RNA binding proteins PTBP1 and HNRNPL regulate CFTR mRNA decay.

Background: CFTR nonsense alleles generate negligible CFTR protein due to the nonsense mutation: 1) triggering CFTR mRNA degradation by nonsense-mediated mRNA decay (NMD), and 2) terminating CFTR mRNA translation prematurely. Thus, people with cystic fibrosis (PwCF) who carry nonsense alleles cannot benefit from current modulator drugs, which target CFTR protein. In this study, we examined whether PTBP1 and HNRNPL, two RNA binding proteins that protect a subset of mRNAs with a long 3' untranslated region (UTR) from NMD, similarly affect CFTR mRNA.Silencing RNAs were used to deplete PTBP1 or HNRNPL in 16HBE14o- human bronchial epithelial cells expressing WT, G542X, or W1282X CFTR. CFTR mRNA abundance was measured relative to controls by quantitative PCR. PTBP1 and HNRNPL were also exogenously expressed in each cell line and CFTR mRNA levels were similarly quantified. Results: PTBP1 depletion reduced CFTR mRNA abundance in all three 16HBE14o- cell lines; HRNPL depletion reduced CFTR mRNA abundance in only the G542X and W1282X cell lines. Notably, decreased CFTR mRNA abundance correlated with increased mRNA decay. Exogenous expression of PTBP1 or HNRNPL increased CFTR mRNA abundance in all three cell lines; HNRNPL overexpression generally increased CFTR to a greater extent in G542X and W1282X 16HBE14o- cells.Our data indicate that PTBP1 and HNRNPL regulate CFTR mRNA abundance by protecting CFTR transcripts from NMD. This suggests that PTBP1 and/or HNRNPL may represent potential therapeutic targets to increase CFTR mRNA abundance and enhance responses to CFTR modulators and other therapeutic approaches in PwCF.

A regulated NMD mouse model supports NMD inhibition as a viable therapeutic option to treat genetic diseases.

Nonsense-mediated mRNA decay (NMD) targets mRNAs that contain a premature termination codon (PTC) for degradation, preventing their translation. By altering the expression of PTC-containing mRNAs, NMD modulates the inheritance pattern and severity of genetic diseases. NMD also limits the efficiency of suppressing translation termination at PTCs, an emerging therapeutic approach to treat genetic diseases caused by in-frame PTCs (nonsense mutations). Inhibiting NMD may help rescue partial levels of protein expression. However, it is unclear whether long-term, global NMD attenuation is safe. We hypothesize that a degree of NMD inhibition can be safely tolerated after completion of prenatal development. To test this hypothesis, we generated a novel transgenic mouse that expresses an inducible, dominant-negative form of human UPF1 (dnUPF1) to inhibit NMD in mouse tissues by different degrees, allowing us to examine the effects of global NMD inhibition in vivo A thorough characterization of these mice indicated that expressing dnUPF1 at levels that promote relatively moderate to strong NMD inhibition in most tissues for a 1-month period produced modest immunological and bone alterations. In contrast, 1 month of dnUPF1 expression to promote more modest NMD inhibition in most tissues did not produce any discernable defects, indicating that moderate global NMD attenuation is generally well tolerated in non-neurological somatic tissues. Importantly, a modest level of NMD inhibition that produced no overt abnormalities was able to significantly enhance in vivo PTC suppression. These results suggest that safe levels of NMD attenuation are likely achievable, and this can help rescue protein deficiencies resulting from PTCs.

Reduced bone length, growth plate thickness, bone content, and IGF-I as a model for poor growth in the CFTR-deficient rat.

