Sequence-specific Oligonucleotide Probes Research Articles

HLA-B allele dropout in PCR sequence-specific oligonucleotide probe typing due to intronic polymorphism in the novel B*58:01:01:02 allele.

Currently, Luminex technology based on the PCR sequence-specific oligonucleotide (SSO) probe method has been widely used for HLA genotyping in the immunogenetics laboratories. Here, we reported a case with HLA-B allele dropout by Luminex technology. The initial HLA-B result of the Luminex method with a commercial agent kit was inconclusive, and then, the result of PCR-SBT technology indicated the dropout as a HLA-B*58 allele. Subsequently, the full-length sequence of HLA-B allele was determined by TOPO-TA cloning, and a novel allele B*58:01:01:02 was identified in the individual. Compared with HLA-B*58:01:01:01, the novel allele showed some nucleotides difference at 509 C>T, 521 T>G and CCC insertion in position 503 of intron 2. According to the full-length sequence, the new mutations of intron 2 were contributed to HLA-B locus allele dropout in the sample. Our results indicated multiplatform should be used to improve the HLA typing accuracy when a conclusive HLA genotype cannot be determined.

Determining performance characteristics of an NGS-based HLA typing method for clinical applications.

This study presents performance specifications of an in-house developed human leukocyte antigen (HLA) typing assay using next-generation sequencing (NGS) on the Illumina MiSeq platform. A total of 253 samples, previously characterized for HLA-A, -B, -C, -DRB1 and -DQB1 were included in this study, which were typed at high-resolution using a combination of Sanger sequencing, sequence-specific primer (SSP) and sequence-specific oligonucleotide probe (SSOP) technologies and recorded at the two-field level. Samples were selected with alleles that cover a high percentage of HLA specificities in each of five different race/ethnic groups: European, African-American, Asian Pacific Islander, Hispanic and Native American. Sequencing data were analyzed by two software programs, Omixon's target and GenDx's NGSengine. A number of metrics including allele balance, sensitivity, specificity, precision, accuracy and remaining ambiguity were assessed. Data analyzed by the two software systems are shown independently. The majority of alleles were identical in the exonic sequences (third field) with both programs for HLA-A, -B, -C and -DQB1 in 97.7% of allele determinations. Among the remaining discrepant genotype calls at least one of the analysis programs agreed with the reference typing. Upon additional manual analysis 100% of the 2530 alleles were concordant with the reference HLA genotypes; the remaining ambiguities did not exceed 0.8%. The results demonstrate the feasibility and significant benefit of HLA typing by NGS as this technology is highly accurate, eliminates virtually all ambiguities, provides complete sequencing information for the length of the HLA gene and forms the basis for utilizing a single methodology for HLA typing in the immunogenetics labs.

A study of the frequencies of HLA-DQA1 alleles and their clinical application values in the donor-recipient HLA-10/10 matched hematopoietic stem cell transplantation

