封闭群斑马鱼的遗传生化标记研究

目的通过对AB、LF、ST（short tail,本地短尾群）三个封闭群斑马鱼的研究比较,建立封闭群斑马鱼的遗传生化标记。方法依照国标GB／T14927.1-2008的遗传操作规程优化实验条件,对三种群斑马鱼的7个遗传生化位点进行研究,以得到其遗传生化图谱。结果6-磷酸葡萄糖脱氢酶（Gpd）、过氧化氢酶（Ce）、苹果酸脱氢酶（Mod）、葡萄糖磷酸异构酶（Gpi）、酯酶（ES）位点表现出多态性。对三种群多态性位点进行卡方分析,Gpd、Gpi位点群间差异显著（P〈0.01）,Ce、Mod有群间差异（P〈0.05）。碱性磷酸酶（AKP）、乳酸脱氢酶（LDH）位点,各品系谱带较一致,未见同工酶多态性。结论Gpd、Ce、Mod、Gpi、ES可作为3种封闭群斑马鱼群间评价的生化标记,通过进一步研究,可建立封闭群斑马鱼的遗传标准。     

Wild Sex in Zebrafish: Loss of the Natural Sex Determinant in Domesticated Strains

Sex determination can be robustly genetic, strongly environmental, or genetic subject to environmental perturbation. The genetic basis of sex determination is unknown for zebrafish (Danio rerio), a model for development and human health. We used RAD-tag population genomics to identify sex-linked polymorphisms. After verifying this “RAD-sex” method on medaka (Oryzias latipes), we studied two domesticated zebrafish strains (AB and TU), two natural laboratory strains (WIK and EKW), and two recent isolates from nature (NA and CB). All four natural strains had a single sex-linked region at the right tip of chromosome 4, enabling sex genotyping by PCR. Genotypes for the single nucleotide polymorphism (SNP) with the strongest statistical association to sex suggested that wild zebrafish have WZ/ZZ sex chromosomes. In natural strains, “male genotypes” became males and some “female genotypes” also became males, suggesting that the environment or genetic background can cause female-to-male sex reversal. Surprisingly, TU and AB lacked detectable sex-linked loci. Phylogenomics rooted on D. nigrofasciatus verified that all strains are monophyletic. Because AB and TU branched as a monophyletic clade, we could not rule out shared loss of the wild sex locus in a common ancestor despite their independent domestication. Mitochondrial DNA sequences showed that investigated strains represent only one of the three identified zebrafish haplogroups. Results suggest that zebrafish in nature possess a WZ/ZZ sex-determination mechanism with a major determinant lying near the right telomere of chromosome 4 that was modified during domestication. Strains providing the zebrafish reference genome lack key components of the natural sex-determination system but may have evolved variant sex-determining mechanisms during two decades in laboratory culture.     

Effects of cis and trans regulatory variations on the expression divergence of heat shock response genes between yeast strains

Recently, two genome-wide association studies in Asia identified gene polymorphisms known as rs4488809, rs9816619 in TP63 and rs2131877, rs952481 in C3orf21. It has been proposed that these polymorphisms are susceptibility loci for non-small cell lung cancer (NSCLC) development among Japanese and Korean populations. We ask whether susceptibility to NSCLC is limited to the Chinese population or whether the environment also affects genetic polymorphisms. We conducted a matched case-control study to explore this question. Results show that polymorphism of TP63 was not associated with NSCLC development, whereas variant genotypes of C3orf21 were nominally associated with a reduced risk of lung adenocarcinoma (OR=0.619, 95% CI=0.390-0.976). These results strongly suggest that environmental agents interact with human genetic polymorphism independent of ethnic background. In addition, the C3orf21 gene may be a potential susceptibility marker for lung adenocarcinoma independent of ethnic background and environmental agents.     