BACKGROUND: Reduced growth and osteopenia are common in individuals with cystic fibrosis (CF). Additionally, improved weight and height are associated with better lung function and overall health in the disease. Mechanisms for this reduction in growth are not understood. We utilized a new CFTR knockout rat to evaluate growth in young CF animals, via femur length, microarchitecture of bone and growth plate, as well as serum IGF-I concentrations. METHODS: Femur length was measured in wild-type (WT) and SD-CFTRtm1sage (Cftr-/-) rats, as a surrogate marker for growth. Quantitative bone parameters in Cftr-/- and WT rats were measured by micro computed tomography (micro-CT). Bone histomorphometry and cartilaginous growth plates were analyzed. Serum IGF-I concentrations were also compared. RESULTS: Femur length was reduced in both Cftr-/- male and female rats compared to WT. Multiple parameters of bone microarchitecture (of both trabecular and cortical bone) were adversely affected in Cftr-/- rats. There was a reduction in overall growth plate thichkness in both male and female Cftr-/- rats, as well as hypertrophic zone thickness and mean hypertrophic cell volume in male rats, indicating abnormal growth characteristics at the plate. Serum IGF-I concentrations were severely reduced in Cftr-/- rats compared to WT littermates. CONCLUSIONS: Despite absence of overt lung or pancreatic disease, reduced growth and bone content were readily detected in young Cftr-/- rats. Reduced size of the growth plate and decreased IGF-I concentrations suggest the mechanistic basis for this phenotype. These findings appear to be intrinsic to the CFTR deficient state and independent of significant clinical confounders, providing substantive evidence for the importance of CFTR on maintinaing normal bone growth.

Increasing the Endoplasmic Reticulum Pool of the F508del Allele of the Cystic Fibrosis Transmembrane Conductance Regulator Leads to Greater Folding Correction by Small Molecule Therapeutics.

Small molecules that correct the folding defects and enhance surface localization of the F508del mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) comprise an important therapeutic strategy for cystic fibrosis lung disease. However, compounds that rescue the F508del mutant protein to wild type (WT) levels have not been identified. In this report, we consider obstacles to obtaining robust and therapeutically relevant levels of F508del CFTR. For example, markedly diminished steady state amounts of F508del CFTR compared to WT CFTR are present in recombinant bronchial epithelial cell lines, even when much higher levels of mutant transcript are present. In human primary airway cells, the paucity of Band B F508del is even more pronounced, although F508del and WT mRNA concentrations are comparable. Therefore, to augment levels of "repairable" F508del CFTR and identify small molecules that then correct this pool, we developed compound library screening protocols based on automated protein detection. First, cell-based imaging measurements were used to semi-quantitatively estimate distribution of F508del CFTR by high content analysis of two-dimensional images. We evaluated ~2,000 known bioactive compounds from the NIH Roadmap Molecular Libraries Small Molecule Repository in a pilot screen and identified agents that increase the F508del protein pool. Second, we analyzed ~10,000 compounds representing diverse chemical scaffolds for effects on total CFTR expression using a multi-plate fluorescence protocol and describe compounds that promote F508del maturation. Together, our findings demonstrate proof of principle that agents identified in this fashion can augment the level of endoplasmic reticulum (ER) resident "Band B" F508del CFTR suitable for pharmacologic correction. As further evidence in support of this strategy, PYR-41-a compound that inhibits the E1 ubiquitin activating enzyme-was shown to synergistically enhance F508del rescue by C18, a small molecule corrector. Our combined results indicate that increasing the levels of ER-localized CFTR available for repair provides a novel route to correct F508del CFTR.

Osteoblast CFTR inactivation reduces differentiation and osteoprotegerin expression in a mouse model of cystic fibrosis-related bone disease.

Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF). CF-related bone disease (CFBD) is characterized by uncoupled bone turnover--impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR), the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr-/-) mouse model. In the murine calvarial organ culture assay, Cftr-/- calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+) littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr-/- compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl) mRNA was detected, significantly less osteoprotegerin (Opg) was expressed in Cftr-/- compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr-/- murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt signaling was defective in Cftr-/- murine calvarial osteoblasts. These results support that genetic inactivation of CFTR in osteoblasts contributes to low bone mass and that targeting osteoblasts may represent an effective strategy to treat CFBD.

The silent codon change I507-ATC->ATT contributes to the severity of the ΔF508 CFTR channel dysfunction.