Objective To analyze the frequencies of HLA-DQA1 alleles and their clinical values in the donor-recipient HLA-A, -B, -C, -DRB1, -DQB1(10/10) matched hematopoietic stem cell transplantation(HSCT). Methods This study recruited 127 patients who received allogeneic HSCT and 127 unrelated donors. High-resolution (High Res) DNA typing for HLA-DQA1 alleles were performed on the 254 subjects by using sequence specific oligonucleotide probes(SSOP) and high resolution of sequence specific primer(High Res SSP). Results The DQA1 allele genotypes of 36 pairs of donor-recipient were directly identified by using SSOP. The ambiguous DQA1 allele genotypes of the rest 91 pairs were identified by using High Res SSP. Among the 127 pairs of donor-recipient, 5 pairs were HLA-DQA1 alleles mismatched, while the others were all matched. No significant differences in the distribution of HLA-DQA1 alleles were observed between the donors and the recipients. Sixteen HLA-DQA1 alleles were detected in the 127 donors, which were DQA1*02∶01 (19.3%), DQA1*01∶02(19.3%), DQA1*03∶02/03(17.0%), DQA1*01∶03 (9.8%), DQA1*06∶01(9.1%), DQA1*05∶01(7.1%), DQA1*05∶05(5.9%), DQA1*03∶01 (4.7%), DQA1*01∶04(2.4%), DQA1*01∶05(2.0%), DQA1*01∶01(1.2%), DQA1*05∶03(0.8%), DQA1*05∶08(0.8%), DQA1*04∶01(0.4%), DQA1*05∶06(0.4%) from high to low frequency. Moreover, a new allele was detected in the patients. The haplotypes' frequencies and linkage disequilibrium(LD) analysis of HLA-DQA1 and HLA-DQB1 showed that the most common haplotype was DQA1*02∶01-DQB1*02∶02(16.1%), followed by DQA1*03∶02/03-DQB1 *03∶03 (11.8%) and DQA1*01∶03-DQB1*06∶01 (9.1%). Stronger LD were observed between DQA1*02∶01 and DQB1*02∶02, DQA1*03∶02 and DQB1*03∶03, DQA1*01∶03 and DQB1*06∶01, HLA-DQA1*06∶01 and DQB1*03∶01, DQA1*05∶01 and DQB1*02∶02(P<0.001). Conclusion There was strong linkage disequilibrium between HLA-DQA1 and HLA-DQB1 genes. The polymorphism of HLA-DQA1 gene was less than that of HLA-DQB1 gene. No more guidance was provided to donor selection in unrelated donor-recipient HLA matched HSCT by adding HLA-DQA1 genotyping, but it might have clinical application values in HSCT with HLAⅡ locus mismatched donor and recipient. Key words: High resolution; HLA-DQA1 gene; HLA-DQB1 gene; Gene frequency; Linkage disequilibrium

Hashimoto tiroidit hastalarında HLA-DRB1 alellerinin dağılımı

Objective: Hashimoto’s thyroiditis (HT) is an autoimmune thyroid disease resulting from complex interactions between genetic and environmental factors. The Human Leukocyte Antigen (HLA) genes have been found to be involved in the susceptibility to HT. In this retrospective study, we aimed to investigate the associations between HLA-DRB1 alleles and patients with HT. Material and Methods: Forty eight patients with HT were included in this study. We analyzed HLA-DRB1 by polymerase chain reaction with sequence specific oligonucleotide probes (PCR-SSOP), and compared them with 126 unrelated healthy controls. Results: The HLA-DRB1*03 allele was higher in the HT patients than in the controls (p=0.006). Also, the HLA-DRB1*01 allele was lower in the HT patients than in the controls (p=0.021). Conclusion: The susceptible and protectable alleles were found as HLA-DRB1*03 and HLA-DRB1*01 alleles respectively in patients with HT in the Kayseri Turkish population, in Turkey. Further studies with a larger cohort are required to confirm the role of HLA-DRB1 alleles in the development of HT in the Turkish population.

Association of genetic variants with atrial fibrillation.

Recent genome-wide association studies (GWASs) identified various genes and loci that confer susceptibility to coronary artery disease or myocardial infarction among Caucasian populations. As myocardial ischemia is an important risk factor for atrial fibrillation, we hypothesized that certain polymorphisms may contribute to the genetic susceptibility to atrial fibrillation through affecting the susceptibility to coronary artery disease. The aim of the present study was to examine the possible association of atrial fibrillation in Japanese individuals with 29 polymorphisms identified as susceptibility loci for coronary artery disease or myocardial infarction in the meta-analyses of GWASs in Caucasian populations. The study subjects comprised 5,470 Japanese individuals (305 subjects with atrial fibrillation and 5,165 controls). Genotypes for 29 polymorphisms 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 χ2 test revealed that rs599839 (G→A) of the proline/serine-rich coiled-coil 1 gene (PSRC1, P=0.0084) and rs11556924 (C→T, Arg363His) of the zinc finger, C3HC-type containing 1 gene (ZC3HC1, P=0.0076) were significantly (P<0.01) associated with atrial fibrillation. Multivariable logistic regression analysis with adjustment for age, gender, body mass index, estimated glomerular filtration rate, and the prevalence of smoking, hypertension, diabetes mellitus, and dyslipidemia revealed that rs599839 (P=0.0043; odds ratio, 1.56; dominant model) and rs11556924 (P=0.0043; odds ratio, 1.93; dominant model) were significantly associated with atrial fibrillation, with the minor G and T alleles, respectively, representing risk factors for this condition. PSRC1 and ZC3HC1 may thus be susceptibility loci for atrial fibrillation in Japanese individuals.

