Detection of novel genetic markers by mismatch analysis.

Chemical mismatch detection has been used to identify previously unknown genomic sequence variations that represent a new source of markers for genetic analysis. The approach detects all types of sequence changes, and therefore overcomes the limitation of restriction analysis, which identifies only a small fraction of the available sequence variations. Three new markers identified at the 3' end of the human dystrophin gene result from variable numbers of exact tandem repeats of 4bp (two examples) or 5bp (one example). None of these would have been detected as restriction fragment length polymorphisms by established procedures.     

Mutations in complement regulatory proteins predispose to preeclampsia: a genetic analysis of the PROMISSE cohort

Background

Pregnancy in women with systemic lupus erythematosus (SLE) or antiphospholipid antibodies (APL Ab)—autoimmune conditions characterized by complement-mediated injury—is associated with increased risk of preeclampsia and miscarriage. Our previous studies in mice indicate that complement activation targeted to the placenta drives angiogenic imbalance and placental insufficiency.

Methods and Findings

We use PROMISSE, a prospective study of 250 pregnant patients with SLE and/or APL Ab, to test the hypothesis in humans that impaired capacity to limit complement activation predisposes to preeclampsia. We sequenced genes encoding three complement regulatory proteins—membrane cofactor protein (MCP), complement factor I (CFI), and complement factor H (CFH)—in 40 patients who had preeclampsia and found heterozygous mutations in seven (18%). Five of these patients had risk variants in MCP or CFI that were previously identified in atypical hemolytic uremic syndrome, a disease characterized by endothelial damage. One had a novel mutation in MCP that impairs regulation of C4b. These findings constitute, to our knowledge, the first genetic defects associated with preeclampsia in SLE and/or APL Ab. We confirmed the association of hypomorphic variants of MCP and CFI in a cohort of non-autoimmune preeclampsia patients in which five of 59 were heterozygous for mutations.

Conclusion

The presence of risk variants in complement regulatory proteins in patients with SLE and/or APL Ab who develop preeclampsia, as well as in preeclampsia patients lacking autoimmune disease, links complement activation to disease pathogenesis and suggests new targets for treatment of this important public health problem.

Study Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00198068","term_id":"NCT00198068"}}NCT00198068 Please see later in the article for the Editors'' Summary     

Biochemical markers in the pathogenesis of preeclampsia: novel link between placental growth factor and interleukin-6

Molecular and Cellular Biochemistry - Preeclampsia (PE) is a multisystem disorder of pregnancy characterized by sudden onset of hypertension and proteinuria. The appearance and diagnosis of the...     

Proteomic analysis of human placental syncytiotrophoblast microvesicles in preeclampsia

Background

Placental syncytiotrophoblast microvesicles (STBM) are shed into the maternal circulation during normal pregnancy. STBM circulate in significantly increased amounts in preeclampsia (PE) and are considered to be among contributors to the exaggerated proinflammatory, procoagulant state of PE. However, protein composition of STBM in normal pregnancy and PE remains unknown. We therefore sought to determine the protein components of STBM and whether STBM protein expressions differ in preeclamptic and normal pregnancies.Patients with PE (n = 3) and normal pregnant controls (n = 6) were recruited. STBM were prepared from placental explant culture supernatant. STBM proteins were analyzed by a combination of 1D Gel-LC-MS/MS. Protein expressions levels were quantified using spectral counts and validated by immunohistochemistry.

Results

Over 400 proteins were identified in the STBM samples. Among these, 25 proteins were found to be differentially expressed in preeclampsia compared to healthy pregnant controls, including integrins, annexins and histones.

Conclusion

STBM proteins include those that are implicated in immune response, coagulation, oxidative stress, apoptosis as well as lipid metabolism pathways. Differential protein expressions of STBM suggest their pathophysiological relevance in PE.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-40) contains supplementary material, which is available to authorized users.     

Development of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae

Key message

We develop a set of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae.

