Investigation of sensitivity,specificity and accuracy of Tetra primer ARMS PCR method in comparison with conventional ARMS PCR,based on sequencing technique outcomes in IVS-II-I genotyping of beta thalassemia patients

Purpose

Beta thalassemia is one of the most important hematic diseases all around the world and solving the problems caused by this abnormality is strongly dependent on precise detection and reliable screening of high-risk couples. The aim of our study was the investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method comparing with conventional ARMS PCR, based on sequencing technique outcomes for genotyping of IVS-II-I mutation in beta thalassemia patients.

Methods

Fifty seven samples including two homozygote, 49 heterozygote and 6 normal specimens were analyzed by Tetra primer ARMS PCR and conventional ARMS PCR methods. DNA was extracted by the standard method of salting out for leukocyte genomic DNA extraction of blood specimens and a high pure PCR template preparation kit was used for DNA purification of CVS samples. The results obtained by Tetra primer ARMS PCR and conventional ARMS PCR methods were compared with gold standard technique, i.e. sequencing.

Results

All three parameters including specificity, sensitivity and accuracy were 100% for Tetra primer ARMS PCR method, while they were 100%, 92.45% and 92.7% for conventional ARMS PCR technique respectively. Comparing with Tetra primer ARMS PCR which represented 100% agreement with sequencing method, conventional ARMS PCR technique only showed 47.1% agreement, because of 4 discordant results.

Conclusion

Tetra primer ARMS PCR method is an almost reliable, sensitive and accurate technique and it is suggested that it can be used as a complementary method for diagnostic cases instead of conventional ARMS PCR method. This suggestion originated with perfect rate of agreement between outcomes of sequencing method, as a gold standard method of detecting the mutations, and Tetra primer ARMS PCR technique comparing with conventional ARMS PCR method.     

Altering protein specificity: techniques and applications

Protein engineering constitutes a powerful tool for generating novel proteins that serve as catalysts to induce selective chemical and biological transformations that would not otherwise be possible. Protocols that are commonly employed for altering the substrate specificity and selectivity profiles by mutating known enzymes include rational and random methods as well as techniques that entail evolution, selection and screening. Proteins identified by these techniques play important roles in a variety of industrial and medicinal applications and in the study of protein structure-function relationships. Herein we present a critical overview of methods for creating new functional proteins having altered specificity profiles and some practical case studies in which these techniques have been applied to solving problems in synthetic and medicinal chemistry and to elucidating enzyme function and biological pathways.     

Transposon Tn10: genetic organization, regulation, and insertion specificity

Transposon Tn10 is a composite element in which two individual insertion sequence (IS)-like sequences cooperate to mediate transposition of the intervening material. The two flanking IS10 elements are not identical; IS10-right is responsible for functions required to promote transposition, and IS10-left is defective in transposition functions. We suggest that the two IS10 elements were originally identical in sequence and have subsequently diverged. IS10-right is compactly organized with structural gene(s), promoters, and sites important for transposition and (presumably) its regulation all closely linked and, in some cases, overlapping. IS10 has a single major coding region that almost certainly encodes an essential transposition function. A pair of opposing promoters flank the start of this coding region. One of these promoters is responsible for expression in vivo of transposon-encoded transposition functions. We propose that the second promoter is involved in modulation of Tn10 transposition. Genetic analysis suggests that transposon-encoded function(s) may be preferentially cis-acting. Insertion of Tn10 into particular preferred target sites is due primarily to the occurrence of a particular six-base pair target DNA sequence. The properties of this sequence suggest that symmetrically disposed subunits of a single protein may be responsible for both recognition and cleavage of target DNA during insertion.     