The most common disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the out-of-frame deletion of 3 nucleotides (CTT). This mutation leads to the loss of phenylalanine-508 (ΔF508) and a silent codon change (SCC) for isoleucine-507 (I507-ATC→ATT). ΔF508 CFTR is misfolded and degraded by endoplasmic reticulum-associated degradation (ERAD). We have demonstrated that the I507-ATC→ATT SCC alters ΔF508 CFTR mRNA structure and translation dynamics. By comparing the biochemical and functional properties of the I507-ATT and I507-ATC ΔF508 CFTR, we establish that the I507-ATC→ATT SCC contributes to the cotranslational misfolding, ERAD, and to the functional defects associated with ΔF508 CFTR. We demonstrate that the I507-ATC ΔF508 CFTR is less susceptible to the ER quality-control machinery during translation than the I507-ATT, although 27°C correction is necessary for sufficient cell-surface expression. Whole-cell patch-clamp recordings indicate sustained, thermally stable cAMP-activated Cl(-) transport through I507-ATC and unstable function of the I507-ATT ΔF508 CFTR. Single-channel recordings reveal improved gating properties of the I507-ATC compared to I507-ATT ΔF508 CFTR (NPo=0.45±0.037 vs. NPo=0.09±0.002; P<0.001). Our results signify the role of the I507-ATC→ATT SCC in the ΔF508 CFTR defects and support the importance of synonymous codon choices in determining the function of gene products.

Association of cystic fibrosis genetic modifiers with congenital bilateral absence of the vas deferens.

OBJECTIVE: To investigate whether genetic modifiers of cystic fibrosis (CF) lung disease also predispose to congenital bilateral absence of the vas deferens (CBAVD) in association with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. We tested the hypothesis that polymorphisms of transforming growth factor (TGF)-ß1 (rs 1982073, rs 1800471) and endothelin receptor type A (EDNRA) (rs 5335, rs 1801708) are associated with the CBAVD phenotype. DESIGN: Genotyping of subjects with clinical CBAVD. SETTING: Outpatient and hospital-based clinical evaluation. PATIENT(S): DNA samples from 80 subjects with CBAVD and 51 healthy male controls from various regions of Europe. This is one of the largest genetic studies of this disease to date. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genotype analysis. RESULT(S): For single nucleotide polymorphism (SNP) rs 5335, we found increased frequency of the CC genotype among subjects with CBAVD. The difference was significant among Turkish patients versus controls (45.2% vs. 19.4%), and between all cases versus controls (36% vs. 15.7%). No associations between CBAVD penetrance and polymorphisms rs 1982073, rs 1800471, or rs 1801708 were observed. CONCLUSION(S): Our findings indicate that endothelin receptor type A polymorphism rs 5335 may be associated with CBAVD penetrance. To our knowledge, this is the first study to investigate genetic modifiers relevant to CBAVD.

Trends in the epidemiology of human G1P[8] rotaviruses: a hungarian study.

Epidemiological trends of the globally most common rotavirus genotype, G1P[8], were investigated in Hungary during a 16-year period by sequencing and phylogenetic analysis of the surface antigens. Antigen shift among epidemiologically major G1P[8] strains was observed in 6 seasons, as indicated by changes in the sublineages of the G1 VP7 and the P[8] VP4 genes. The temporal clustering of some rotavirus VP4 and VP7 gene sublineages and the periodic emergence and/or resurgence of previously unrecognized rotavirus sublineages in the study population suggest a dynamic nature for these common strains. Recently established international strain surveillance networks may help to identify and track the spread of epidemiologically important rotavirus strains across countries and continents.

The role of the F508C mutation in congenital bilateral absence of the vas deferens.

