Recent genome-wide association studies (GWASs) and their meta-analyses have identified various genes and loci underlying the predisposition to ischemic stroke or coronary artery disease in Caucasian populations. Given that ischemic stroke and coronary artery disease may have a shared genetic archite
cture, certain polymorphisms may confer genetic susceptibility to these two diseases. The aim of the present study was to examine the possible association of ischemic stroke with 29 single-nucleotide polymorphisms (SNPs) previously identified by the meta-analyses of GWASs as susceptibility loci for coronary artery disease. The study population comprised 3,187 Japanese individuals, including 894 subjects with ischemic stroke and 2,293 controls. The genotypes for the 29 SNPs of the 28 genes were determined by a method that combines the polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. Comparisons of the allele frequencies by the ¿2 test between subjects with ischemic stroke and controls revealed that rs9319428 (G->A) of the fms-related tyrosine kinase 1 gene (P=0.0471), rs2075650 (G->A) of the translocase of outer mitochondrial membrane 40 homolog gene (TOMM40, P=0.0102) and rs273909 (T->C) of the solute carrier family 22, member 4 gene (SLC22A4, P=0.0097) were significantly (P<0.05) associated with the prevalence of ischemic stroke. Multivariable logistic regression analysis with adjustment for age, gender, body mass index, smoking status and the prevalence of hypertension, diabetes mellitus and dyslipidemia revealed that rs2075650 of TOMM40 (P=0.0443; recessive model; odds ratio=0.50) and rs273909 of SLC22A4 (P=0.0123; dominant model; odds ratio=0.45) were significantly associated with ischemic stroke with the minor G and C allele, respectively, being protective against this condition. TOMM40 and SLC22A4 may thus be susceptibility loci for ischemic stroke in Japanese individuals.
OCTN1 was immuno-detected in the cervical cancer cell HeLa, in which the complete pattern of acetylcholine metabolizing enzymes is expressed. Comparison of immuno-staining intensity of HeLa OCTN1 with the purified recombinant human OCTN1 allowed measuring the specific OCTN1 concentration in the HeLa
cell extract and, hence calculating the HeLa OCTN1 specific transport activity that was about 10 nmolxmin(-1)xmg protein(-1), measured as uptake of [(3)H]acetylcholine in proteoliposomes reconstituted with HeLa extract. This value was very similar to the specific activity of the recombinant protein. Acetylcholine transport was suppressed by incubation of the protein or proteoliposomes with the anti-OCTN1 antibody and was strongly inhibited by PLP and MTSEA, known inhibitors of OCTN1. The absence of ATP in the internal side of proteoliposomes strongly impaired transport function of both the HeLa and, as expected, the recombinant OCTN1. HeLa OCTN1 was inhibited by spermine, NaCl (Na(+)), TEA, gamma-butyrobetaine, choline, acetylcarnitine and ipratropium but not by neostigmine. Besides acetylcholine, choline was taken up by HeLa OCTN1 proteoliposomes. The transporter catalyzed also acetylcholine and choline efflux which, differently from uptake, was not inhibited by MTSEA. Time course of [(3)H]acetylcholine uptake in intact HeLa cells was measured. As in proteoliposomes, acetylcholine transport in intact cells was inhibited by TEA and NaCl. Efflux of [(3)H]acetylcholine occurred in intact cells, as well. The experimental data concur in demonstrating a role of OCTN1 in transporting acetylcholine and choline in HeLa cells.
A suitable experimental tool based on proteoliposomes for assaying Organic Cation Transporter Novel member 1 (OCTN1) of peritoneum was pointed out. OCTN1, recently acknowledged as acetylcholine transporter, was immunodetected in rat peritoneum. Transport was assayed following flux of radiolabelled
TEA, acetylcholine or acetylcarnitine in proteoliposomes reconstituted with peritoneum extract. OCTN1 mediated, besides TEA, also acetylcholine and a slower acetylcarnitine transport. External sodium inhibited acetylcholine uptake but not its release from proteoliposomes. Differently, sodium did not affect acetylcarnitine uptake. These results suggested that physiologically, acetylcholine should be released while acetylcarnitine was taken up by peritoneum cells. Transport was impaired by OCTN1 inhibitors, butyrobetaine, spermine, and choline. Biotin was also found as acetylcholine transport inhibitor. Anti-OCTN1 antibody specifically inhibited acetylcholine transport confirming the involvement of OCTN1. The transporter was also immunodetected in human mesothelial primary cells. Extract from these cells was reconstituted in proteoliposomes. Transport features very similar to those found with rat peritoneum were observed. Validation of the proteoliposome model for peritoneal transport study was then achieved assaying transport in intact mesothelial cells. TEA, butyrobetaine and Na(+) inhibited acetylcholine transport in intact cells while efflux was Na(+) insensitive. Therefore transport features in intact cells overlapped those found in proteoliposomes.