Abstract

Being evolutionary conserved, single-copy orthologous (COSII) genes are particularly useful in comparative mapping and phylogenetic investigation among species. In this study, we identified 2,684 COSII genes based on five sequenced Poaceae genomes including rice, maize, sorghum, foxtail millet, and brachypodium, and then developed 1,072 COSII markers whose transferability and polymorphism among five bamboo species were further evaluated with 46 pairs of randomly selected primers. 91.3 % of the 46 primers obtained clear amplification in at least one bamboo species, and 65.2 % of them produced polymorphism in more than one species. We also used 42 of them to construct the phylogeny for the five bamboo species, and it might reflect more precise evolutionary relationship than the one based on the vegetative morphology. The results indicated a promising prospect of applying these markers to the investigation of genetic diversity and the classification of Poaceae. To ease and facilitate access of the information of common interest to readers, a web-based database of the COSII markers is provided (http://www.sicau.edu.cn/web/yms/PCOSWeb/PCOS.html).     

A genetic map of Blumeria graminis based on functional genes, avirulence genes, and molecular markers

A genetic map of the powdery mildew fungus, Blumeria graminis f. sp. hordei, an obligate biotrophic pathogen of barley, is presented. The linkage analysis was conducted on 81 segregating haploid progeny isolates from a cross between 2 isolates differing in seven avirulence genes. A total of 359 loci were mapped, comprising 182 amplified fragment length polymorphism markers, 168 restriction fragment length polymorphism markers including 42 LTR-retrotransposon loci and 99 expressed sequence tags (ESTs), all the seven avirulence genes, and a marker closely linked to the mating type gene. The markers are distributed over 34 linkage groups covering a total of 2114 cM. Five avirulence genes were found to be linked and mapped in clusters of three and two, and two were unlinked. The Avr(a6) gene was found to be closely linked to markers suitable for a map-based cloning approach. A linkage between ESTs allowed us to demonstrate examples of synteny between genes in B. graminis and Neurospora crassa.     

Comparative analysis of genetic relationships in barley based on RFLP and RAPD markers

Genetic relationships have seldom been analyzed with different types of molecular markers in order to compare the information provided by each marker class. We investigated genetic relationships among nine barley cultivars using separate cluster analyses based on restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs). Genomic DNA restricted with three enzymes and hybridized with 68 probes revealed 415 RFLPs (74.2% of all bands). Among the 128 primers used for RAPD analysis, 100 provided a reproducible profile, 89 of which revealed 202 polymorphic and 561 monomorphic bands (26.5 and 73.5%, respectively). A nonrandom distribution of 62 RAPDs with a tendency to cluster near centromeric regions was produced when these RAPDs were mapped using 76 doubled-haploid lines derived from a cross between two of the nine cultivars. The correlation between the RFLP and RAPD similarity matrices computed for the 36 pairwise comparisons among the nine cultivars was equal to 0.83. The dendrograms obtained by cluster analyses of the RFLP and RAPD data differed. These results indicate that in barley the information provided by RFLPs and RAPDs is not equivalent, most likely as a consequence of the fact that the two marker classes explore, at least in part, different portions of the genome.     

Leakiness of genetic markers and susceptibility to post-plating mutagenesis inEscherichia coli

WhenEscherichia coli strain AB1157 is subjected to starvation for threonine or leucine on solid media, threonine-independent or leucine-independent colonies continue to emerge for several days after plating. This process is strongly streptomycin dependent. Under identical conditions arginine-independent colonies do not arise when arginine starvation is imposed. Since thethr1 andleuB6 alleles of AB1157 could be classified as ‘leaky’ while theargE3 allele cannot be so classified, there seems to be a correlation between leakiness of mutant genetic markers and post-plating mutagenesis which counters the effect of the mutations. Some of the threonine-independent variants acquired the ability to increase the leakiness of otherwise nonleaky markers such asargE3 and permit development of arginine independence in arecA-dependent,lexA-independent manner. I show that these variants harbour a mutation, tentatively namedadi (adaptation inducer), at around 72 min on the genetic map, and that theadi mutation increases the intrinsic leakiness of alacZ (ochre) mutation, perhaps by enhanced translational error. These observations are discussed in relation to the phenomenon of ‘adaptive’ mutagenesis, its possible mechanism, and its specificity.     