Computation vs. cloning: evaluation of two methods for haplotype determination

Nuclear sequence data, often from multiple loci, are increasingly being employed in analyses of population structure and history, yet there has been relatively little evaluation of methods for accurately and efficiently separating the alleles or haplotypes in heterozygous individuals. We compared the performance of a computational method of haplotype reconstruction and standard cloning methods using a highly variable intron (ornithine decarboxylase, intron 6) in three closely related species of dabbling ducks (genus Anas). Cloned sequences from 32 individuals were compared to results obtained from phase 2.1.1 . phase correctly identified haplotypes in 28 of 30 heterozygous individuals when the underlying model assumed no recombination. Haplotypes of the remaining two individuals were also inferred correctly except for unique polymorphisms, the phase of which was appropriately indicated as uncertain (phase probability = 0.5). For a larger set of 232 individuals, results were essentially identical regardless of the recombination model used and haplotypes for all 30 of the tested heterozygotes were correctly inferred, with the exception of uncertain phase for unique polymorphisms in one individual. In contrast, initial sequences of one clone per sample yielded accurate haplotype determination in only 26 of 30 individuals; polymerase chain reaction (PCR)/cloning errors resulting from misincorporation of individual nucleotides could be recognized and avoided by comparison to direct sequences, but errors due to PCR recombination resulted in incorrect haplotype reconstruction in four individuals. The accuracy of haplotypes reconstructed by phase , even when dealing with a relatively small number of samples and numerous variable sites, suggests broad utility of computational approaches for reducing the cost and improving the efficiency of data collection from nuclear sequence loci.     

Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value

The diagnosis of rheumatoid arthritis (RA) is primarily based on clinical symptoms, so it is often difficult to diagnose RA in very early stages of the disease. A disease-specific autoantibody that could be used as a serological marker would therefore be very useful. Most autoimmune diseases are characterized by a polyclonal B-cell response targeting multiple autoantigens. These immune responses are often not specific for a single disease. In this review, the most important autoantibody/autoantigen systems associated with RA are described and their utility as a diagnostic and prognostic tool, including their specificity, sensitivity and practical application, is discussed. We conclude that, at present, the antibody response directed to citrullinated antigens has the most valuable diagnostic and prognostic potential for RA.     

Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value

The diagnosis of rheumatoid arthritis (RA) is primarily based on clinical symptoms, so it is often difficult to diagnose RA in very early stages of the disease. A disease-specific autoantibody that could be used as a serological marker would therefore be very useful. Most autoimmune diseases are characterized by a polyclonal B-cell response targeting multiple autoantigens. These immune responses are often not specific for a single disease. In this review, the most important autoantibody/autoantigen systems associated with RA are described and their utility as a diagnostic and prognostic tool, including their specificity, sensitivity and practical application, is discussed. We conclude that, at present, the antibody response directed to citrullinated antigens has the most valuable diagnostic and prognostic potential for RA.     

Genetic ecological monitoring in human populations: heterozygosity, mtDNA haplotype variation, and genetic load

Yu. P. Altukhov suggested that heterozygosity is an indicator of the state of the gene pool. The idea and a linked concept of genetic ecological monitoring were applied to a new dataset on mtDNA variation in East European ethnic groups. Haplotype diversity (an analog of the average heterozygosity) was shown to gradually decrease northwards. Since a similar trend is known for population density, interlinked changes were assumed for a set of parameters, which were ordered to form a causative chain: latitude increases, land productivity decreases, population density decreases, effective population size decreases, isolation of subpopulations increases, genetic drift increases, and mtDNA haplotype diversity decreases. An increase in genetic drift increases the random inbreeding rate and, consequently, the genetic load. This was confirmed by a significant correlation observed between the incidence of autosomal recessive hereditary diseases and mtDNA haplotype diversity. Based on the findings, mtDNA was assumed to provide an informative genetic system for genetic ecological monitoring; e.g., analyzing the ecology-driven changes in the gene pool.     

A double scanning microphotometer for image analysis: hardware, software and biomedical applications.

A new image processing system designed for densitometry and pattern analysis of microscopic specimens is described with special regard to the hardware, the software and the biologic applications. The data acquisition procedure involves the combination between the scanning of the preparation by means of a motorized stage and the scanning of successive fields by a mechanical device. The signal provided by the photomultiplier is converted into digital values which are directed to an on-line computer. The data processing is based on a one-pass computation involving automata theory and therefore it avoids the storage of the image in the computer memory. In so doing, an entire and continuous image of the whole preparation can be processed at the highest magnification of the microscope whatever the size of the analyzed specimen may be. A biologic application of the system is reported and concerns the automatic identification and counting of cells in the various phases of the mitotic cycle.     