PURPOSE: Congenital bilateral absence of the vas deferens is a pathologic condition associated with normal spermatogenesis, azoospermia, and lack of both vasa deferentia. A significant association between mutations in the cystic fibrosis transmembrane conductance regulator gene among men with congenital bilateral absence of the vas deferens has been established. The objective of this study was to determine whether the F508C variant in the cystic fibrosis transmembrane conductance regulator gene has a significant effect on congenital bilateral absence of the vas deferens prevalence, when present in conjunction with a second cystic fibrosis transmembrane conductance regulator disease causing mutation. METHODS AND RESULTS: We compared the frequency of F508C in male subjects submitted for diagnostic testing on suspicion of cystic fibrosis or during cystic fibrosis carrier screening, to men with a clinical diagnosis of congenital bilateral absence of the vas deferens. Although frequencies of F508C did not vary significantly between 850 individuals undergoing cystic fibrosis carrier screening and those submitted for diagnostic testing on suspicion of cystic fibrosis, the frequency of F508C in the congenital bilateral absence of the vas deferens population was significantly higher than expected (chi2 = 6.95, corrected P = 0.0486). CONCLUSION: We conclude that the F508C variant in cystic fibrosis transmembrane conductance regulator may represent a pathogenic defect and lead to congenital bilateral absence of the vas deferens when combined with a second cystic fibrosis transmembrane conductance regulator mutation.

Inhibitory effects of hypertonic saline on P. aeruginosa motility.

Salt transport defects in CF lungs predispose to overwhelming and fatal respiratory infection caused by Pseudomonas aeruginosa. Motility of this organism is central to pathogenesis in a number of settings. Incubation of numerous strains of P. aeruginosa with hypertonic saline caused a concentration-dependent decrease in bacterial motility. Reduction of P. aeruginosa virulence through this effect may contribute to clinical efficacy of hypertonic saline aerosols in CF patients.

Does glycoprotein IIIa gene (Pl(A)) polymorphism influence clopidogrel resistance? : a study in older patients.

BACKGROUND: Clopidogrel is a potent antiplatelet drug used for secondary prevention after ischaemic cardiovascular or cerebrovascular events. In patients with aspirin (acetylsalicylic acid) intolerance or resistance, it is used as monotherapy. Recent data report that Pl(A) polymorphism of the glycoprotein IIIa gene may account for differences in aspirin-induced antiplatelet effects. An increased degree of platelet reactivity was also reported in Pl(A2) carriers compared with Pl(A1/A1) patients after administration of a clopidogrel 300mg loading dose. OBJECTIVES: The aim of this study was to assess the modulatory effect of the Pl(A2) allele on platelet aggregation in patients taking long-term clopidogrel. M ETHODS: The prevalence of the Pl(A2) allele was assessed in 38 (21 males, 17 females; mean age 63 +/- 13 years) clopidogrel-resistant and 59 (26 males, 33 females; mean age 63 +/- 11 years) clopidogrel-responsive patients. The polymerase chain reaction-restriction fragment length polymorphism method was utilised to evaluate Pl(A) polymorphism. A Carat TX4 optical platelet aggregometer (Carat Diagnostics Ltd, Budapest, Hungary) was used to measure 5 and 10 micromol/L adenosine diphosphate-induced platelet aggregation. RESULTS: Significantly more patients were taking combination antiplatelet therapy in the clopidogrel-resistant group than in the clopidogrel-responsive group (50% vs 30%, respectively). The prevalence of the Pl(A2) allele did not differ significantly between the two groups (0.09 vs 0.13), even after adjustment for combination therapy and various risk factors. CONCLUSIONS: Our results show that carriers of the Pl(A2) allele do not have an increased risk of clopidogrel resistance. These findings and data from our previous studies suggest that patients with a Pl(A2) allele homozygosity may benefit from antiplatelet therapy based on clopidogrel rather than aspirin.

Histopathologic abnormalities of the lymphoreticular tissues in organic cation transporter 2 deficiency: evidence for impaired B cell maturation.

Immunohistology of lymphoreticular tissues of a fatal case of organic cation transporter 2 deficiency revealed inhibited proliferation with increased apoptosis in the germinal centers, resulting in "burned out" follicles. This is indicative of impaired antigen driven B cell affinity maturation. Defective humoral immune response might explain the recurrent infections in untreated organic cation transporter 2 deficiency.

Apolipoprotein A5 gene promoter region T-1131C polymorphism associates with elevated circulating triglyceride levels and confers susceptibility for development of ischemic stroke.