Significant association of the KIR2DL3/HLA-C1 genotype with susceptibility to Crohn’s disease

We aimed to analyze the possible association of KIR/HLA-C genotypes with the susceptibility to Crohn’s disease (CD) in a Spanish population. A total of 125 patients with CD and 339 healthy controls were selected for this study. KIR and HLA-C typing were developed by sequence-specific oligonucleotide probing. We found that the centromeric A/A genotype and HLA-C1 combination was significantly increased in CD patients (P<10−3). The KIR2DL3/2DL3 genotype was significantly increased in CD patients (P<0.0005). Moreover, we also observed a highly significant increase of KIR2DL3-HLA-C1 homozygosis in CD patients (P<0.0005). Our results confirm the relevance of the KIR2DL2/KIR2DL3 genes and their interaction with HLA-C to CD. We show that the contribution of the KIR genes to CD susceptibility extends beyond the association with individual KIRs, with an imbalance between activating and inhibitory KIR genes seeming to influence the susceptibility to CD.

Rapid KRAS Mutation Detection via Hybridization-Induced Aggregation of Microbeads.

Using hybridization-induced aggregation (HIA), a unique bead-based DNA detection technology scalable for a microchip platform, we describe a simplistic, low-cost method for rapid mutation testing. HIA utilizes a pair of sequence-specific oligonucleotide probes bound to magnetic microbeads. Hybridization to a target DNA strand tethers the beads together, inducing bead aggregation. By simply using the extent of bead aggregation as a measure of the hybridization efficiency, we avoid the need for additional labels and sophisticated analytical equipment. Through strategic manipulation of the assay design and experimental parameters, we use HIA to facilitate, for the first time, the detection of single base mutations in a gene segment and, specifically, the detection of activating KRAS mutations. Following the development and optimization of the assay, we apply it for KRAS mutation analysis of four human cancer cell lines. Ultimately, we present a proof-of-principle method for detecting any of the common KRAS mutations in a single-step, 2 min assay, using only one set of oligonucleotide probes, for a total analysis time of less than 10 min post-PCR. The assay is performed at room temperature and uses simple, inexpensive instrumentation that permits multiplexed analysis.

C∗08:02/16:02 versus C∗05:01/16:01, ambiguous pairs particularly susceptible to typing error

We present four individuals initially determined to be C∗08:02:01:01 & 16:02:01 but subsequently typed as C∗05:01:01:01 & 16:01:01. These consist of a patient typed in our laboratory as well as three unrelated donors (URDs). The URDs had initial typing performed at National Marrow Donor Program (NMDP) laboratories and confirmatory typing performed in our lab. Typing for our patient was performed by sequence specific oligonucleotide probe (SSOP, One Lambda) and sequence based typing (SBT, Conexio) techniques. SBT included both the C∗08:02/16:02 and C∗05:01/16:01 types as ambiguous pairs. These were resolved by SSOP showing the apparent patient C∗ locus types as 08:02 and 16:02. The patient had a diagnosis of mantle cell lymphoma and an unrelated donor search was conducted based on the type of A∗02:01/29:02, B∗44:02/44:03, C∗08:02/16:02, DRB1∗04:01/07:01. Samples from three potentially matched URDs were received. All were listed in the NMDP registry as C∗08:02/16:02. Confirmatory typing on the URDs in our laboratory by SBT showed the same ambiguous pair as expected. However, SSOP appeared to rule out the C∗08:02/16:02 combination listed by the NMDP and indicated that C∗05:01/16:01 was the correct type for all three donors. To better confirm the C∗ locus types, sequencing with an allele-specific sequencing primer (HARP) was performed on all four individuals. C∗ locus typing by sequence specific primers was also performed for three. These methods identified C∗05:01 and 16:01 as the correct type in the patient and all three URDs. Review of the initial patient typing showed a single false negative DNA probe. This probe in the One Lambda SSOP kit separates the ambiguous pairs and is susceptible to false negative results. It was near the cut-off value in our patient as well as one of the donors. Laboratories using the techniques of One Lambda SSOP with SBT for high resolution typing should be aware of the possibility of an incorrect type when the C∗08:02/16:02 pair is identified. Close attention to B∗/C∗ associations is also warranted as the C∗05:01/16:01 pair is expected to be considerably more frequent than C∗08:02/16:02 in association with B∗44:02/44:03. Additional potential C∗08:02/16:02 URDs were listed in the initial search. The NMDP was notified that investigation of those donors might be useful.

