Polymorphism in the P-selectin and interleukin-4 genes as determinants of stroke: a population-based, prospective genetic analysis

Candidate gene polymorphisms related to inflammation, thrombosis and lipid metabolism have been implicated in the development of ischemic stroke. Using DNA samples collected at baseline in a prospective cohort of 14 916 initially healthy American men, we genotyped 92 polymorphisms from 56 candidate genes among 319 individuals who subsequently developed ischemic stroke and among 2092 individuals who remained free of reported cardiovascular disease over a mean follow-up period of 13.2 years to prospectively determine whether candidate gene polymorphisms contribute to stroke risk. After adjustment for multiple comparisons and age, smoking, body mass index, hypertension, hyperlipidemia and diabetes, two related to inflammation [a val640leu polymorphism in the P-selectin gene (OR=1.63, 95% CI 1.22-2.17, P=0.001) and a C582T polymorphism in the interleukin-4 gene (OR=1.40, 95% CI 1.13-1.73, P=0.003)] were found to be independent predictors of thrombo-embolic stroke. In bootstrap replications, the inclusion of genetic information from these two polymorphisms improved prediction models for stroke based upon traditional risk factors alone (ROC 0.67 versus 0.64). Two polymorphisms related to thrombosis (an arg353gln polymorphism in the factor VII gene and a T11053G polymorphism in the plasminogen activator inhibitor type-1 gene) and one related to lipid metabolism [a C(-482)T polymorphism in the apolipoprotein CIII gene] achieved nominal significance, but were not found to be independent predictors after multiple comparison adjustment. Two inflammatory candidate gene polymorphisms were identified which were independently associated with incident stroke. These population-based data demonstrate the ability of prospective, epidemiological studies to test candidate gene associations for athero-thrombotic disease.     

Human population-based identification of CD4(+) T-cell peptide epitope determinants

A human cell-based method to identify functional CD4(+) T-cell epitopes in any protein has been developed. Proteins are tested as synthetic 15-mer peptides offset by three amino acids. Percent responses within a large donor population are tabulated for each peptide in the set. Peptide epitope regions are designated by difference in response frequency from the overall background response rate for the compiled dataset. Epitope peptide responses are reproducible, with a median coefficient of variance of 21% when tested on multiple random-donor sets. The overall average response rate within the dataset increases with increasing putative human population antigenic exposure to a given protein. The background rate was high for HPV16 E6, and was low for human-derived cytokine proteins. The assay identified recall epitope regions within the donor population for the protein staphylokinase. For an industrial protease with minimal presumed population exposure, immunodominant epitope peptides were identified that were found to bind promiscuously to many HLA class II molecules in vitro. The peptide epitope regions identified in presumably unexposed donors represent a subset of the total recall epitopes. Finally, as a negative control, the assay found no peptide epitope regions in human beta2-microglobulin. This method identifies functional CD4(+) T-cell epitopes in any protein without pre-selection for HLA class II, suggests whether a donor population is pre-exposed to a protein of interest, and does not require sensitized donors for in vitro testing.     

Linkage between stature and a region on chromosome 20 and analysis of a candidate gene,bone morphogenetic protein 2

Sib-pair linkage analysis of the quantitative trait, stature, in over 500 Pima Indians indicates that a genetic determinant of governing stature is located on chromosome 20. Analysis of 10 short tandem repeat polymorphisms localized this linkage to a 3.2cM region that includes D20S98 and D20S66. Using all possible sib-pair combinations, linkage was detected to both stature (P = 0.0001) and to leg length (P = 0.001), but not to sitting height. Single-strand conformational polymorphism analysis of exon 3 of the bone morphogenetic protein 2 (BMP2) gene, a candidate gene in this region, in genomic DNA of 20 of the tallest and 20 of the shortest individuals did not show any consistent differences associated with leg length or height. Sequence analysis of the region encoding the mature protein revealed a single nucleotide substitution, a T to G transversion, not detected by single-strand conformational polymorphism (SSCP) analysis. This transversion results in a conservative amino acid substitution of glycine for valine at codon 80 of BMP2. The frequency of this allele was 0.23 in the sample. No significant differences in height were noted in persons carrying either allele. This indicates that this structural alteration in the mature BMP2 protein does not contribute to the differences in stature observed in the Pima Indians, nor is this structural change in the mature protein likely to be responsible for the linkage observed with stature on chromosome 20. © 1995 Wiley-Liss, Inc.