Ben Said M, etal., Hum Genet. 2016 May;135(5):513-24. doi: 10.1007/s00439-016-1657-7. Epub 2016 Mar 29.
The high prevalence/incidence of hearing loss (HL) in humans makes it the most common sensory defect. The majority of the cases are of genetic origin. Non-syndromic hereditary HL is extremely heterogeneous. Genetic approaches have been instrumental in deciphering genes that are crucial for auditory
function. In this study, we first used NADf chip to exclude the implication of known North-African mutations in HL in a large consanguineous Tunisian family (FT13) affected by autosomal recessive non-syndromic HL (ARNSHL). We then performed genome-wide linkage analysis and assigned the deafness gene locus to ch:5q23.2-31.1, corresponding to the DFNB60 ARNSHL locus. Moreover, we performed whole exome sequencing on FT13 patient DNA and uncovered amino acid substitution p.Cys113Tyr in SLC22A4, a transporter of organic cations, cosegregating with HL in FT13 and therefore the cause of ARNSHL DFNB60. We also screened a cohort of small Tunisian HL families and uncovered an additional deaf proband of consanguineous parents that is homozygous for p.Cys113Tyr carried by the same microsatellite marker haplotype as in FT13, indicating that this mutation is ancestral. Using immunofluorescence, we found that Slc22a4 is expressed in stria vascularis (SV) endothelial cells of rodent cochlea and targets their apical plasma membrane. We also found Slc22a4 transcripts in our RNA-seq library from purified primary culture of mouse SV endothelial cells. Interestingly, p.Cys113Tyr mutation affects the trafficking of the transporter and severely alters ergothioneine uptake. We conclude that SLC22A4 is an organic cation transporter of the SV endothelium that is essential for hearing, and its mutation causes DFNB60 form of HL.
Han TU, etal., Autoimmunity. 2015;48(5):313-7. doi: 10.3109/08916934.2015.1016219. Epub 2015 Feb 24.
Two single-nucleotide polymorphisms (SNPs) in SLC22A4 encoding an organic cation/zwitterion transporter protein, rs2073838 (commonly called slc2F1) and rs3792876 (slc2F2), had been associated with susceptibility to rheumatoid arthritis (RA) in two Japanese and o
ne recent Chinese studies but not in other two Japanese and six Caucasian studies. In this study, the two SNPs were genotyped for 2313 Korean participants and their associations with RA susceptibility and severity were examined. SNP association with RA susceptibility was tested among 1304 RA patients and 1009 healthy controls, and association with joint erosion among 1063 erosive and 241 non-erosive RA patients. Meta-analysis for RA susceptibility association was additionally performed using 10 previous studies and the current one. The two SNPs were almost perfectly correlated with each other (r(2 )= 0.98), and therefore only slc2F1 was tested for association. RA susceptibility association was not found in Koreans (p = 0.93), but still significant in meta-analysis of six Asian studies including this Korean study (p = 0.00036, odds ratio = 1.1) or all 11 studies additionally including five Caucasian studies (p = 0.00021, odds ratio = 1.1). In contrast, an association was found for RA severity in Koreans. The minor allele A was marginally associated with 1.5-fold increased risk of joint erosion among RA patients afflicted for SLC22A4 was associated with joint erosion in not-very-longstanding RA, although RA susceptibility association was weak and its clinical significance was uncertain.
It has been reported that organic cation/carnitine transporter 1 (OCTN1) is associated with rheumatoid arthritis and Crohn's disease. Additionally, we reported that OCTN1 is expressed in hematopoietic cells, and is associated with proliferation and differentiation of erythroid cells. However, physio
logical role of OCTN1 is still unclear. Ergothioneine, an anti-oxidant, was recently reported to be a good substrate of human OCTN1. However, the transport characteristics of ergothioneine in rat remains to be clarified. The present study, is to further investigate the role of rat Octn1 on transport of ergothioneine in rat Octn1 transfected cells and natively expressing cell line PC12 derived from rat adrenal pheochromocytoma. [(3)H]Ergothioneine uptake by rat Octn1 stably transfected HEK293 cells was saturable, sodium dependent with 1 : 1 stoichiometry of ergothioneine, and pH dependent. Since ergothioneine was reported to presumably play a protective role against oxidative stress-induced apoptosis in PC12 cells, its transport in this cell line was investigated. The expression of rat Octn1 and a saturable and Na(+)-dependent transport of ergothioneine were observed in PC12 cells, suggesting that ergothioneine transport in this cell line may be mediated by rat Octn1. These findings suggested that rat Octn1 may act as a survival factor by taking up ergothioneine to suppress oxidative stress in this cell line. In conclusion, functional characteristics of ergothioneine transport by rat Octn1 is similar to that of human OCTN1 and it is suggested that rat Octn1 is important by transporting anti-oxidant ergothioneine in PC12 cells, though its role in vivo is to be investigated.