Genetics of preeclampsia: an approach to genetic linkage studies

Due to its high prevalence during pregnancies, preeclampsia is considered an important public health problem. Many investigators agree in that its expression is related to the interaction between genetic and environmental factors. Many studies have searched for genetic factors, attempting to identify chromosomal regions or candidate genes whose variants may be related to high preeclampsia susceptibility. Several studies have associated a number of susceptibility genes to preeclampsia, but the results have not been replicated consistently in all populations. Mapping of genes and chromosomal regions by linkage analysis has located potential markers on chromosomes 2 and 4. Identification of the genes located in these candidate regions will pinpoint the genetic risk factors, will lead to a better understanding of the syndrome, and will provide clues for its prevention and treatment.     

Association analysis,genetic diversity and structure analysis of tobacco based on AFLP markers

Knowledge in the area of genetic diversity could aid in providing useful information in the selection of material for breeding such as hybridization programs and quantitative trait loci mapping. To this end, 50 Nicotiana tabacum genotypes were genotyped with 21 primer combination of amplified fragment length polymorphism (AFLP). A total of 480 unambiguous DNA fragments and 373 polymorphic bands were produced with an average of 17.76 per primer combination. Also, the results revealed high polymorphic rate varing from 52.63 to 92.59 %, demonstrating that AFLP technique utilized in this research can be a powerful and valuable tool in the breeding program of N. tabacum. Cluster analysis based on complete linkage method using Jaccard’s genetic distance, grouped the 50 tobacco genotypes into eight clusters including three relatively big clusters, one cluster including Golden gift, Burly 7022 and Burly Kreuzung, one cluster consisting of two individuals (Pereg234, R9) and three single-member clusters (Pennbel69, Coker176 and Budisher Burley E), Recent genotypes showed high genetic distance from other genotypes belonging to cluster I and II. Association analysis between seven important traits and AFLP markers were performed using four statistical models. The results revealed the model containing both the factors, population structure (Q) and general similarity in genetic background arising from shared kinship (K), reduces false positive associations between markers and phenotype. According to the results nine markers were determined that could be considered to be the most interesting candidates for further studies.     

The metabolome of human placental tissue: investigation of first trimester tissue and changes related to preeclampsia in late pregnancy

Unique biochemical and physical challenges to both mother and fetus are observed during human pregnancy, and the placenta plays an important role in protecting the fetus and supporting its development. Consequently, many pregnancy complications are associated with altered placental biochemistry and structure. Here we have further developed a combination of analytical tools for determining the tissue metabolome of placental tissue by applying a methanol/water/chloroform extraction method followed by analysis of the polar fraction (methanol/water) using GC?CToF?CMS and of the non-polar fraction (chloroform) using UPLC?CLTQ?COrbitrap?CMS. This combination maximises the number of different metabolites detected and is the first holistic investigation of placental tissue applying UPLC?CMS. Placental tissue differs between early and late first trimester pregnancies in that the developing placenta is exposed to significantly different oxygen tensions and undergoes a change from histiotrophic to haemotrophic nutrition. Application of these metabolomic methods detected 156 unique and chemically identified metabolites that showed statistically significant differences (P?<?0.05). These included changes in di- and triglycerides, phospholipids, sphingolipids, fatty acids and fatty acid carnitines. This is the first metabolomics study to identify these changes that potentially show the initiation or switch to fatty acid beta-oxidation for mitochondrial ATP production. A separate study showed a small number of changes that were related to the position of sampling of the placental tissue and to the type of delivery from pregnancy. This result indicates that variations associated with sampling position and delivery type are small compared to between-subject variation. However, the authors recommend robust experimental design which may include sampling from the same position of the placenta and from the same delivery type. When comparing tissue from term-uncomplicated pregnancies with those exhibiting preeclampsia at term, 86 unique and chemically identified metabolites showed statistically significant differences (P?<?0.05). Potential changes in metabolism operating in the mitochondria, in vitamin D metabolism and in oxidative and nitrative stress were observed. These proof-of-principle studies demonstrate the sensitivity of placental tissue metabolomics to define changes related to alterations in environment and perfusion and related to diseases of pregnancy including preeclampsia. Data are available on request.     