1 This article is a U.S. Government work and, as such, is in the public domain in the United States of America.

     

Linkage disequilibrium, haplotype and association studies of a chromosome 4 GABA receptor gene cluster: candidate gene variants for addictions.

Strong genetic contributions to individual differences in vulnerability to addictions are well supported by classical genetic studies. Linkage and association genome scans for addiction vulnerability have provided converging evidence for several chromosomal regions which are likely to harbor allelic variants that contribute to such vulnerability. We and others have delineated a candidate addiction-associated chromosome 4p12 "rSA3" region based on convergent data from association genome scanning studies in polysubstance abusers [Uhl et al. (2001); Am J Hum Genet 69(6):1290-1300], linkage-based studies in alcoholism [Long et al. (1998); Am J Med Genet 81(3):216-221; Reich et al. (1998); Am J Med Genet 81(3):207-215] and association-based studies for alcoholism and association-based studies for individual differences in electroencephalographic (EEG) spectral power phenotypes [Porjesz et al. (2002); Proc Natl Acad Sci USA 99(6):3729-3733; Edenberg et al. (2004); Am J Hum Genet 74(4):705-714]. The rSA3 region contains interesting candidate genes that encode the alpha 2, alpha 4, beta 1, and gamma 1 receptor subunits for the principal brain inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) [Covault et al. (2004); Am J Med Genet Part B 129B:104-109; Edenberg et al. (2004); Am J Hum Genet 74(4):705-714; Lappalainen et al. (2005); Alcohol Clin Exp Res 29(4):493-498]. We now report assessment of single nucleotide polymorphism (SNP) genotypes in this region in three samples of substance abusers and controls. These results delineate the haplotypes and patterns of linkage disequilibrium in this region, focus attention of the GABRA2 gene and identify modest associations between GABRA2 genotypes and addiction phenotypes. These results are consistent with modest roles for GABRA2 variants in addiction vulnerabilities.     

Linkage and candidate gene analysis of 14q22-24 in bipolar disorder: support for GCHI as a novel susceptibility gene.

Using a collection of Irish sib-pair nuclear families, we previously obtained modest evidence of linkage implicating 14q22-24 in bipolar disorder (BPD). To follow-up on this preliminary finding, an extended linkage analysis was performed which employed thirteen microsatellite markers, spanning a total distance of 85 cM on 14q. Effectively, P-values <0.05 were observed for a region extending over 41.88 cM, with the marker D14S281 displaying a peak multipoint non-parametric lod (NPL) score of 2.72 and an associated P-value of 0.003. Support for this finding was also obtained from flanking markers indicating excess allele sharing at 14q22-24 in Irish bipolar sib-pairs. A web-based candidate gene search of 14q22-24 resulted in the selection of GTP cyclohydrolase I (GCHI), located 200 kb 3' of D14S281, as the best plausible candidate gene for involvement in BPD. GCHI is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin (BH(4)), a natural cofactor for tyrosine and tryptophan hydroxylases. These enzymes play an essential role in the biosynthesis of various hormones and neurotransmitters such as dopamine, noradrenaline, adrenaline, and serotonin. Numerous studies have also suggested that the clinical symptoms of depression might be related to a deficiency of BH(4). An association study between BPD and a novel single nucleotide polymorphism (SNP) in GCHI (G to A at position -959 bp, upstream of the ATG codon), is also presented here. This study revealed that the variant A allele is preferentially transmitted to BPI probands (chi(2) = 4.54, P = 0.033) suggesting that variants within GCHI may contribute to BPD in the Irish population.     

Evaluation of RGS4 as a candidate gene for schizophrenia.

Several studies have suggested that the regulator of G-protein signaling 4 (RGS4) may be a positional and functional candidate gene for schizophrenia. Three single nucleotide polymorphisms (SNP) located at the promoter region (SNP4 and SNP7) and the intron 1 (SNP18) of RGS4 have been verified in different ethnic groups. Positive results have been reported in these SNPs with different numbers of SNP combinatory haplotypes. In this study, these three SNP markers were genotyped in 218 schizophrenia pedigrees of Taiwan (864 individuals) for association analysis. Among these three SNPs, neither SNP4, SNP7, SNP18 has shown significant association with schizophrenia in single locus association analysis, nor any compositions of the three SNP haplotypes has shown significantly associations with the DSM-IV diagnosed schizophrenia. Our results fail to support the RGS4 as a candidate gene for schizophrenia when evaluated from these three SNP markers.     