Functional Assessment of Human Coding Mutations Affecting Skin Pigmentation Using Zebrafish

A major challenge in personalized medicine is the lack of a standard way to define the functional significance of the numerous nonsynonymous, single nucleotide coding variants that are present in each human individual. To begin to address this problem, we have used pigmentation as a model polygenic trait, three common human polymorphisms thought to influence pigmentation, and the zebrafish as a model system. The approach is based on the rescue of embryonic zebrafish mutant phenotypes by “humanized” zebrafish orthologous mRNA. Two hypomorphic polymorphisms, L374F in SLC45A2, and A111T in SLC24A5, have been linked to lighter skin color in Europeans. The phenotypic effect of a second coding polymorphism in SLC45A2, E272K, is unclear. None of these polymorphisms had been tested in the context of a model organism. We have confirmed that zebrafish albino fish are mutant in slc45a2; wild-type slc45a2 mRNA rescued the albino mutant phenotype. Introduction of the L374F polymorphism into albino or the A111T polymorphism into slc24a5 (golden) abolished mRNA rescue of the respective mutant phenotypes, consistent with their known contributions to European skin color. In contrast, the E272K polymorphism had no effect on phenotypic rescue. The experimental conclusion that E272K is unlikely to affect pigmentation is consistent with a lack of correlation between this polymorphism and quantitatively measured skin color in 59 East Asian humans. A survey of mutations causing human oculocutaneous albinism yielded 257 missense mutations, 82% of which are theoretically testable in zebrafish. The developed approach may be extended to other model systems and may potentially contribute to our understanding the functional relationships between DNA sequence variation, human biology, and disease.     

Influence of the -308 TNF-alpha and -174 IL-6 polymorphisms on lipid profile in Mexican subjects

Polymorphisms in the promoter region of several cytokine genes have been associated with differential cytokine production. Several reports indicate that polymorphisms in the tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) genes are associated with lipid abnormalities. The aim of this study was to identify the genotype frequencies for -308G/ATNF-alpha and -174G/CIL-6 polymorphisms in Mexican subjects and to determine the influence of both polymorphisms on serum lipid levels. Serum lipid concentrations were measured in 100 healthy Mexican subjects. Screening of the -308G/ATNF-alpha and -174G/CIL-6 polymorphisms was performed in all participants using PCR-RFLPs. Genotype frequency for TNF-alpha polymorphism was: 87% GG and 13% GA, whereas IL-6 polymorphism was: 77% GG and 23% GC. The polymorphism frequencies obtained in this study were significantly different to Caucasian populations. High serum levels of triglycerides and total cholesterol were associated with GG genotype of the -308 TNF-alpha polymorphism, as well as low HDL-c levels, but no association was found between the -174 IL-6 polymorphism and serum lipid concentrations. We observed a significant association of the -308 TNF-alpha polymorphism with lipid profile in Mexican subjects. Furthermore, the genotype distribution of -308 TNF-alpha and -174 IL-6 polymorphisms in Mexican Mestizo population similar to populations in different continents may be due to our genetic background influenced by the mixture of Spaniards, Indian and black genes.     

Coupled mutagenesis screens and genetic mapping in zebrafish

Forward genetic analysis is one of the principal advantages of the zebrafish model system. However, managing zebrafish mutant lines derived from mutagenesis screens and mapping the corresponding mutations and integrating them into the larger collection of mutations remain arduous tasks. To simplify and focus these endeavors, we developed an approach that facilitates the rapid mapping of new zebrafish mutations as they are generated through mutagenesis screens. We selected a minimal panel of 149 simple sequence length polymorphism markers for a first-pass genome scan in crosses involving C32 and SJD inbred lines. We also conducted a small chemical mutagenesis screen that identified several new mutations affecting zebrafish embryonic melanocyte development. Using our first-pass marker panel in bulked-segregant analysis, we were able to identify the genetic map positions of these mutations as they were isolated in our screen. Rapid mapping of the mutations facilitated stock management, helped direct allelism tests, and should accelerate identification of the affected genes. These results demonstrate the efficacy of coupling mutagenesis screens with genetic mapping.     