Effect of HLA on development of asthma

Background Asthma is a heterogeneous disease characterized by a chronic inflammation of the airways caused by the interaction of genetic susceptibility with environmental factors. Inflammation and remodeling are critical components of asthma. It is shown that many genes are involved in the pathogenesis of asthma. Aim of study: The association between HLA-DRB1 alleles and development of asthma in a sample of Iraqi Arab Muslims. Patients and methods A case-control study was carried out in Medical city Teaching Hospital, Baghdad-Iraq. Patients with asthma attended this hospital in the period September – 2013 to January – 2015. HLA-DRB1 genotyping was done using a panel of sequence-specific oligonucleotide probes (SSOP) using HLA-DRB1 amplification and hybridization kits (SSO HLA type DRB1 plus and Mastermix for HLA type DRB1 Amp plus kits-Innogenetics-Belgium) using automated method by AutoLipa – 48 Innogenetics-Belgium. Results There was an increased frequency of HLADRB1∗03:01:01 in control group compared with patients group ( P = 0.009, Odds ratio = 0.1228, 95% CI: 0.0254–0.5930). Other allele like HLA-DRB1∗070101was significantly increased in asthmatic patients in comparisom with control group ( P = 0.005, Odds ratio = 6.641, 95% CI: 1.7319–25.4657). Conclusions HLA alleles have an effect on development asthma in patients with HLA-DRB1∗070101 while HLADRB1∗030101 is a protective allele in Iraqi Arab Muslims individuals against development of asthma.

Concordance of next generation sequence-based and sequence specific oligonucleotide probe-based HLA-DRB1 genotyping

Next generation sequencing (NGS) of clonally amplified DNA, using Roche 454 technology, was used to genotype HLA-DRB1, DRB3, DRB4, and DRB5 loci (exon 2 only) from a set of 993 samples from newborns with maternally-reported African American ancestry. DRB1 exon 2 was genotyped previously on the same sample set using sequence-specific oligonucleotide probe (SSOP) technology. Comparison of the genotype calls from both methods indicated concordance of 92.3%. Some discordance was expected due to the higher resolution of NGS data, compared to SSOP data. This resulted from selection of the incorrect allele from the ambiguity string produced by SSOP genotyping. Of 76 discordant genotypes, only three were due to resolution of ambiguity with the NGS method. The low percent of changes due to the increased resolution of the NGS method instills confidence in the overall value of previous data genotyped with moderate resolution methods, i.e., the vast majority of alleles present in a population are those that are detectable at moderate resolution. The remaining 73 discordant genotypes resulted from preventable errors in sample handling, data interpretation, and data entry. These results underscore the potential for error that can result from factors such as low quality genomic DNA, manual data entry, and interpretation of marginal genotyping results. Optimization of genomic DNA quality, automation of genotyping steps wherever possible, and use of the highest resolution technology available can lead to dramatic improvements in HLA genotype data quality. NGS-based methodology generated data of superior quality and accuracy compared to the SSOP system.

Relationships of Human Leukocyte Antigen-A, -B, -DRB1 Alleles, and Haplotypes in 129 Ethnic Turkish Patients With Acute Myeloblastic Leukemia.