中国人群中抗结核药物引发肝损伤的易感基因标记研究

为系统性研究中国人群中抗结核药物引发肝损伤的易感基因标记,本研究以41例抗结核药物引发肝损伤的病人和39例健康对照为研究对象,采用Haloplex捕获测序的方法对其基因组中药物代谢、转运和免疫相关通路的109个基因进行靶向测序。用Plink软件对DNA突变位点与肝损伤的发生进行关联分析,以千人基因组计划东亚人群作为对照组,对显著性位点进行验证,并用SIFT和Polyphen2软件对预测显著关联的位点进行功能预测。结果发现UGT1A4 rs2011404(X^2=4.6809,P=0.0305)是抗结核药物引发的肝损伤的易感基因标记,且rs2011404突变可能引起UGT1A4蛋白的功能障碍。本研究为临床上对抗结核药物的合理用药提供了有益的参考。     

Ethical implications of genetic analysis of individual susceptibility to diseases

Ethics can be regarded as a reflection or reconsideration of existing moral codes in the search of good and goes beyond moral conduct. This means that ethics is a never-ending process, which in science must develop with the development of science itself. Thus, the process of seeking better ethics is as integral within science as the development of new methods. Along these lines of thought it can be argued that (1) poor science cannot be ethically sound, (2) every scientist has a personal responsibility to develop ethics in his area of expertise, (3) the development of solid ethical background in science requires education in ethics as well as in methodology and scientific thinking and (4) research ethics cannot develop in solitude, but needs input from other scientists, other fields (including philosophy) and society. Several burning questions can be identified within genetic analysis for individual susceptibility. These ethical aspects can be viewed from three different perspectives: practice of research, patient/research subject personally and long-term implications in society. This paper tries more to awaken thoughts than give clear answers.     

Detection and analysis of genetic variation in Hordeum spontaneum populations from Israel using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to analyse genetic diversity within and between Hordeum spontaneum populations sampled from Israel. Nei's index of genetic differentiation was used to partition diversity into within and between population components. Fifty-seven per cent of the variation detected was partitioned within 10 H. spontaneum populations. Using principal component and multiple regression analysis, part of the variation detected between populations was seen to be associated with certain ecogeographical factors. Fifty-eight per cent of the distribution of the phenotypic frequencies of three RAPD phenotypes detected using a single primer in 20 H. spontaneum populations could be accounted for by four ecogeographical variables, suggesting adaptive variation at certain RAPD loci.     

A novel approach to locate Phytophthora infestans resistance genes on the potato genetic map

Mapping resistance genes is usually accomplished by phenotyping a segregating population for the resistance trait and genotyping it using a large number of markers. Most resistance genes are of the NBS-LRR type, of which an increasing number is sequenced. These genes and their analogs (RGAs) are often organized in clusters. Clusters tend to be rather homogenous, viz. containing genes that show high sequence similarity with each other. From many of these clusters the map position is known. In this study we present and test a novel method to quickly identify to which cluster a new resistance gene belongs and to produce markers that can be used for introgression breeding. We used NBS profiling to identify markers in bulked DNA samples prepared from resistant and susceptible genotypes of small segregating populations. Markers co-segregating with resistance can be tested on individual plants and directly used for breeding. To identify the resistance gene cluster a gene belongs to, the fragments were sequenced and the sequences analyzed using bioinformatics tools. Putative map positions arising from this analysis were validated using markers mapped in the segregating population. The versatility of the approach is demonstrated with a number of populations derived from wild Solanum species segregating for P. infestans resistance. Newly identified P. infestans resistance genes originating from S. verrucosum, S. schenckii, and S. capsicibaccatum could be mapped to potato chromosomes 6, 4, and 11, respectively.     