Failure to Replicate a Genetic Association May Provide Important Clues About Genetic Architecture

Replication has become the gold standard for assessing statistical results from genome-wide association studies. Unfortunately this replication requirement may cause real genetic effects to be missed. A real result can fail to replicate for numerous reasons including inadequate sample size or variability in phenotype definitions across independent samples. In genome-wide association studies the allele frequencies of polymorphisms may differ due to sampling error or population differences. We hypothesize that some statistically significant independent genetic effects may fail to replicate in an independent dataset when allele frequencies differ and the functional polymorphism interacts with one or more other functional polymorphisms. To test this hypothesis, we designed a simulation study in which case-control status was determined by two interacting polymorphisms with heritabilities ranging from 0.025 to 0.4 with replication sample sizes ranging from 400 to 1600 individuals. We show that the power to replicate the statistically significant independent main effect of one polymorphism can drop dramatically with a change of allele frequency of less than 0.1 at a second interacting polymorphism. We also show that differences in allele frequency can result in a reversal of allelic effects where a protective allele becomes a risk factor in replication studies. These results suggest that failure to replicate an independent genetic effect may provide important clues about the complexity of the underlying genetic architecture. We recommend that polymorphisms that fail to replicate be checked for interactions with other polymorphisms, particularly when samples are collected from groups with distinct ethnic backgrounds or different geographic regions.     

Human lymphocyte polymorphisms detected by quantitative two-dimensional electrophoresis.

A survey of 186 soluble lymphocyte proteins for genetic polymorphism was carried out utilizing two-dimensional electrophoresis of 14C-labeled phytohemagglutinin (PHA)-stimulated human lymphocyte proteins. Nineteen of these proteins exhibited positional variation consistent with independent genetic polymorphism in a primary sample of 28 individuals. Each of these polymorphisms was characterized by quantitative gene-dosage dependence insofar as the heterozygous phenotype expressed approximately 50% of each allelic gene product as was seen in homozygotes. Patterns observed were also identical in monozygotic twins, replicate samples, and replicate gels. The three expected phenotypes (two homozygotes and a heterozygote) were observed in each of 10 of these polymorphisms while the remaining nine had one of the homozygous classes absent. The presence of the three phenotypes, the demonstration of gene-dosage dependence, and our own and previous pedigree analysis of certain of these polymorphisms supports the genetic basis of these variants. Based on this data, the frequency of polymorphic loci for man is: P = 19/186 = .102, and the average heterozygosity is .024. This estimate is approximately 1/3 to 1/2 the rate of polymorphism previously estimated for man in other studies using one-dimensional electrophoresis of isozyme loci. The newly described polymorphisms and others which should be detectable in larger protein surveys with two-dimensional electrophoresis hold promise as genetic markers of the human genome for use in gene mapping and pedigree analyses.     

Genetic polymorphisms in lung disease: bandwagon or breakthrough?

The study of genetic polymorphisms has touched every aspect of pulmonary and critical care medicine. We review recent progress made using genetic polymorphisms to define pathophysiology, to identify persons at risk for pulmonary disease and to predict treatment response. Several pitfalls are commonly encountered in studying genetic polymorphisms, and this article points out criteria that should be applied to design high-quality genetic polymorphism studies.     

The role of surfactant in asthma

The study of genetic polymorphisms has touched every aspect of pulmonary and critical care medicine. We review recent progress made using genetic polymorphisms to define pathophysiology, to identify persons at risk for pulmonary disease and to predict treatment response. Several pitfalls are commonly encountered in studying genetic polymorphisms, and this article points out criteria that should be applied to design high-quality genetic polymorphism studies.     