Mapping of constitutional translocation breakpoints in renal cell cancer patients: identification of KCNIP4 as a candidate gene

Our group and others had previously developed a high throughput procedure to map translocation breakpoints using chromosome flow sorting in conjunction with microarray-based comparative genomic hybridization (arrayCGH). Here we applied both conventional positional cloning and integrated arrayCGH procedures to the mapping of constitutional chromosome anomalies in four patients with renal cell cancer (RCC), three with a chromosome 3 translocation, and one with an insertion involving chromosome 3. In one of these patients, who was carrying a t(3;4)(p13;p15), the KCNIP4 gene was found to be disrupted. KCNIP4 belongs to a family of potassium channel-interacting proteins and is highly expressed in normal kidney cells. In addition, KCNIP4 splice variants have specifically been encountered in RCC.     

Linkage between bronchial responsiveness to methacholine and gene markers of IL-4 cytokine gene cluster and T-cell receptor α/δ gene complex in Korean nuclear families

BACKGROUND: Several candidate genes have been reported to be linked to intermediate phenotypes of asthma in Caucasian populations. OBJECTIVE: To evaluate linkage between phenotypes of asthma and gene markers of high affinity IgE receptor-beta gene (D11S97), IL-4 cytokine gene cluster (IL-4R1), and T-cell receptor alpha/delta gene complex (D14S50) in Korean nuclear families. METHODS: Nuclear families (127 probands and their 130 siblings) for the linkage analysis were ascertained through asthmatic children. Linkages between total serum IgE response, skin responses to common aeroallergens, and bronchial responsiveness to methacholine were performed using a sib-pair approach. RESULTS: The square difference of the slope of the dose-response curve (DRS) between sib-pairs with two IL-4R1 identical alleles was smaller than with one or with neither IL-4R1 identical allele (P = 0.004). As for D14S50, the differences of DRS between sib-pairs with two identical alleles and with one identical allele were smaller than with neither identical alleles (P = 0.01). As for D11S97, no significant differences were observed among the groups with identical alleles of two, one or zero. With regard to total serum IgE levels, no significant linkage was found between this phenotype and the above three gene markers. As for skin responses to common aeroallergens, significant evidence was obtained to establish a linkage between this phenotype and the marker IL-4R1 (P = 0.01). However, no significant linkage was found between this phenotype and the markers D11S97 and D14S50. CONCLUSION: The expression of bronchial responsiveness to methacholine may be influenced by genetic factors in the IL-4 cytokine gene cluster and/or T-cell receptor alpha/delta gene complex, but the genetic influence of the FcepsilonRI-beta gene may be minimal in the expression of bronchial responsiveness in Korean nuclear families.     

Regulation of expression of IL-4 alleles: analysis using a chimeric GFP/IL-4 gene

CD4 cells from mice heterozygous for an IL-4 and a GFP/IL-4 gene frequently express a single allele. Analysis of IL-4 or GFP production by cells from recently primed Th2 cells indicates that essentially all are competent to transcribe either allele but have a low probability of doing so. By contrast, long-term Th2 clones show distinct and heritable ratios in the proportion of cells that express IL-4 or GFP. We conclude that in the course of Th2 priming an early efficient event renders both alleles capable of being inefficiently transcribed; a second, less frequent event occurs that renders one allele more competent, accounting for the differential expression of IL-4 and GFP in different clones.     