To evaluate the frequencies of HLA class I (A, B) and class II (DRB1) alleles in acute myeloblastic leukemia (AML) and to compare them with the frequencies of those alleles in unrelated, healthy ethnic Turkish control subjects. We investigated the relationship of HLA alleles in 129 ethnic Turkish patients with AML and 126 unrelated, healthy, ethnic Turkish controls using the polymerase chain reaction with sequence-specific oligonucleotide probes (PCR-SSOP) method via Luminex technology. Allele frequencies of HLA-A*23, HLA-A*68, HLA-B*13, HLA-B*40, and HLA-DRB1*01 were lower in patients with AML compared with control individuals (P =.04, P =.02, P =.005, P = 02, and P =.02, respectively). In contrast, the HLA-DRB1*15 allele frequency was higher than in the controls (P =.01). The most commonly observed haplotype was A*01/B*08/DRB1*03 (5.4% vs 0.8%; P =.03) in patients with AML. In contrast, the most commonly observed haplotype was A*02/B*35/DRB1*04 (2.3% vs 3.2%) in controls. We could not find any haplotypes negatively associated with AML. Also, the homozygosity of HLA-A*01 and HLA-A*02 alleles were higher in patients with AML compared with controls (P =.046; P =.001, respectively). The HLA-DRB1*15 allele, the A*01/B*08/DRB1*03 haplotypes, and the homozygosity of HLA-A*01 and HLA-A*02 may play a presumptive predisposing factor in AML. Also, the HLA-A*23, HLA-A*68, HLA-B*13, HLA-B*40, and HLA-DRB1*01 alleles have been found to be negatively associated with AML.

Candidate HLA genes for prediction of co-trimoxazole-induced severe cutaneous reactions.

Co-trimoxazole is a sulfonamide-containing antibiotic that is effective in the treatment of several infections and for prophylaxis of Pneumocystis jiroveci pneumonia. This drug has been reported as a common culprit drug for the Stevens-Johnson syndrome (SJS) and for toxic epidermal necrolysis (TEN). Human leukocyte antigens (HLAs) play a key role in the immunopathogenesis of severe cutaneous reactions induced by several drugs. This study investigated the association between the HLA class I and HLA-DRB1 polymorphisms and co-trimoxazole-induced SJS/TEN in a Thai population. Forty-three patients with co-trimoxazole-induced SJS/TEN and 91 co-trimoxazole-tolerant patients were enrolled in the study. HLA class I and HLA-DRB1 were genotyped using the reverse sequence-specific oligonucleotide probe method. The frequencies of three alleles of HLA, namely HLA-B*15:02, HLA-C*06:02, and HLA-C*08:01, were significantly higher in the co-trimoxazole-induced SJS/TEN group compared with controls. The risks for co-trimoxazole-induced SJS/TEN in patients with the HLA-B*15:02, HLA-C*06:02, or HLA-C*08:01 allele were about 3-11-fold higher when compared with those who did not carry one of these alleles. Individuals who carried the HLA-B*15:02-C*08:01 haplotype had a 14-fold higher risk for co-trimoxazole-induced SJS/TEN. Evidence of associations between co-trimoxazole-induced SJS/TEN and HLA alleles including HLA-B*15:02, HLA-C*06:02, and HLA-C*08:01 were found in the study population. These findings may suggest that apart from the HLA molecules, other molecules involved in the molecular pathogenesis of these severe cutaneous adverse drug reactions may play an important role in the susceptibility of individuals to SJS/TEN caused by co-trimoxazole.

ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues.

Elucidating the dynamic organization of nuclear RNA foci is important for understanding and manipulating these functional sites of gene expression in both physiological and pathological states. However, such studies have been difficult to establish in vivo as a result of the absence of suitable RNA imaging methods. Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks. Upon in vivo electroporation, exciton-controlled sequence-specific oligonucleotide probes revealed focally concentrated endogenous 28S rRNA and U3 snoRNA at nucleoli and poly(A) RNA at nuclear speckles. Time-lapse imaging reveals steady-state stability of these RNA foci and dynamic dissipation of 28S rRNA concentrations upon polymerase I inhibition in native brain tissue. Confirming the validity of this technique in a physiological context, the in vivo RNA labeling did not interfere with the function of target RNA nor cause noticeable cytotoxicity or perturbation of cellular behavior.

HLA-Class I genes are associated with a higher burden of atherosclerosis in patients with psoriatic disease