Plasminogen Activator Inhibitor-2 Polymorphism Associates with Recurrent Coronary Event Risk in Patients with High HDL and C-Reactive Protein Levels

The objective of this work was to investigate whether fibrinolysis plays a role in establishing recurrent coronary event risk in a previously identified group of postinfarction patients. This group of patients was defined as having concurrently high levels of high-density lipoprotein cholesterol (HDL-C) and C-reactive protein (CRP) and was previously demonstrated to be at high-risk for recurrent coronary events. Potential risk associations of a genetic polymorphism of plasminogen activator inhibitor-2 (PAI-2) were probed as well as potential modulatory effects on such risk of a polymorphism of low-density lipoprotein receptor related protein (LRP-1), a scavenger receptor known to be involved in fibrinolysis in the context of cellular internalization of plasminogen activator/plansminogen activator inhibitor complexes. To this end, Cox multivariable modeling was performed as a function of genetic polymorphisms of PAI-2 (SERPINB, rs6095) and LRP-1 (LRP1, rs1800156) as well as a set of clinical parameters, blood biomarkers, and genetic polymorphisms previously demonstrated to be significantly and independently associated with risk in the study population including cholesteryl ester transfer protein (CETP, rs708272), p22phox (CYBA, rs4673), and thrombospondin-4 (THBS4, rs1866389). Risk association was demonstrated for the reference allele of the PAI-2 polymorphism (hazard ratio 0.41 per allele, 95% CI 0.20-0.84, p=0.014) along with continued significant risk associations for the p22phox and thrombospondin-4 polymorphisms. Additionally, further analysis revealed interaction of the LRP-1 and PAI-2 polymorphisms in generating differential risk that was illustrated using Kaplan-Meier survival analysis. We conclude from the study that fibrinolysis likely plays a role in establishing recurrent coronary risk in postinfarction patients with concurrently high levels of HDL-C and CRP as manifested by differential effects on risk by polymorphisms of several genes linked to key actions involved in the fibrinolytic process.     

Common MCL1 polymorphisms associated with risk of tuberculosis

MCL1 expression has been found to be up-regulated during infection with virulent Mycobacterium tuberculosis. We investigated the genetic polymorphisms in MCL1 as potential candidate gene for a host genetic study of clinical TB infection. We have sequenced exons and their boundaries of MCL1, including the 1.5 kb promoter region, to identify polymorphisms, and eight polymorphisms were identified. The genetic associations of polymorphisms in MCL1 with clinical TB patients (n=486) and normal controls (n=370) were analyzed. Using statistical analyses, one common promoter polymorphism (MCL1- 324C > A) which is absolutely linked with three other SNPs in the promoter and 3'UTR regions, were found to be significantly associated with increased risk of clinical TB disease. The frequency of the A-bearing genotype of -324C > A was higher in clinical TB patients than in normal controls (P=0.0008, OR= 1.68). Our findings suggest that polymorphisms in MCL1 might be one of genetic factors for the risk of clinical tuberculosis development.     

Entamoeba histolytica: genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene.

The varied organ tropisms and clinical presentations of infection by Entamoeba histolytica have stimulated interest in the role of parasite genetic diversity in virulence. We investigated genetic diversity among 54 E. histolytica isolates from Bangladesh by analyzing polymorphism in the serine-rich gene by nested PCR on DNA extracted from stool and liver aspirate pus. We detected both size and restriction site polymorphisms among the isolates within this endemic area. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded 25 distinct DNA banding patterns among the 42 stool isolates and an additional 9 distinct patterns among the 12 liver abscess isolates. Approximately half of the isolates had unique polymorphisms. Interestingly, the majority of E. histolytica from the liver had polymorphisms which were not present in intestinal isolates from the same geographic area. These data are consistent with the existence of genetic differences between E. histolytica which cause intestinal and those which cause hepatic disease. We conclude that there is genetic diversity within E. histolytica isolates from an endemic population as reflected in serine-rich E. histolytica protein gene polymorphism. The correlation of genetic differences with the pathogenic potential of E. histolytica strains and the implications of genetic diversity for the immunoprophylaxis of amebiasis require further study.     