Multiple candidate gene analysis identifies alpha-synuclein as a susceptibility gene for sporadic Parkinson's disease

Parkinson's disease (PD), one of the most common human neurodegenerative diseases, is characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. PD is a complex disorder with multiple genetic and environmental factors influencing disease risk. To identify susceptible genes for sporadic PD, we performed case-control association studies of 268 single nucleotide polymorphisms (SNPs) in 121 candidate genes. In two independent case-control populations, we found that a SNP in alpha-synuclein (SNCA), rs7684318, showed the strongest association with PD (P=5.0 x 10(-10)). Linkage disequilibrium (LD) analysis using 29 SNPs in a region around rs7684318 revealed that the entire SNCA gene lies within a single LD block (D'>0.9) spanning approximately 120 kb. A tight LD group (r2>0.85) of six SNPs, including rs7684318, associated most strongly with PD (P=2.0 x 10(-9)-1.7 x 10(-11)). Haplotype association analysis did not show lower P-values than any single SNP within this group. SNCA is a major component of Lewy bodies, the pathological hallmark of PD. Aggregation of SNCA is thought to play a crucial role in PD. SNCA expression levels tended to be positively correlated with the number of the associated allele in autopsied frontal cortices. These findings establish SNCA as a definite susceptibility gene for sporadic PD.     

Promoter polymorphism in the candidate genes, IL-4, IL-9, TGF-beta1, for atopy and asthma

This paper will review genetic variations in the structure of three important candidate genes with varying effects in atopy and asthma that may have significant overall susceptibility associations in relation to the development of atopy and asthma. The three cytokine genes involved are interleukin (IL)-4, IL-9 and transforming growth factor beta1, and the nature of the polymorphisms may be related to significantly higher outputs of these cytokines in atopy and asthma.     

Linkage and candidate gene studies of autism spectrum disorders in European populations

Over the past decade, research on the genetic variants underlying susceptibility to autism and autism spectrum disorders (ASDs) has focused on linkage and candidate gene studies. This research has implicated various chromosomal loci and genes. Candidate gene studies have proven to be particularly intractable, with many studies failing to replicate previously reported associations. In this paper, we investigate previously implicated genomic regions for a role in ASD susceptibility, using four cohorts of European ancestry. Initially, a 384 SNP Illumina GoldenGate array was used to examine linkage at six previously implicated loci. We identify linkage approaching genome-wide suggestive levels on chromosome 2 (rs2885116, MLOD=1.89). Association analysis showed significant associations in MKL2 with ASD (rs756472, P=4.31 × 10⁻⁵) and between SND1 and strict autism (rs1881084, P=7.76 × 10⁻⁵) in the Finnish and Northern Dutch populations, respectively. Subsequently, we used a second 384 SNP Illumina GoldenGate array to examine the association in seven candidate genes, and evidence for association was found in RELN (rs362780, P=0.00165). Further increasing the sample size strengthened the association with RELN (rs362780, P=0.001) and produced a second significant result in GRIK2 (rs2518261, P=0.008). Our results strengthen the case for a more detailed study of the role of RELN and GRIK2 in autism susceptibility, as well as identifying two new potential candidate genes, MKL2 and SND1.     

IL-4 production by allergen-stimulated primary cultures: identification of basophils as the major IL-4-producing cell type

As a potent inducing agent for IgE production and differentiationfactor for allergen-specific T_h2 cells, IL-4 is a key regulatorycytokine both in the pathogenesis of allergic disease and inthe ongoing allergic response. The assay of In vitro IL-4 productionhas often been used to compare the allergen responses of T cellsIsolated from atopic and non-atopic subjects. Because peripheralblood basophils also have the capacity to respond to specificallergen by producing IL-4, we investigated the relative contributionof these two cell types to IL-4 production in allergen-stimulatedprimary cultures. Among unfractionated peripheral blood mononuclearcells (PBMC), the major producers of detectable IL-4 In primaryIn vitro cultures were found to be basophils based on: (i) anallergen dose-response corresponding closely to that requiredfor basophll histamlne release and lower than that requiredfor T cell activation; (II) a rapid time course for IL-4 production(detectable at 3 h), Inconsistent with the typical activationrequirements of fresh T cells; (iii) the production of comparablelevels of IL-4 In cultures stimulated with allergen or anti-lgE;and (Iv) the complete loss of detectable IL-4 production followingspecific depletion of basophils from PBMC. The T cells In thesecultures were functionally able to produce IL-4, as demonstratedby mitogen activation of basophll-depleted PBMC. These findingsdemonstrate that although IL-4 production In primary In vitrocultures can be used as a sensitive Indicator of allergen responsiveness,the accurate Interpretation of this result requires Identificationof the responding cell type. Furthermore, these findings raisethe possibility that basophll production of IL-4 early in thecourse of allergen stimulation may shape subsequent T cell responsesboth In vivo and In vitro.     

