Genetic stratification of pathogen-response-related and other variants within a homogeneous Caucasian Irish population.

Selection pressures from pathogens impact on the worldwide geographic distribution of polymorphisms in certain pathogen-response-associated genes. Such gene-specific effects could lead to confounding by geographic disease associations. We wished to determine if such constraints impinge on the genetic structure of a population of Irish patients and whether variants associated with responses to pathogens showed greater stratification. The counties of origin of each subject's grandparents were used as the geographic variable. F(st), proportional to the extent of population structure, was low (mean F(st)=0.004 across 25 SNPs, range 0.001-0.008) and it was not significantly higher for pathogen response SNPs (F(st)=0.004) than for other SNPs (F(st)=0.003, P=0.21). Correspondence analysis revealed weak trends primarily in approximately northeast to southwest and secondarily in northwest to southeast directions. One-dimensional spatial autocorrelation analysis revealed a weak (Moran's I autocorrelation of -0.10) tendency for SNP frequencies to diverge with greater distance. Two-dimensional autocorrelation indicated a northeast to southwest gradient that was similar for both the pathogen response and other SNPs. The southeastern county, Wexford, showed a distinctive pattern, perhaps consistent with Anglo-Norman settlements. In conclusion, these results indicate that pathogen response SNPs do not exhibit significantly more population structure than other SNPs within this Caucasian population. This suggests that the specific population structure of particular genes may not typically be a cause of strong confounding in genetic studies where population structure is controlled.

Polymorphisms of the Flavin containing monooxygenase 3 (FMO3) gene do not predispose to essential hypertension in Caucasians.

BACKGROUND: The recessive disorder trimethylaminuria is caused by defects in the FMO3 gene, and may be associated with hypertension. We investigated whether common polymorphisms of the FMO3 gene confer an increased risk for elevated blood pressure and/or essential hypertension. METHODS: FMO3 genotypes (E158K, V257M, E308G) were determined in 387 healthy subjects with ambulatory systolic and diastolic blood pressure measurements, and in a cardiovascular disease population of 1649 individuals, 691(41.9%) of whom had a history of hypertension requiring drug treatment. Haplotypes were determined and their distribution noted. RESULTS: There was no statistically significant association found between any of the 4 common haplotypes and daytime systolic blood pressure in the healthy population (p = 0.65). Neither was a statistically significant association found between the 4 common haplotypes and hypertension status among the cardiovascular disease patients (p = 0.80). CONCLUSION: These results suggest that the variants in the FMO3 gene do not predispose to essential hypertension in this population.

Honeybee (Apis mellifera L.) mrjp gene family: computational analysis of putative promoters and genomic structure of mrjp1, the gene coding for the most abundant protein of larval food.

Mrjp1 gene belongs to the honeybee mrjp gene family encoding the major royal jelly proteins (MRJPs), secreted by nurse bees into the royal jelly. In this study, we have isolated the genomic clone containing the entire mrjp1 gene and determined its sequence. The mrjp1 gene sequence spans over 3038 bp and contains six exons separated by five introns. Seven mismatches between the mrjp1 gene sequence and two previously independently published cDNA sequences were found, but these differences do not lead to any change in the deduced amino acid sequence of MRJP1. With the aid of inverse polymerase chain reaction we obtained sequences flanking the 5' ends of other mrjp genes (mrjp2, mrjp3, mrjp4 and mrjp5). Putative promoters were predicted upstream of all mrjp genes (including mrjp1). The predicted promoters contain the TATA motif (TATATATT), highly conserved both in sequence and position. Ultraspiracle (USP) transcription factor (TF) binding sites in putative promoter regions and clusters of dead ringer TF binding sites upstream of these promoters were predicted computationally. We propose that USP, as a juvenile hormone (JH) binding TF, might possibly act as a mediator of mrjp expression in response to JH. Mrjp1's genomic locus is predicted to encode an antisense transcript, partially overlapping with five mrjp1 exons and entirely overlapping with the putative promoter and predicted transcriptional start point of mrjp1. This finding may shed light on the mechanisms of regulation of mrjps expression. Southern blot analysis of genomic DNA revealed that all so far known members of mrjp gene family (mrjp1, mrjp2, mrjp3, mrjp4 and mrjp5) are present as single-copy genes per haploid honeybee genome. Although MRJPs and the yellow protein of Drosophila melanogaster share a certain degree of similarity in aa sequence and although it has been shown that they share a common evolutionary origin, neither structural similarities in the gene organization, nor significant similarities between intron sequences of mrjp1 gene and fourteen yellow-like genes of D. melanogaster were found.

