Sequence-specific Oligonucleotide Probes Research Articles

P248 Next generation sequencing reveals a novel hla-a*24 allele associated with hla-dqb1*06:10 in asians

Aim Next Generation Sequencing (NGS) for HLA typing improves not only the resolution of typing results but also aids in the discovery of novel alleles. Here we present a novel HLA-A∗24 allele associated with HLA-DQB1∗06:10, which was found in an Asian family and an unrelated donor. Methods Genomic DNA samples isolated from a patient, family members and potential unrelated donors were tested by reverse sequence specific oligonucleotide probes (rSSOP), sequence based typing (SBT) and NGS technology. In NGS typing, genomic DNA was amplified using the Illumina TruSight HLA v2 Sequencing Panel kit. After normalization, fragmentation/tagmentation and dual-indexing procedures, the amplicons were pooled onto the MiSeq sequencer to facilitate cluster generation and sequencing by synthesis on the flow cell. The generated data was then analyzed with Illumina TruSight HLA Assign software. Results A patient being evaluated for a potential hematopoietic stem cell transplant (HSCT) was typed by Sanger sequencing and found to be homozygous at HLA-A, B, C and DRB1. By Luminex LabType, the patient was heterozygous at DQA1, DQB1 and DPB1. However, subsequent NGS typing revealed heterozygousity for HLA-A, with an expected A∗24:02:01 and a novel A∗24 with a mutation in exon 6 which resulted in an amino acid change from lysine to glutamic acid. The same mutation was also found in one of his children and one of four unrelated donors. The patient, child and unrelated donor who had the HLA-A∗24:NEW allele all self-identified as Asian. In all three cases, the HLA-A∗24:NEW allele was associated with HLA-DQB1∗06:10; this association is found only in Asians at low frequency. Conclusions This HLA-A∗24:NEW allele may be associated with DQB1∗06:10 in Asians. Implementing NGS technology in clinical practice will not only allow us to choose the best donor from many potentially matched donors but also will fill in gaps in HLA databases to improve our typing.

Relationship between pemphigus and American tegumentary leishmaniasis: insights from serological and genetic profiles.

Antibodies against Leishmania peptides (Lbr-peps) and desmogleins (Dsgs) have been reported in pemphigus foliaceus (PF) and leishmaniasis patients, respectively. We aimed to compare serological and genetic features in a Brazilian region endemic for American tegumentary leishmaniasis (ATL) and pemphigus. Commercial anti-Dsg ELISA and in-house ELISA with Lbr-peps were used to determine the serological profile, in addition to immunoblotting (IB) and indirect immunofluorescence (IIF) assays. HLA-DRB1 and -DQA1/DQB1 alleles were characterized by PCR combined with sequence-specific oligonucleotide probes (PCR-SSOP). The serological and genetic profiles were compared using 78 PF, 62 pemphigus vulgaris (PV) and 58 ATL patients against 163 and 1592 healthy controls, respectively. Some ATL patients showed positive results for anti-Dsg1 and/or anti-Dsg3 antibodies. They also revealed 130, 160 and/or 230 kDa epidermal peptides in IB. Moreover, some ATL samples exhibited pemphigus or a bullous pemphigoid pattern in IIF. ELISA and IB assays showed Lbr-peps in pemphigus patients. HLA-DQA1*01 and -DQA1*01:02 were protective and susceptibility alleles for ATL, respectively, but the opposite for pemphigus. Anti-Dsgs in ATL may represent epiphenomena. Anti-Lbr-pep antibodies in pemphigus suggest a previous infection. A differential association of the HLA profile may contribute to the lack of co-association between pemphigus and ATL.

Development of HLA-B*57:01 Genotyping Real-Time PCR with Optimized Hydrolysis Probe Design

HLA-B*57:01 genotyping before abacavir (ABC) administration is a standard of care to avoid ABC-driven hypersensitivity reactions. Several HLA-B*57:01 tests have been developed, each with advantages and disadvantages. Some have limited accuracy, require special instrumentation, and/or are labor intensive and expensive. We developed a novel hydrolysis probe-based real-time PCR method of HLA-B*57:01 genotyping. Primer and probes were designed based on published sequence variations in exon 3 of HLA-B that distinguish HLA-B*57:01 from ABC-insensitive alleles such as HLA-B*57:03 and HLA-B*58:01. We designed PCR primers to amplify HLA-B*57:01 along with closely related alleles, such as HLA-B*57:03, directly from genomic DNA. Most ABC-insensitive alleles, including HLA-B*58:01, would not produce any products in the PCR reaction. Our hydrolysis probes enable differentiation of HLA-B*57:01 from the other amplified, but ABC-insensitive, alleles. In addition to using real-time PCR, we used restriction enzymes to generate differential digestion patterns that led to the development of an HLA-B*57:01 PCR-restriction fragment length polymorphism marker. When used to genotype a set of 75 selected clinical samples, our real-time PCR assay demonstrated 100% accuracy in distinguishing between the HLA-B*57:01-positive and -negative alleles when results were compared to those of sequence-specific oligonucleotide probe typing and reference laboratory testing. Our newly developed test will allow clinical laboratories with real-time PCR capabilities to perform HLA-B*57:01 genotyping in a timely and economical manner.