Assessment of linkage disequilibrium by the decay of haplotype sharing, with application to fine-scale genetic mapping.

Linkage disequilibrium (LD) is of great interest for gene mapping and the study of population history. We propose a multilocus model for LD, based on the decay of haplotype sharing (DHS). The DHS model is most appropriate when the LD in which one is interested is due to the introduction of a variant on an ancestral haplotype, with recombinations in succeeding generations resulting in preservation of only a small region of the ancestral haplotype around the variant. This is generally the scenario of interest for gene mapping by LD. The DHS parameter is a measure of LD that can be interpreted as the expected genetic distance to which the ancestral haplotype is preserved, or, equivalently, 1/(time in generations to the ancestral haplotype). The method allows for multiple origins of alleles and for mutations, and it takes into account missing observations and ambiguities in haplotype determination, via a hidden Markov model. Whereas most commonly used measures of LD apply to pairs of loci, the DHS measure is designed for application to the densely mapped haplotype data that are increasingly available. The DHS method explicitly models the dependence among multiple tightly linked loci on a chromosome. When the assumptions about population structure are sufficiently tractable, the estimate of LD is obtained by maximum likelihood. For more-complicated models of population history, we find means and covariances based on the model and solve a quasi-score estimating equation. Simulations show that this approach works extremely well both for estimation of LD and for fine mapping. We apply the DHS method to published data sets for cystic fibrosis and progressive myoclonus epilepsy.     

Insulin-specific human T cells. Epitope specificity, major histocompatibility complex restriction, and alloreactivity to a diabetes-associated haplotype

T cells from an insulin-treated diabetic (ML, HLA DR1, w6) were stimulated in vitro with insulin, cloned at limiting dilution, and examined for their fine specificity and genetic restriction. T cell lines (TCL) derived from beef insulin stimulation were highly specific for epitopes on beef insulin, whereas pork insulin stimulation generated T cells that recognized determinants shared with beef insulin. Included among TCL reactive with pork insulin is one line (P2/9) that is autoreactive with human insulin. Antigen-presenting cells of known HLA type and monoclonal antibodies directed at class II major histocompatibility complex antigens were used to confirm the role of HLA-DR in restricting the response of insulin immune T cells. No preference or determinant selection within the donor's haplotypes was identified because either DR1 or DRw6 antigen-presenting cells could present the A loop of beef insulin. A TCL that recognized the A loop of beef insulin in association with DR1 was also alloreactive to HLA DR3, or a molecule closely linked to it, in the absence of insulin. A second T cell clone with insulin specificity and alloreactivity was also derived by allo stimulation of the donor's cells with DR3+ cells. When tested with a series of DR3+ stimulator cells, the alloreactivity was directed at diabetes-associated haplotypes. These data show that the T cell repertoire for insulin of a single diabetic donor encompasses that of multiple inbred animal strains and includes fine specificity for one to two amino acids, recognition of autologous insulin, and cross-reactivity with an allogeneic major histocompatibility complex antigen.     

Macrophage sensitivity to endotoxin: genetic control by a single codominant gene.

The effects of LPS on macrophages in vitro have been examined. LPS triggers macrophages to produce LAF and PGE2 in vitro. LPS is also cytotoxic for macrophages derived from LPS-sensitive mice and will significantly inhibit their phagocytic ability. Both LAF production and cytotoxicity are due to the direct effects of LPS on the macrophage and do not require the particpation of lymphocytes. Each of these functions is abnormal in C3H/HeJ mice. The nature of the gene(s) controlling these macrophage responses to LPS has been determined. The response of (C3H/HeJ X C3H/HeN)F1 macrophages was intermediate when compared to the parental responses and no sex linkage was found. Backcross linkage analysis suggested that the same autosomal codominant gene controls both macrophage and B lymphocyte-LPS sensitivity.     