Epitope presentation is an important determinant of the utility of antigens identified from protein arrays in the development of autoantibody diagnostic assays.

Autoantibodies represent an attractive biomarker for diagnostic assays principally due to the stability of immunoglobulin in patient serum facilitating measurement with conventional assays. Immune responses to tumorigenesis may facilitate detection of ovarian cancer in the early stages of the disease with identification of a panel of tumour specific autoantibodies. Despite the reporting of many tumour associated autoantibodies using arrays of tumour antigens, this has not led to the advance in diagnostic capability as rapidly as was initially expected. Here we examine the potential diagnostic utility of candidate autoantibody biomarkers identified via screening of serum samples on a high content human protein array from a unique cohort of early stage and late stage ovarian cancer patients. We analyse the performance of autoantibodies to the tumour suppressor protein p53 and the novel autoantigens alpha adducin and endosulfine alpha identified in our array screen. Each antigen has different performance characteristics using conventional ELISA format and Western blot immunoassay. The high attrition rate of promising autoantigens identified by array screening can in part be explained by the presentation of the epitope of the antigen in the subsequent method of validation and this study provides directions on maximising the potential of candidate biomarkers. This article is part of a Special Issue entitled: Translational Proteomics.

Optimized autoantibody profiling on protein arrays.

Profiling the autoantibody (AAb) repertoire in serum has been routinely used for many years for the diagnosis of autoimmune diseases, including rheumatoid arthritis, scleroderma, and lupus. In recent years, AAb profiling of cancers has become a prominent field in oncology research. Protein arrays enable high-throughput screening of clinical samples, characterising the serum profile using low volumes of samples. This chapter describes the use of a protein array comprising 37,200 redundant proteins (containing over 10,000 non-redundant human recombinant proteins) for identification of the proteins bound by the antibodies in human sera using a test set of serum samples. The proteins identified have the potential to be candidate biomarkers. These recombinant proteins are expressed, purified, and robotically spotted on microarrays or chips to facilitate the screening of additional serum samples with the aim of identifying a candidate biomarker or panel of potential biomarkers for applications in disease diagnosis, stage, progression, or response to therapy.

Diagnostic and prognostic biomarker discovery strategies for autoimmune disorders.

Current clinical, laboratory or radiological parameters cannot accurately diagnose or predict disease outcomes in a range of autoimmune disorders. Biomarkers which can diagnose at an earlier time point, predict outcome or help guide therapeutic strategies in autoimmune diseases could improve clinical management of this broad group of debilitating disorders. Additionally, there is a growing need for a deeper understanding of multi-factorial autoimmune disorders. Proteomic platforms offering a multiplex approach are more likely to reflect the complexity of autoimmune disease processes. Findings from proteomic based studies of three distinct autoimmune diseases are presented and strategies compared. It is the authors' view that such approaches are likely to be fruitful in the movement of autoimmune disease treatment away from reactive decisions and towards a preventative stand point.

Renin gene polymorphisms and haplotypes, blood pressure, and responses to renin-angiotensin system inhibition.