Resistance of the target islet tissue to autoimmune destruction contributes to genetic susceptibility in Type 1 diabetes

   

Type 1 diabetes occurs when self-reactive T lymphocytes destroy the insulin-producing islet β cells of the pancreas. The defects causing this disease have often been assumed to occur exclusively in the immune system. We present evidence that genetic variation at the Idd9 diabetes susceptibility locus determines the resilience of the targets of autoimmunity, the islets, to destruction. Susceptible islets exhibit hyper-responsiveness to inflammatory cytokines resulting in enhanced cell death and increased expression of the death receptor Fas. Fas upregulation in β cells is mediated by TNFR2, and colocalization of TNFR2 with the adaptor TRAF2 in NOD β cells is altered. TNFR2 lies within the candidate Idd9 interval and the diabetes-associated variant contains a mutation adjacent to the TRAF2 binding site. A component of diabetes susceptibility may therefore be determined by the target of the autoimmune response, and protective TNFR2 signaling in islets inhibit early cytokine-induced damage required for the development of destructive autoimmunity.     

Multivariate analysis of noise in genetic regulatory networks

Stochasticity is an intrinsic property of genetic regulatory networks due to the low copy numbers of the major molecular species, such as, DNA, mRNA, and regulatory proteins. Therefore, investigation of the mechanisms that reduce the stochastic noise is essential in understanding the reproducible behaviors of real organisms and is also a key to design synthetic genetic regulatory networks that can reliably work. We use an analytical and systematic method, the linear noise approximation of the chemical master equation along with the decoupling of a stoichiometric matrix. In the analysis of fluctuations of multiple molecular species, the covariance is an important measure of noise. However, usually the representation of a covariance matrix in the natural coordinate system, i.e. the copy numbers of the molecular species, is intractably complicated because reactions change copy numbers of more than one molecular species simultaneously. Decoupling of a stoichiometric matrix, which is a transformation of variables, significantly simplifies the representation of a covariance matrix and elucidates the mechanisms behind the observed fluctuations in the copy numbers. We apply our method to three types of fundamental genetic regulatory networks, that is, a single-gene autoregulatory network, a two-gene autoregulatory network, and a mutually repressive network. We have found that there are multiple noise components differently originating. Each noise component produces fluctuation in the characteristic direction. The resulting fluctuations in the copy numbers of the molecular species are the sum of these fluctuations. In the examples, the limitation of the negative feedback in noise reduction and the trade-off of fluctuations in multiple molecular species are clearly explained. The analytical representations show the full parameter dependence. Additionally, the validity of our method is tested by stochastic simulations.     

Utility of RAPD markers in identifying genetic linkages to genes of economic interest in peach

The identification of molecular markers linked to economically important traits for use in crop improvement is very important in long-lived perennial species. Three-hundred-and-sixty RAPD primers were used with bulked segregant analysis to identify markers linked to loci of specific interest in peach [(Prunus persica) L. Batch] and peach x almond [(Prunus dulcis) Batch] crosses. The traits analyzed included flesh color, adhesion, and texture; pollen fertility; plant stature; and three isozyme loci. The Mendelian behavior of the RAPD loci was established, and RAPD markers were mapped relative to the loci controlling flesh color, adhesion, and texture, and the isozyme loci Mdh-1, 6Pgd-2 and Aat-1, as well as the existing RFLP genetic linkage map constructed previously using a peach x almond F₂ population. This technique has facilitated rapid identification of RAPD and RFLP markers that are linked to the traits under study. Loci controlling these traits mapped predominantly to linkage groups 2 and 3 of the peach genetic linkage map. Linkages to genes with both dominant and co-dominant alleles were identified, but linkages to dominant genes were more difficult to find. In several crosses, RAPD marker bands proved to be allelic. One co-dominant RAPD formed a heteroduplex band in heterozygous individuals and in mixtures of alternate homozygotes. The Mendelian behavior of the RAPD loci studied was established and the results suggest that RAPD markers will be useful for plant improvement in peach.