Primary cultured cortical neurons and PC12 cell lines were exposed to acidosis-permissive (pH 6.5) or standard (pH 7.2) media for 20h. HIF-1alpha and its target genes were assayed by Western blot, real-time PCR, HIF-1 DNA-binding assay and chloramphenicol acetyltransferase reporter gene assay. HIF-1alpha protein level and HIF-1 DNA-binding activities were increased in cerebral cortices of rats treated with acetazolamide. Moreover, the mRNA levels of erythropoietin, vascular endothelial growth factor, and glucose transporter-1 also increased. The HIF-1alpha protein level and activity of HIF-driven chloramphenicol acetyltransferase reporters of cortical neurons and PC12 cells treated with acidosis media were significantly enhanced. We conclude that the normoxic induction of HIF-1alpha and HIF-1 mediated genes by acetazolamide may mediate the effect of acetazolamide in the reduction of symptoms of acute mountain sickness.

Transcriptome profiling and pathway analysis of the effects of mono-(2-ethylhexyl) phthalate in mouse Sertoli cells.

Phthalates are confirmed to have toxic effects on the reproductive system and are likely to have further damaging actions in humans. The present study explored the molecular mechanisms of the toxic effect of mono-(2-ethylhexyl) phthalate (MEHP) on mouse Sertoli cells. Cell apoptosis and proliferation assays were used to assess the effects of MEHP on the TM4 Sertoli cell line derived from mouse testes. TM4 cells were treated with two doses of MEHP or left untreated as a control group, followed by RNA extraction and analysis using high-throughput transcriptome sequencing technology. The gene expression profile obtained was then subjected to a bioinformatics analysis to explore the molecular mechanisms of reproductive toxicity. The results revealed that 528 and 269 genes were upregulated in the high- and low-dose MEHP groups of cells compared with the control group, while 148 and 173 genes were downregulated. Gene ontology (GO) analysis indicated that the differently expressed genes were associated with the GO term 'extracellular region' of the cellular component domain in the high and low MEHP groups. Compared with the control group, eight common pathway changes were identified in the high- and low-dose MEHP groups, including 'terpenoid backbone biosynthesis'. Reverse transcription-quantitative polymerase chain reaction analysis was used to validation, and hermetic effects were observed for certain genes. These results provide an important basis and experimental data for further research into the mechanisms of phthalate-induced toxicity.

Co-evolutionary analysis of insulin/insulin like growth factor 1 signal pathway in vertebrate species.

Insulin/insulin like growth factor 1 signaling pathway plays an essential role on the regulation of variant, interrelated and fundamental processes, such as metabolism, growth, reproduction and aging. This pathway is highly conserved during evolution. We analyzed eight vertebrate species' insulin/insulin like growth factor 1 pathway and examined the co-evolutionary relationship between different protein components by quantifying similarity of phylogenetic trees. The collected species include Takifugu rubripes, Danio rerio, Xenopus laevis, Gallus gallus, Mus musculus, Rattous norvegicus, Pan troglodytes and Homo sapiens. Results show that interacting proteins in this pathway share highly co-evolutionary relationship while contrarily the proteins without interaction have low co-evolutionary relationship. We also predict some receptor and ligand partners enjoy highly binding potential in corresponding species' pathway.

Cloning and characterization of a novel splice variant of human U2AF1L3 gene.

Pre-mRNA splicing allows individual genes to produce multiple protein isoforms with diverse functions. Recognition of functional splice sites in pre-mRNAs is very important in this splicing process and requires some protein auxiliary factors such as U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF35, encoded by U2AF1). By its RNA binding domains, U2AF35 interacts with U2AF65 to bind 3' splice site of pre-mRNA and initiates splicing. Another protein, which is named as U2AF1-like3 (U2AF1L3), shows high similarity with U2AF35 and may have related function in pre-mRNA splicing. Here, we report a splice variant of U2AF1L3, which is 767 bp in length and has an open reading frame (ORF) coding a predicted 181 amino acids protein. Reverse transcription-PCR (RT-PCR) shows that this isoform has different expression pattern with U2AF1L3 and is highly expressed in heart, brain and lung.

Cloning and characterization of a novel splice variant of human Rab18 gene (RAB18).

Rab GTPase proteins are a kind of small GTP-binding proteins, which functions mainly focus on regulating interacellular trafficking pathways during vesicular transport. To date, 60 distinct human RAB proteins have been identified. RAB18 gene is discovered from endothelial cells. Its function is considered as endosomes and plasma membrane recycling. Research indicates RAB18 may relate to inflammation and some kinds of tumor. Here we report a splice variant of RAB18, which is 2571 bp in length and has an open reading frame coding a predicted 235 amino-acids protein. RT-PCR shows that the cDNA has different expression pattern with RAB18 and is highly expressed in testis.