Renin catalyzes the rate-limiting step of the renin-angiotensin system. A T allele variant at position -5312 within a distal enhancer region has been reported to increase in vitro renin gene transcription. Among 387 White bank employees, ambulatory blood pressures were higher in 133 -5312T allele carriers than in 254 CC homozygotes-mean differences [99% confidence interval] between carriers and homozygotes for daytime and night-time systolic/diastolic pressure were 2.5[0.4,4.6]/1.7[0.2,3.2] and 2.4[0.5,4.4]/1.5[0.1,2.9] respectively. Ambulatory pressure estimates for the only common renin haplotype including the -5312T variant (-5312T, 5090C, 5912A, 9479A, 10194G), were statistically significantly higher than estimates for all other haplotypes. Among 259 White hypertensive participants in a randomized double-blind clinical trial comparing a renin antagonist, aliskiren, with an angiotensin receptor blocker, losartan, plasma renin activity did not differ with renin -5312C/T genotype. Nocturnal blood pressure reductions with losartan 100 mg daily were significantly greater in -5312T allele carriers than in CC homozygotes (mean[standard error]; -12.9[3.7]/-7.9[2.4] versus -7.1[2.5]/-4.2[1.6]) whereas with aliskiren 150 and 300 mg daily, lesser reductions were observed in -5312T allele carriers than in CC homozygotes (-5.4[2.0]/-4.1[1.3] versus -10.1[1.4]/-6.5[1.1]; P<0.03 for treatmentxgenotype interaction for night-time systolic and diastolic pressures). Hence, the -5312 renin C/T enhancer polymorphism does contribute to blood pressure variation in Whites and also appears to predict responses to inhibition of the renin-angiotensin system. These findings suggest that genotyping at this locus may aid in the identification of susceptibility to hypertension and in the selection of optimal therapy for individual hypertensive patients.

The impact of genetic variation in the region of the GPIIIa gene, on Pl expression bias and GPIIb/IIIa receptor density in platelets.

Some studies have suggested that genetic variability in the glycoprotein (GP) IIIa gene modulates expression of platelet GPIIb/IIIa (alpha(2b)beta(3)). We sought to determine as to whether combinations of genetic variants within the GPIIIa gene (haplotypes) influenced the expression of GPIIIa RNA and protein levels in human platelets. Three promoter polymorphisms, Pl(A1/A2) genotype and platelet receptor densities were determined in 207 acute coronary syndrome (ACS) patients. Allele-specific quantitative reverse transcription-polymerase chain reaction of platelet RNA from Pl(A1/A2) heterozygotes identified a greater expression of Pl(A2) bearing transcripts among heterozygotes. Among the patients studied, the ratio of Pl(A1)/Pl(A2) RNA expression was significantly influenced by promoter haplotype (P < 0.01). However, this effect reflected carriership of rare not common haplotypes (P = 0.2). There was a threefold variation between subjects in the number of GPIIb/IIIa receptors expressed per platelet, although no association between receptor density and the Pl(A2) (P = 0.93) or promoter polymorphisms was demonstrated (-468A, P = 0.52; -425C, P = 0.59; -400A, P = 0.52). Among common haplotypes, Pl(A1)/Pl(A2) RNA expression was negatively correlated with adjusted GPIIb/IIIa receptor density (P = 0.04). The overall trend towards higher expression of Pl(A2) bearing message in Pl(A1/A2) heterozygotes, and the existence of rare haplotypes with more pronounced changes indicate the existence of cis-acting genetic factors that remain to be identified.

DEFOG: a practical scheme for deciphering families of genes.

We developed a novel efficient scheme, DEFOG (for "deciphering families of genes"), for determining sequences of numerous genes from a family of interest. The scheme provides a powerful means to obtain a gene family composition in species for which high-throughput genomic sequencing data are not available. DEFOG uses two key procedures. The first is a novel algorithm for designing highly degenerate primers based on a set of known genes from the family of interest. These primers are used in PCR reactions to amplify the members of the gene family. The second combines oligofingerprinting of the cloned PCR products with clustering of the clones based on their fingerprints. By selecting members from each cluster, a low-redundancy clone subset is chosen for sequencing. We applied the scheme to the human olfactory receptor (OR) genes. OR genes constitute the largest gene superfamily in the human genome, as well as in the genomes of other vertebrate species. DEFOG almost tripled the size of the initial repertoire of human ORs in a single experiment, and only 7% of the PCR clones had to be sequenced. Extremely high degeneracies, reaching over a billion combinations of distinct PCR primer pairs, proved to be very effective and yielded only 0.4% nonspecific products.