s / Atherosclerosis 241 (2015) e32ee71 e53 Inflammation and atherosclerosis EAS-0065. HLA-CLASS I GENES ARE ASSOCIATED WITH A HIGHER BURDEN OF ATHEROSCLEROSIS IN PATIENTS WITH PSORIATIC DISEASE L. Eder , F. Abji , C.F. Rosen , V. Chandran , D.D. Gladman . Rheumtology, Toronto Western Hospital, Toronto, Canada; Dermatology, Toronto Western Hospital, Toronto, Canada Aim: Psoriasis is a chronic inflammatory skin disease. The extent of inflammation is linked with cardiovascular risk in these patients. We aimed to investigate the association between Human-Leukocyte Antigen (HLA) markers of severe phenotype of psoriatic disease (PsD) and the extent of atherosclerosis in these patients. Methods: 411 Caucasian patients with PsD were analyzed. An ultrasound of the carotid arteries was performed and the presence of atherosclerotic plaques was recorded. The size of each plaque was measured and the resulting score, total plaque area (TPA), represented the extent of atherosclerosis. TPA was classified to 4 categories (No plaques, mild, moderate and severe atherosclerosis). HLA genotyping was performed by sequencespecific oligonucleotide probes. The association between each HLA allele and the severity of atherosclerosis was assessed by ordinal logistic regression models adjusted for age, sex and traditional cardiovascular risk factors. Results: 62% of patients had at least one atherosclerotic plaque. HLAB*1302 and HLA-C*0602 were associated with more severe atherosclerosis (ageand sex-adjusted OR 2.3 (95% Confidence Interval (CI) 1.2, 4.3) and OR 1.7 (95% CI 1.1, 2.5), respectively), while HLA-B*3801 was associated with a lower extent of atherosclerosis (ageand sex-adjusted OR 0.49, 95% CI 0.28, 0.86). These associations remained statistically significant after adjusting for traditional cardiovascular risk factors. Higher levels of erythrocyte sedimentation rate over time were associated with HLA-B*1302 (p1⁄40.01) and HLA-C*0602 (p1⁄40.001). No association was found between these alleles and traditional cardiovascular risk factors. Conclusion: HLA-C*0602 and B*1302 are markers of more severe atherosclerosis in patients with PsD. EAS-0089. MODULATION OF TLR2-MEDIATED INFLAMMATION BY AZAPEPTIDES AS SELECTIVE LIGANDS OF CD36 IN ATHEROSCLEROSIS K. Mellal , K. Zoccal , M. Mulumba , G.Y. Ramos , D.W. Lubell , J.F. Cailhier , S. Marleau , H. Ong . 1 Faculty of Pharmacy, University of Montreal, Montreal, Canada; 2 Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil; Department of Chemistry, University of Montreal, Montreal, Canada; CHUM, University of Montreal,

HLA-A allele dropout in sequence-specific oligonucleotide probe typing due to intronic polymorphism in the novel A*31:01:02:02 allele.

HLA-A*31:01:02:02 differs from A*31:01:02 in a single nucleotide mutation at intron 3, nucleotide position 1000 (G > A).

Association of TOMM40 and SLC22A4 polymorphisms with ischemic stroke.

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 architecture, 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 χ2 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.

HLA associations in South Asian multiple sclerosis

Background: Previous efforts to identify Human Leukocyte Antigen (HLA) gene associations with multiple sclerosis (MS) in the South Asian population have been underpowered. Aim: To identify the primary HLA class II alleles associated with MS in Indians. Methods: We typed HLA-DRB1, -DQA1 and -DQB1 in 419 patients and 451 unrelated controls by polymerase chain reaction using sequence specific oligonucleotide probes (PCR-SSOP). Results: At the gene level DRB1 showed significant evidence of association (p=0.0000012), DQA1 showed only marginal evidence of association (p=0.04) and there was no evidence for association at DQB1 (p=0.26). At the DRB1 locus association is confirmed with the *15:01 (p=0.00002) and the *03 (p=0.00005) alleles. Conclusion: Our study confirms that the risk effects attributable to the HLA- DRB1*15:01and DRB1*03 alleles seen in Europeans are also seen in Indians. The absence of any evidence of association with DQB1 alleles reflects the lower linkage disequilibrium between DQB1 alleles and DRB1 risk alleles present in this population, and illustrates the potential value of fine mapping signals of association in different ethnic groups.

HLA-B*1502 and carbamazepine-induced severe cutaneous adverse drug reactions in Vietnamese.