Linkage analysis and molecular haplotyping of the dopamine D4 receptor gene promoter region

OBJECTIVES: Polymorphic regions of the dopamine D4 receptor gene and its promoter region are in the focus of psychogenetic association studies. Besides the accurate phenotype characterization, highly reliable genotyping methods are also of outstanding importance in these works. METHODS: DNA samples of 598 healthy unrelated Caucasian individuals were used to validate the described molecular haplotyping methods and to determine the allele, genotype and haplotype frequencies and the linkage disequilibrium between the polymorphisms of the dopamine D4 receptor promoter region. RESULTS: We described a double genotyping system for the -521CT and -616CG polymorphisms, using a polymerase chain reaction restriction fragment length polymorphism or an allele-specific amplification. Allele and genotype frequencies of the novel -615AG single-nucleotide polymorphism are also determined (-615G=13.21%). For molecular haplotyping of the three single-nucleotide polymorphisms and a 120-bp duplication polymorphism, the allele-specific amplification was combined with restriction digestion. The results of the elaborated haplotyping methods were validated by molecular haplotyping of cloned fragments. CONCLUSIONS: The developed methods have been arranged into an 'economic' protocol that might be extended for higher reliability with a double haplotyping ('full mode'). Despite the close proximity of these sites, only a moderate linkage was found between the -615AG and -616CG (Delta(2)=0.162), between the -616AG and -521CT (Delta(2)=0.0221) and between the -615AG and -521CT single-nucleotide polymorphisms (Delta(2)=0.0346). The 120-bp duplication was shown to be in linkage equilibrium with any of the three single-nucleotide polymorphisms. Applications of these results should accelerate psychogenetic association studies of the dopamine D4 receptor gene.     

Organisation of the human PAX4 gene and its exclusion as a candidate for the Wolcott-Rallison syndrome.

Neonatal diabetes mellitus is a rare condition, the causes of which are mostly unknown. One well defined though very rare entity is the autosomal recessive Wolcott-Rallison syndrome, in which permanent neonatal diabetes, osteopenia, and epiphyseal dysplasia occur. Only five previous families have been reported, and here we describe the second in which parental consanguinity was present. The proband was born to first cousin parents and died at 2 years from the sequelae of poorly controlled diabetes. To test the hypothesis that mutation of PAX4, required in the mouse for pancreatic islet beta cell development, might cause WRS, the structure of the human PAX4 gene was deduced and DNA from two unrelated WRS patients sequenced. No PAX4 mutation was present, though the entire coding region was sequenced in both patients. It therefore appears unlikely that PAX4 is involved in the aetiology of Wolcott-Rallison syndrome, though it remains a good candidate for other forms of neonatal diabetes mellitus.     

Southern blot analysis of T-cell receptor gene rearrangements in cynomolgus monkeys,and identification of a progenitor cell HPRT mutation

Increases in peripheral blood T-lymphocyte HPRT mutant frequency may reflect either a number of independent HPRT gene mutational events or clonal proliferation of a single HPRT mutant. Sequence analysis of HPRT mutations in conjunction with T-cell receptor (TCR) gene rearrangement pattern analysis can distinguish these possibilities. Our laboratory previously characterized a nonhuman primate model for in vivo mutation studies using the clonal HPRT mutation assay. In the present study we report the use of probes for human TCR B and y genes to characterize TCR rearrangements in cynomolgus monkeys. Together, these methods were used to examine a monkey which exhibited a mean spontaneous HPRT mutant frequency (MF) of 16.4 × 10^?6, compared to the normal mean MF of 3.03 × 10^?6. The elevated MF resulted from the occurrence of a single HPRT mutation in a lymphocyte progenitor cell or stem cell, since T-cell clones isolated from the monkey exhibited a G to T transversion at base pair 539 in the HPRT coding region, and had unique rearrangements of TCR y along with an apparent germline TCR B configuration. In a preliminary in vivo mutation study, the animal was treated with the investigational potent mutagen and antitumor agent adozelesin (U-73975). No increase in HPRT mutant frequency was observed. The HPRT mutant clones isolated after treatment showed rearrangement of both TCR y and B genes. Possible explanations for these findings are discussed. © 1995 Wiley-Liss, Inc.