ATP-dependent reactions catalyzed by inner membrane vesicles of rat liver mitochondria. Kinetics, substrate specificity, and bicarbonate sensitivity.

Three ATP-dependent reactions catalyzed by the inner membrane of rat liver mitochondria and the ATPase reaction catalyzed by purified mitochondrial ATPase (F1), were studied with respect to kinetic properties, substrates specificity, and sensitivity to bicarbonate. The ATP-dependent transhydrogenase reaction (reduction of NADP+ by NADH) catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers, with Km (ATP) values of 0.035 mM and 0.054 mM respectively. The Vmax of transhydrogenase activity (25 nmol min-1 mg-1) is the same in Tris-bicarbonate or Tris-Cl buffer. ITP and GTP readily substitute for ATP in the transhydrogenase reaction. The ATP-P1 exchange reaction catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers with Km (ATP) values of 1.0 mM and 1.4 mM respectively. The Vmax of exchange (200 nmol min-1 mg-1) is the same in either buffer. ITP and GTP do not effectively replace ATP in the exchange reaction.     

Long-range, high-throughput haplotype determination via haplotype-fusion PCR and ligation haplotyping

Ligation Haplotyping is a robust, novel method for experimental determination of haplotypes over long distances, which can be applied to assaying both sequence and structural variation. The simplicity and efficacy of the method for genotyping large chromosomal rearrangements and haplotyping SNPs over long distances make it a valuable and powerful addition to the methodological repertoire, which will be beneficial to studies of population genetics and evolution, disease association and inheritance, and genomic variation. We illustrate the versatility of the method both by genotyping a Yp paracentric inversion, found in approximately 60% of Northwest European males, that strongly influences the germline rate of infertility-causing XY translocations and by haplotyping two autosomal SNPs that lie 16.4 kb apart on chromosome 7, and which influence an individual's susceptibility to systemic lupus erythematosus.     

CARD15 genetic variation in a Quebec population: prevalence,genotype-phenotype relationship,and haplotype structure

The caspase recruitment domain gene (CARD15) was recently identified as the underlying gene associated with the IBD1 locus that confers susceptibility to Crohn disease (CD). CARD15 is related to the NOD1/Apaf-1 family of apoptosis regulators, and three sequence variants (Arg702Trp, Gly908Arg, and Leu1007fsinsC) in the gene were demonstrated to be associated with CD. We collected a cohort of 231 patients with CD and 71 healthy control individuals from the Canadian province of Quebec, to determine the prevalence of these sequence variants in an independent population. Clinical records of all patients were systematically reviewed, and detailed phenotypic information was obtained. All patient DNA samples were genotyped for the three variants, thus enabling an analysis of genotype-phenotype correlations. In this cohort, 45.0% of patients with CD carried at least one variant in the CARD15 gene, compared with 9.0% of control individuals (P<10-7). Allele frequencies of Arg702Trp, Gly908Arg, and Leu1007fsinsC were 12.9%, 5.2%, and 10.3% in patients with CD, compared with 4.2%, 0.7%, and 0.7% in control individuals, respectively. Importantly, CARD15 mutants were seen with equal frequency in patients with familial and sporadic CD. Analysis of the relationship between genotype and phenotype convincingly demonstrates that CARD15 variants are significantly associated with ileal disease involvement, as opposed to strictly colonic disease (P<.001). Moreover, we were able to determine the haplotype structure surrounding this disease gene by genotyping 45 single-nucleotide polymorphisms (SNPs) in a 177-kb region that contained the CARD15 gene. This structure helps clarify the history of these causal mutations. Finally, this analysis shows that CARD15 involvement with CD is detectable by use of publicly available SNPs alone.     