BackgroundIn Vietnam, we observed a high incidence of carbamazepine (CBZ)-induced severe cutaneous adverse drug reactions (SCARs)-Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN), and drug-induced hypersensitivity rash with eosinophilia and systemic symptoms (DRESS). In other Asian countries, HLA-B*1502 is an established risk factor for SCARs.ObjectiveThe aim of our study was to determine the frequency of HLA-B*1502 in SCARs patients at a large University Medical Center in Hanoi, Vietnam.MethodsThirty-eight cases of SCARs caused by CBZ and 25 patients with epilepsy tolerating CBZ were enrolled in a case-controlled study. Clinical manifestations and laboratory findings were recorded for each subject. Genomic DNA was isolated using the QIAamp DNA purification system. The combination of polymerase chain reaction and sequence specific oligonucleotide probes with the Luminex 100×MAP flow cytometry dual laser system was then used to quantitate fluorescently labelled oligonucleotides attached to colour-coded microbeads.ResultsCases comprised 20 SJS (52.6%), 7 TEN (18.4%), 8 overlap syndrome (21.1%), and 3 DRESS patients (7.9%). A strong association between HLA B*1502 and bullous skin reactions such as SJS/TEN and overlap was confirmed with an odds ratio (OR) of 33.78 (95% confidence interval [CI], 7.55-151.03), p < 0.0001, Sensitivity 91.4%, Specificity 76.0%, positive predictive value 84.2%, and negative predictive value 86.4%. We did not, however, observe any correlation between the presence of this allele and CBZ-induced nonbullous skin reactions (DRESS) (OR, 6.33; 95% CI, 0.48-82.74; p = 0.1592).ConclusionOur results indicate the presence of HLA-B*1502 in Vietnamese is a pharmacogenetic risk factor for developing CBZ-induced SJS/TEN.

Association between HLA-DRB1*0405, -DQB1*0401 and -DQA1*0303 alleles and lamotrigine-induced cutaneous adverse drug reactions. A pilot case-control study from Japan

BackgroundHuman leukocyte antigen (HLA) genotypes in lamotrigine -induced (LTG-induced) cutaneous adverse drug reactions (cADRs) have been described in several reports but controversy remains even for a given ethnic group. We attempted to clarify a possible association between LTG-induced cADRs and HLA alleles in Japanese patients. MethodSixteen subjects, including eight patients with LTG-induced cADRs and eight LTG-tolerant controls were included in this study. All eight patients with LTG-induced cADRs gave positive results in a drug-induced lymphocyte stimulation test (DLST) with LTG. We performed HLA-typing for HLA-A, -B, -C, -DRB1, -DQA1, -DQB1, -DPA1 and -DPB1, using PCR with sequence-specific oligonucleotide probes and multiple analyte profiling (xMAP) technology (Luminex System; Luminex Corporation, Austin, TX).We examined differences between allele frequencies in our two groups of subjects and the allele frequencies in the general Japanese population. ResultsThe frequencies of HLA-DRB1*0405, and HLA-DQB1*0401 alleles were higher in our LTG-cADRs patients than the reference frequencies in the general Japanese population. We also detected HLA-DQA1*0303 frequently in our LTG-cADRs patients, but data for this allele in the Japanese population was not available. Our observation was presumably due to the linkage disequilibrium among the three alleles. The haplotype frequency of HLA-DRB1*0405, DQB1*0401 and DQA1*0303 in our LTG-cADRs subjects was also different from the corresponding haplotype frequency in the database for the Japanese population and the difference was statistically significant. One patient with the HLA-DRB1*0405, -DQB1*0401 and DQA1*0303 haplotype was safely re-treated with LTG after results of a DLST with LTG ceased to be positive about 4 months after discontinuation of LTG. LimitationsOur analysis included only 16 patients. Associations between LTG-induced cADRs and specific HLA loci will have to be confirmed in larger studies. ConclusionsLTG-induced cADRs are associated with HLA-DRB1*0405, -DQB1*0401 and -DQA1*0303.

HLA-A, -B, -C, and -DRB1 genotyping of 1000 Northern Irish individuals from Belfast, Northern Ireland in the United Kingdom

One thousand individuals from Belfast, Northern Ireland were genotyped at the HLA-A, -B, -C and -DRB1 loci using sequence-specific oligonucleotide probe methods. HLA-A locus genotypes display a minor Hardy–Weinberg (HW) deviation (p=0.0375); HLA-B, -C and -DRB1 genotypes are consistent with expected HW proportions. These genotype data are available in the Allele Frequencies Net Database under identifier AFND 1243.