OBJECTIVE: In a Japanese study, the C6607T SNP mapping to intron 1 of the SLC22A4 gene encoding the OCTN1 protein was found to be associated with rheumatoid arthritis. Similarly, a G24658C transversion in intron 6 of the gene encoding the RUNX1 transcription fac
tor that regulates OCTN1 and also likely OCTN2 expression was also found to confer susceptibility to the disease. METHODS: We investigated the prevalence of these two SNPs by RFLP analysis in a cohort of 209 Hungarian rheumatoid arthritis patients, and 217 healthy controls. Since both the OCTN1 and OCTN2 play a central role in the transmembrane transport of carnitine, we also determined the quantitative serum carnitine ester profile by ESI tandem mass spectrometry. RESULTS: No statistically significant differences were found comparing the genotype prevalence rates between the patients and the controls for either the SLC22A4 genotypes or for the RUNX1 SNPs. There was no significant difference in the serum carnitine ester profile when the rheumatoid arthritis patients were compared with the controls; furthermore, no significant difference in the carnitine esters could be detected when genotype specific subgroups of the patients and the controls were studied. CONCLUSION: Data of the current study do not confirm the universal and population independent susceptibility role of the SLC22A4 C6607T and RUNX1 G24658C variants for rheumatoid arthritis; furthermore, the data presented here show, that there are no significant carnitine-metabolism associated functional consequences of the different genotypes evidenced by the lack of detectable differences in the carnitine ester profiles.
Takata Y, etal., J Hum Genet. 2008;53(2):163-73. Epub 2007 Dec 18.
We conducted population-based association tests for the four selected SNPs (rs2240340/padi4_94, rs7528684/fcrl3_3, rs3792876/slc2F2 and rs2268277/runx1) previously reported to be associated with rheumatoid arthritis (RA). The study population consisted of 950 unrelated Japanese subjects with RA and
507 controls, none of whom had previously been tested for these variants. Only the SNP rs2240340/padi4_94 was modestly associated with RA [allele odds ratio (OR) 1.22, 95% confidence interval (CI) 1.04-1.43, P=0.012]. The most significant association effect was found for genotype contrast between minor and major allele homozygotes (OR 1.53, 95% CI 1.10-2.12, P=0.010). No other SNPs showed a statistically significant association with RA in our population. Meta-analysis of published studies and our new data confirmed a highly significant association between PADI4 gene SNPs and increased risk of RA in East Asian populations (allele fixed-effects summary OR 1.31, 95% CI 1.22-1.41, P<0.0001). We found some evidence for an association of either rs7528684/fcrl3_3 or rs3792876/slc2F2 with RA; however, because the magnitudes of effects were apparently much weaker than those reported in the initial positive reports, and there were substantial levels of inter-study OR heterogeneity, we concluded that additional studies are needed to fully understand the present results.
OBJECTIVE: To replicate the association reported in Japanese individuals of functional SLC22A4 and RUNX1 polymorphisms with rheumatoid arthritis (RA), and to test the possible role in this trait of a functional variant of the SUMO4 gene that was shown to be asso
ciated with another related autoimmune disease, type 1 diabetes (T1D). METHODS: Our study population consisted of 886 patients with RA and 987 healthy controls. All subjects were of Spanish Caucasian origin. We conducted a case-control association study with 6 single-nucleotide polymorphisms (SNP) spanning the SLC22A4 gene. SNP mapping in the RUNX1 gene associated with RA in a Japanese population and a SUMO4 polymorphism associated with T1D were also studied. RESULTS: No statistically significant differences between patients with RA and healthy controls were observed when comparing the distribution of the genotypes or alleles of any of the SLC22A4 polymorphisms tested. Similarly, no evidence of association between RA and the SLC22A4 haplotype previously reported to be associated in a Japanese population was found. With regard to the RUNX1 and SUMO4 SNP, we did not observe statistically significant differences in the distribution of genotypes or alleles between patients with RA and healthy controls. CONCLUSION: These results suggest that the SLC22A4, RUNX1, and SUMO4 polymorphisms analyzed do not confer a relevant role in susceptibility to RA in the Spanish population.