Genetic polymorphisms of Fas (CD95) and FasL (CD178) in human longevity: studies on centenarians

Apoptosis plays a crucial role in immunosenescence, as also evidenced by the increased expression of Fas in lymphocytes from aged people. However, little is known about the genetic regulation of Fas and its ligand, FasL. We have studied their polymorphisms in 50 centenarians and 86 young donors living in Northern Italy. The first Fas polymorphism, at position -670, has in Caucasian a heterozigosity of 51%; the second, at -1377 position, has the wild type allele (G) with a very high frequency (83%) respect to the mutant allele. Genotype and allele distribution for both polymorphisms were similar in controls and centenarians. Similar results were found as far as two FasL polymorphisms (IVS2nt-124 and IVS3nt169) are concerned. On the whole, our data suggest that Fas and FasL polymorphisms, as well as their haplotypes, are unlikely to be associated with successful human longevity.     

Genetic polymorphism of interleukin-8 (IL-8) is associated with Helicobacter pylori-induced duodenal ulcer

BACKGROUND AND AIMS: Helicobacter pylori infection almost invariably causes chronic gastritis, but only a proportion of the infected subjects develop peptic ulcers. The local inflammation associated with H. pylori infection is characterized by an increased production of the proinflammatory cytokines IL-1-B, IL-6, IL-8 and TNF-alpha. Since such cytokine production is often determined by the genetic polymorphism of regions regulating cytokine gene expression, we investigated the relationship between TNF-alpha and IL-8 polymorphisms and the development of duodenal ulcer disease. We also sought a correlation between the promoter polymorphism of the lipopolysaccharide (LPS) receptor CD14 and the formation of peptic ulcer, because CD14 plays a crucial role in the initiation of the cytokine cascade. METHODS: Genomic DNA extracted from the peripheral blood of 69 patients with H. pylori-positive duodenal ulcer disease and 47 H. pylori-positive healthy controls was analyzed for TNF-alpha -308 promoter polymorphism by RFLP, and for IL-8 -251 polymorphism by ARMS. Genetic polymorphism within the promoter of the CD14 gene was identified using the LightCycler instrument via melting point analysis. RESULTS: No significant correlation could be revealed between the TNF-alpha and CD14 promoter polymorphisms and the clinical outcome of H. pylori infection. The IL-8 A/T heterozygote mutant variant was detected with a significantly higher frequency (65.22%) among the ulcer patients than among the healthy, H. pylori-positive blood donors (36.17%), while the frequency of the normal allelic genotype (TT) was significantly higher in the control group (44.6% vs 15.9%). CONCLUSION: Analysis of the genetic predisposition to enhanced cytokine production revealed a significant association only for the IL-8 polymorphism. This observation draws attention to the possible importance of IL-8 polymorphism as a genetic predisposing factor in the pathomechanism of H. pylori-induced duodenal ulcer disease, and to the relative protection from duodenal ulcer disease that is associated with the TT genotype.     

Identification of polymorphisms within the vascular endothelial growth factor (VEGF) gene: correlation with variation in VEGF protein production

Dysregulated vascular endothelial growth factor (VEGF) expression has been implicated as a major contributor to the development of a number of common disease pathologies. The aim of this study was to establish the extent of genetic variability within the VEGF gene and to determine whether this genetic variation influenced levels of VEGF protein expression. The promoter region and exon 1 of the VEGF gene were screened for polymorphisms using single-stranded conformation (SSCP) polymorphism analysis and direct PCR-sequencing. We identified 15 novel sequence polymorphisms most of which were rare. Eleven of these polymorphisms were single base substitutions, three were single base insertions and one was a two base deletion. Thirteen of the polymorphisms were located within the promoter and two in the 5' untranslated region (5'UTR) of the gene. We established PCR-RFLP typing systems for ten of the polymorphisms. For the two common polymorphisms at -460 and +405, we developed a combined sequence specific priming (SSP) PCR typing system to determine the cis/trans orientation of each allele and hence, ascertain haplotypes. A significant correlation was observed between lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cell (PBMC) VEGF protein production and genotype for the +405 polymorphism.     