Signatures of positive selection at hemopexin (PEX) domain of matrix metalloproteinase-9 (MMP-9) gene.

Matrix metalloproteinases-9 (MMP-9) is an important cancer-associated, zinc-dependent endopeptidase. To investigate the natural selection hypothesis of MMP-9, the orthologous sequences from 12 vertebrates were compared and a molecular evolution analysis was performed. Results suggest that amino acid residues present in the middle region of the protein are more selectively constrained, whereas amino acid residues in the C-terminal region of the MMP-9 protein including exon 13 showed lowest conservation level in non-primate species, suggesting that it is an exon with fast evolving rate compared to the others analyzed. InterProScan analysis shows that exon 13 was located in hemopexin (PEX) domain of MMP-9. Positive selection was detected in PEX domain of MMP-9 protein between human and other species, which indicates that selective pressure may play a role in shaping the function of MMP-9 in the course of evolution.

rs17501976 polymorphism of CLDN1 gene is associated with decreased risk of colorectal cancer in a Chinese population.

The purpose of this study was to determine the relationship between polymorphisms in Claudin-1 (CLDN1) and the risk of colorectal cancer in a Chinese population. In this study, a case-control study was conducted in which polymorphisms in CLDN1 were analyzed in 50 patients with colorectal cancer (CRC) and 50 healthy individuals as controls. No rs16865344 and rs17429833 polymorphism were found among all analyzed samples. For the rs17501976 polymorphism, the TC genotype (OR = 0. 41, 95% CI = 0.18-0.91, and P = 0.045) was closely associated with the risk of colorectal cancer compared with the more common TT genotype. And the TC + CC genotypes (OR = 0.41, 95% CI = 0.18-0.91, and P = 0.045) were also significantly associated with the risk of CRC compared with the TT genotype. However, a C > T change of the rs17501976 polymorphism did not show a difference in transcription factor binding to the promoter region of CLDN1. For rs12696600 polymorphism, no significant difference was found in colorectal cancer risk between cases and controls in corresponding genotypes. Collectively, our data suggest that rs17501976 polymorphism significantly associated with a decreased susceptibility to CRC in a Chinese population.

A Comprehensive Analysis of the Transcriptomes of Marssonina brunnea and Infected Poplar Leaves to Capture Vital Events in Host-Pathogen Interactions.

BACKGROUND: Understanding host-pathogen interaction mechanisms helps to elucidate the entire infection process and focus on important events, and it is a promising approach for improvement of disease control and selection of treatment strategy. Time-course host-pathogen transcriptome analyses and network inference have been applied to unravel the direct or indirect relationships of gene expression alterations. However, time series analyses can suffer from absent time points due to technical problems such as RNA degradation, which limits the application of algorithms that require strict sequential sampling. Here, we introduce an efficient method using independence test to infer an independent network that is exclusively concerned with the frequency of gene expression changes. RESULTS: Highly resistant NL895 poplar leaves and weakly resistant NL214 leaves were infected with highly active and weakly active Marssonina brunnea, respectively, and were harvested at different time points. The independent network inference illustrated the top 1,000 vital fungus-poplar relationships, which contained 768 fungal genes and 54 poplar genes. These genes could be classified into three categories: a fungal gene surrounded by many poplar genes; a poplar gene connected to many fungal genes; and other genes (possessing low degrees of connectivity). Notably, the fungal gene M6_08342 (a metalloprotease) was connected to 10 poplar genes, particularly including two disease-resistance genes. These core genes, which are surrounded by other genes, may be of particular importance in complicated infection processes and worthy of further investigation. CONCLUSIONS: We provide a clear framework of the interaction network and identify a number of candidate key effectors in this process, which might assist in functional tests, resistant clone selection, and disease control in the future.

Molecular cloning and characterization of a novel human BTBD8 gene containing double BTB/POZ domains.

BTB/POZ domain is an evolutionarily conserved protein-protein interaction domain often found in developmentally regulated transcription factors. Previous studies have shown that many additional conserved motifs have been found in association with BTB/POZ domain, including kelch repeats, zinc finger domains, FYVE fingers and ankyrin repeats. Here we report a novel human gene containing double BTB/POZ domains, named BTBD8 in agreement with the HUGO Nomenclature Committee. The cDNA sequence contains an open reading frame of 918 bp encoding a putative protein of 305 amino acid residues with a predicted molecular mass of 34.6 kDa. Protein pattern analysis shows that it contains double BTB/POZ domains. Weak expression was detected in adult brain and prostate of the 16 adult tissues examined, while it had a more abundant expression pattern in human fetal brain. The expression pattern of BTBD8 shows it may have a function related to brain development.