Estimation of genetic variance for macro- and micro-environmental sensitivity using double hierarchical generalized linear models

Background

Genetic variation for environmental sensitivity indicates that animals are genetically different in their response to environmental factors. Environmental factors are either identifiable (e.g. temperature) and called macro-environmental or unknown and called micro-environmental. The objectives of this study were to develop a statistical method to estimate genetic parameters for macro- and micro-environmental sensitivities simultaneously, to investigate bias and precision of resulting estimates of genetic parameters and to develop and evaluate use of Akaike’s information criterion using h-likelihood to select the best fitting model.

Methods

We assumed that genetic variation in macro- and micro-environmental sensitivities is expressed as genetic variance in the slope of a linear reaction norm and environmental variance, respectively. A reaction norm model to estimate genetic variance for macro-environmental sensitivity was combined with a structural model for residual variance to estimate genetic variance for micro-environmental sensitivity using a double hierarchical generalized linear model in ASReml. Akaike’s information criterion was constructed as model selection criterion using approximated h-likelihood. Populations of sires with large half-sib offspring groups were simulated to investigate bias and precision of estimated genetic parameters.

Results

Designs with 100 sires, each with at least 100 offspring, are required to have standard deviations of estimated variances lower than 50% of the true value. When the number of offspring increased, standard deviations of estimates across replicates decreased substantially, especially for genetic variances of macro- and micro-environmental sensitivities. Standard deviations of estimated genetic correlations across replicates were quite large (between 0.1 and 0.4), especially when sires had few offspring. Practically, no bias was observed for estimates of any of the parameters. Using Akaike’s information criterion the true genetic model was selected as the best statistical model in at least 90% of 100 replicates when the number of offspring per sire was 100. Application of the model to lactation milk yield in dairy cattle showed that genetic variance for micro- and macro-environmental sensitivities existed.

Conclusion

The algorithm and model selection criterion presented here can contribute to better understand genetic control of macro- and micro-environmental sensitivities. Designs or datasets should have at least 100 sires each with 100 offspring.     

Parameters for evaluation of viability assays: accuracy, precision, specificity, sensitivity, and standardization

The selection of appropriate viability assays is critical in evaluating the efficacy of any cryopreservation procedure. The appropriateness of a given assay depends on the specific tissue and the function which is being optimized. Although a broad range of "viability" assays have been used, these assays can be classified in seven principle groups: (i) Morphological procedures, including routine histology, surface antigen localization, and transmission electron or scanning microscopy; (ii) proliferation studies; (iii) metabolic assays; (iv) implantation; (v) mechanical assays; (vi) motility; and (vii) DNA or RNA synthetic assays. Regardless of the class of assay, each assay may be further characterized as qualitative, quantitative, or quantal and each type may vary in the degree of subjectivity. In selecting a specific viability assay, biological variability, assay bias, and the statistical probability of both Type I and Type II errors should be considered crucial. Here we discuss a number of critical factors involved in validating viability assays, including accuracy, precision, standardization, specificity, sensitivity, selection of statistical methodology, and range of the assay.     

Melting curves, denaturation maps, and genetic map of phiX174: their relations and applications.

In this paper we analyze theoretically the observable details of the differential melting curves (DMC) and the denaturation maps (DM) of a DNA. With the help of a mathematical model, we explore their implications, their relation with each other and with the genetic map of the molecule, and discuss possible future applications. ?X174 is used as the example, since its sequence and genetic map are available. We find that each gene section of ?X174 has a characteristic DMC. A reconstruction scheme to get the DMC of a whole piece from those of its constituent genes is shown to be fairly successuful. The relations between the melting curve and the denaturation maps are clarified. We observe that nearly always, the beginning and end of a gene melt at lower temperatures. The sharp features in the DM indicate that despite the long-range cooperative interactions, the DM do reflect the local sequence effect. Denaturation maps (theoretical) of ?X174 and SV40 are presented. From available data of other authors, we estimate that the dependence of the melting temperature t_m on GC, the fraction of (G+C)-content, and on x, the ionic concentration in fractions of the standard saline citrate solution, can be expressed as t_m(x, GC) = -5.2 (log x)GC + 18.4 log x + 41.0GC + 69.4. The first two coefficients are less certain.