The possible pathogenic role of triglycerides (TG) in the development of ischemic stroke is still under extensive investigation. Recently, apolipoprotein (apo)A5 gene promoter region T-1131C polymorphism has been shown to associate with elevated serum TG levels. In the current work, a total of 302 subjects were classified as being large vessel-associated, small vessel-associated, or belonging to a mixed group of ischemic stroke-affected patients. The level of TG was increased in all groups (p < 0.01). The apoA5-1131C allele frequency was approximately twofold in all groups of stroke patients compared with the controls (5 vs 10-12%; p < 0.05); and the apoA5-1131C allele itself was also found to associate with increased TG levels in all groups. In a multivariate logistic regression analysis model adjusted for differences in age, gender, serum cholesterol, hypertension, presence of diabetes mellitus, smoking and drinking habits, and ischemic heart disease, a significantly increased risk of developing stroke disease was found in patients carrying the apoA5-1131C allele (p < 0.05; odds ratio OR = 2.1 [1.3-4.7]); this association was also proven for all subtypes of the stroke. The results presented here suggest that the apoA5-1131C allele is an independent risk factor for the development of stroke. Being that apoA5 gene is under the control of the peroxisome proliferator-activated receptor alpha, theoretically, the current observations also can have long-term therapeutic consequences.

Genetic analysis of candidate genes modifying the age-at-onset in Huntington's disease.

The expansion of a polymorphic CAG repeat in the HD gene encoding huntingtin has been identified as the major cause of Huntington's disease (HD) and determines 42-73% of the variance in the age-at-onset of the disease. Polymorphisms in huntingtin interacting or associated genes are thought to modify the course of the disease. To identify genetic modifiers influencing the age at disease onset, we searched for polymorphic markers in the GRIK2, TBP, BDNF, HIP1 and ZDHHC17 genes and analysed seven of them by association studies in 980 independent European HD patients. Screening for unknown sequence variations we found besides several silent variations three polymorphisms in the ZDHHC17 gene. These and polymorphisms in the GRIK2, TBP and BDNF genes were analysed with respect to their association with the HD age-at-onset. Although some of the factors have been defined as genetic modifier factors in previous studies, none of the genes encoding GRIK2, TBP, BDNF and ZDHHC17 could be identified as a genetic modifier for HD.

Angiotensin II type-1 receptor A1166C polymorphism is associated with increased risk of ischemic stroke in hypertensive smokers.

Recent observations revealed a novel role of angiotensin-converting enzyme 2 and the angiotensin II type-1 receptor (AT1R) in lung injury, thereby extending knowledge about the functions of the angiotensin system. Angiotensin II, whose target is the AT1R, is a potent vasoconstrictor. Accordingly, an imbalance leading to enhanced activity of the angiotensin II-AT1R axis is postulated to contribute to both circulatory disturbances and lung injury. In this context, a functional single-nucleotide polymorphism, AT1R A1166C, which leads to enhanced responsiveness of the AT1R, has been postulated as a candidate susceptibility factor for ischemic stroke. The aim of our study was to investigate its occurrence in ischemic stroke and to analyze its possible synergistic associations with clinical risk factors. Genetic and clinical data on 308 consecutive patients with acutely developing ischemic stroke were analyzed. A total of 272 stroke and neuroimaging alteration-free subjects served as a control group. Univariate and logistic regression statistical approaches were used. Alone, the AT1R 1166C allele did not pose a risk of stroke. In hypertensive smokers, however, it was associated with an increased risk of ischemic stroke (OR 22.3, 95% CI 5.8-110.2, p<0.001). Further subgroup analysis revealed the same association for both small-vessel (OR 24.3, 95% CI 6.1-121.1, p<0.001) and large-vessel (OR 21.3, 95% CI 4.6-81.1, p<0.001) infarction. On a pathophysiological basis, our results suggest the possibility that the AT1R A1166C polymorphism might give rise to ischemic stroke indirectly via an unfavorable effect on the cardiorespiratory function.

Changes of plasma fasting carnitine ester profile in patients with ulcerative colitis.