Colour polymorphism is likely to be disadvantageous to some populations and species due to genetic architecture and morph interactions

Polymorphism describes two or more distinct, genetically determined, phenotypes that co‐occur in the same population, where the rarest morph is maintained at a frequency above the mutation rate (Ford 1945; Huxley 1955). In a recent opinion piece, we explored a new idea regarding the role of genetic architectures and morph interactions in colour polymorphisms and how this can negatively affect population performance (Bolton et al. 2015). In this issue of Molecular Ecology, Forsman (2016) thoroughly discusses the current evidence for polymorphisms enhancing population performance and critiques the validity of the definitions of polymorphism we use in our original paper. We respond by clarifying that the negative consequences of polymorphisms that we discussed are likely to be most pertinent in species that have a particular set of characteristics, such as strong sexual or social interactions between morphs and discrete genetic architectures. Although it was not our intention to redefine polymorphism, we do believe that there should be further discussion about refining or characterizing balanced polymorphisms with respect to the degree of morph sympatry, discreteness of traits and their underlying genetic architecture, and the types of selection that drive and maintain the variation. The latter describes whether polymorphism is primarily maintained by external factors such as predation pressure or internal factors such as interactions with members of the same species. The contribution of Forsman (2016) is useful to this discussion, and we hope that our exchange of opinions will inspire new empirical and theoretical ideas on the origin and maintenance of colour polymorphisms.     

DNA polymorphisms amplified by arbitrary primers are useful as genetic markers.

Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which amplify from one parent but not the other, are inherited in a Mendelian fashion and can be used to construct genetic maps in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.     

Predicting the functional consequences of non-synonymous single nucleotide polymorphisms: structure-based assessment of amino acid variation

We have developed a formalism and a computational method for analyzing the potential functional consequences of non-synonymous single nucleotide polymorphisms. Our approach uses a structural model and phylogenetic information to derive a selection of structure and sequence-based features serving as indicators of an amino acid polymorphim's effect on function. The feature values can be integrated into a probabilistic assessment of whether an amino acid polymorphism will affect the function or stability of a target protein. The method has been validated with data sets of unbiased mutations in the lac repressor and lysoyzyme. Applying our methodology to recent surveys of genetic variation in the coding regions of clinically important genes, we estimate that approximately 26-32 % of the natural non-synonymous single nucleotide polymorphisms have effects on function. This estimate suggests that a typical person will have about 6240-12,800 heterozygous loci that encode proteins with functional variation due to natural amino acid polymorphism.     

Genetic variation in RTN4 3'-UTR and susceptibility to cervical squamous cell carcinoma

Recent studies have suggested that RTN4 is a multifunctional gene, including inhibition of axonal regeneration, vascular remodeling, apoptosis, and tumor suppression. The TATC and CAA insertion/deletion polymorphisms of RTN4 3'-UTR have been linked to schizophrenia, depression, and dilated cardiomyopathy. To test whether these two polymorphisms are associated with cervical squamous cell carcinoma (CSCC), in this research, by using polymerase chain reaction-polyacrylamide gel electrophoresis, we determined the genotypes of the TATC and CAA polymorphisms in 336 CSCC patients and 450 unrelated control subjects. Allele frequencies of TATC and CAA polymorphisms were not significantly different between CSCC patients and control subjects (odds ratio [OR]=1.22, 95% confidence interval [CI]=0.98-1.50 for TATC; OR=0.95, 95% CI=0.76-1.18 for CAA). Decreased CSCC risk was associated with TATC polymorphism in a recessive model (OR=0.49, 95% CI=0.30-0.77), while no significant association was observed between CAA polymorphism and CSCC in different genetic models. Results of stratified analysis revealed that both TATC and CAA polymorphisms were associated with high clinical stage, and CAA polymorphism was also associated with positive parametrial invasion (OR=0.69, 95% CI=0.48-0.98). The present study provides evidence that TATC and CAA insertion/deletion polymorphisms are associated with CSCC, indicating that genetic variation in RTN4 3'-UTR contributes to the susceptibility to CSCC. It is necessary to confirm these findings in ethnically different populations and with a larger sample.