AIM: To determine the plasma carnitine ester profile in adult patients with ulcerative culitis (UC) and compared with healthy control subjects. METHOD: Using ESI triple quadrupole tandem mass spectrometry, the carnitine ester profile was measured in 44 patients with UC and 44 age- and sex-matched healthy controls. RESULTS: There was no significant difference in the fasting free carnitine level between the patients with UC and the healthy controls. The fasting propionyl- (0.331 +/- 0.019 vs 0.392 +/- 0.017 micromol/L), butyryl- (0.219 +/- 0.014 vs 0.265 +/- 0.012), and isovalerylcarnitine (0.111 +/- 0.008 vs 0.134 +/- 0.008) levels were decreased in the UC patients. By contrast, the level of octanoyl-(0.147 +/- 0.009 vs 0.114 +/- 0.008), decanoyl- (0.180 +/- 0.012 vs 0.137 +/- 0.008), myristoyl- (0.048 +/- 0.003 vs 0.039 +/- 0.003), palmitoyl- (0.128 +/- 0.006 vs 0.109 +/- 0.004), palmitoleyl- (0.042 +/- 0.003 vs 0.031 +/- 0.002) and oleylcarnitine (0.183 +/- 0.007 vs 0.163 +/- 0.007; P < 0.05 in all comparisons) were increased in the patients with UC. CONCLUSION: Our data suggest selective involvement of the carnitine esters in UC patients, probably due to their altered metabolism.

The goose reovirus genome segment encoding the minor outer capsid protein, sigma1/sigmaC, is bicistronic and shares structural similarities with its counterpart in Muscovy duck reovirus.

Reoviruses have recently been shown to be associated with disease in young geese and to be involved in epizooties of severe outcome in Hungary. To assess the genetic variability among these pathogenic goose reoviruses (GRVs), we sequenced the S4 genome segment of five GRV strains isolated from different diseased flocks. We found that the GRV S4 genome segment, consisting of two partially overlapping open reading frames (ORFs), shares substantial structural similarity with its counterpart in muscovy duck reoviruses (DRVs). ORF1 is predicted to encode a polypeptide highly similar to the p10 polypeptide of DRV, and ORF2 supposedly encodes the minor outer capsid protein, sigma1/sigmaC. In one of the five GRV strains examined, we identified a single uracil base insertion close to the middle of ORF2. This insertion resulted in a frameshift and in concomitant acquisition of a termination codon (UAA) a few codons downstream, apparently causing truncation of the C-terminal part of the protein. The functional consequences of this assumed mutation, which would result in loss of more than a half of the protein, have yet to be determined. Nonetheless, the sequence and structural similarities between the genome segment encoding sigmal/sigmaC in GRVs and DRVs suggest that these viruses belong to a species distinct from other established species within subgroup 2 of orthoreoviruses.

Lymphotoxin-alpha gene 252G allelic variant is a risk factor for large-vessel-associated ischemic stroke.

A direct role of lymphotoxin-alpha (LTA) in promoting atherosclerotic plaque growth has been demonstrated recently. The different protein transcripts of the naturally occurring genetic variants of the LTA gene have been demonstrated to exhibit affected functions, and an allelic difference in binding to transcription factor(s) has also been suggested. The homozygous variant of LTA characterized by the intron 1 252A-->G (252G) transition, which naturally coexists with an exon 3 804C-->A (804A) single-nucleotide polymorphism (SNP), has been reported as a susceptibility gene for myocardial infarction. Because the atherosclerotic process is also an integral component in the pathogenesis of certain types of vascular stroke, we investigated the possible significance of the above SNPs in 353 ischemic stroke patients and 180 healthy controls. The homozygous LTA allele with the 252G and 804C SNPs occurred more frequently in stroke patients (13.9%) than in controls (7.20%, p<0.025). Specific subclassification of the patients revealed an accumulation of these SNPs in large-vessel, pathology-associated cerebral infarction (18.2%); multivariate logistic regression analysis of the data confirmed this association, with an odds ratio of 2.1 (95% confidence interval, 1.3-6.2; p<0.005). Elimination of all subjects with a history or evidence of ischemic heart disease, including myocardial infarction, did not affect this association. These data show that besides the role in the development of myocardial infarction, the homozygous carriage of the LTA allele with 252G and 804A SNPs is a novel susceptibility factor for largevesselassociated ischemic stroke.