ERAP1 and HLA-C interaction in inflammatory bowel disease in the Spanish population.

Large genome-wide analysis studies (GWAS) and meta-analyses have dramatically increased our knowledge of the genetic risk factors of inflammatory bowel disease (IBD), identifying at least 163 loci. The endoplasmic reticulum aminopeptidase-2 ( ERAP2) gene has been reported as a potential candidate gene for IBD. GWAS have also shown the potential associations between ERAP single nucleotide polymorphisms (SNP) loci and susceptibility to several autoimmune diseases, and ERAP1 and ERAP2 polymorphisms are related to HLA class I-associated diseases, including ankylosing spondylitis and Behçet's disease. Interestingly, these associations were confined to individuals carrying HLA class I-risk alleles. The aim of this study was to investigate the association of ERAP1 and ERAP2 SNPs with IBD in a Spanish population, analysing their possible interaction with specific HLA-C alleles to IBD susceptibility. A total of 367 individuals were divided into 216 IBD cases and 151 controls. SNP genotyping was performed using TaqMan® genotyping assays, whereas HLA-C typing was analysed by sequence-specific oligonucleotide probing. Herein, we report an association of the ERAP1 SNP rs30187 with the HLA-C*07 allele. The existence of shared inflammatory pathways in immunologically related diseases together with the understanding of ERAP1 function may offer clues to novel treatment strategies.

HLA-A, B, DRB1, DQA1, DQB1 alleles and haplotype frequencies in Dene and Cree cohorts in Manitoba, Canada

BackgroundFirst Nations in the Canadian province of Manitoba have disproportionately high rates of epidemic and endemic TB. Gene polymorphisms that modulate HLA Class I and II antigens are among the risk markers for TB, along with other biologic, and social determinants of health. HLA-A, B, DRB1, DQA1, DQB1 were typed in two Manitoba First Nation indigenous groups to identify and compare the frequency of gene polymorphisms that may influence susceptibility or resistance to TB. MethodsParticipants who self-identified as either Dene or Cree enrolled into the study from two First Nation communities in Manitoba, Canada. Genomic DNA was extracted from blood samples collected with informed consent from Dene (N=63) and Cree (N=42) First Nation study participants. Participants self-reported having treated active TB, treated latent TB or no TB. HLA Class I and II molecules were typed using sequence-specific oligonucleotide (SSO) probes from commercially available kits. ResultsThe rates of treated active and latent TB were marginally higher among the Dene than the Cree participants (p=0.112). Class I and II HLA loci were in Hardy-Weinberg equilibrium in both the Dene and Cree groups. In this exploratory analysis of TB and HLA allele frequencies in Dene and Cree cohorts HLA-A*03 and HLA-DQB1*05:03 were significantly associated with TB. ConclusionsThe high incidence of TB in both Dene and Cree populations in Canada requires both biomedical and socioeconomic prevention and control measures. Among the former, an understanding of HLA diversity among First Nations groups may aid the development of new effective vaccine and therapeutic modalities that depend on the interaction between small molecules and specific HLA epitopes.

Is HLA the cause of the high incidence of type 1 diabetes in the Canary Islands? Results from the Type 1 Diabetes Genetics Consortium (T1DGC)

IntroductionIncidence of childhood-onset type 1 diabetes mellitus in the Canary Islands is the highest reported so far in Spain, and among the highest worldwide. The HLA region accounts for approximately half the genetic risk of type 1 diabetes. Our aim was to assess distribution of high-risk and protective HLA haplotypes in the Canarian families included in the T1DGC, as compared to the rest of Spain. MethodsThe T1DGC study, an international project to study the genetics and pathogenesis of type 1 diabetes, enrolled more than 3000 families with type 1 diabetes worldwide. Spain provided 149 of these families, of whom 42 were from Tenerife and Gran Canaria. HLA was genotyped centrally using a PCR-based, sequence-specific oligonucleotide probe system. Haplotypes were reconstructed using the deterministic algorithm alleHap in the R programming environment. Based on prior T1DGC results in Caucasian population, haplotypes DRB1*0405-DQA1*0301-DQB1*0302, DRB1*0401-DQA1*0301-DQB1*0302, DRB1*0301-DQA1*0501-DQB1*0201, DRB1*0402-DQA1*0301-DQB1*0302 and DRB1*0404-DQA1*0301-DQB1*0302 were considered high-risk. DRB1*0701-DQA1*0201-DQB1*0303, DRB1*1401-DQA1*0101-DQB1*0503, DRB1*1501-DQA1*0102-DQB1*0602, DRB1*1101-DQA1*0501-DQB1*0301, DRB1*1104-DQA1*0501-DQB1*0301, DRB1*1303-DQA1*0501-DQB1*0301, DRB1*1301-DQA1*0103-DQB1*0603 and DRB1*0403-DQA1*0301-DQB1*0302 were considered protective. The distribution of protective, high-risk, and other haplotypes in the (first two) affected siblings and unaffected parents from Canarian and non-Canarian Spanish families was compared (Chi-square test). ResultsNo significant differences were found between the regions in distribution of the HLA haplotypes in the affected siblings or in the non-affected parents. ConclusionsThe high incidence of childhood-onset type 1 diabetes in the Canarian population does not appear to be explained by a greater prevalence of high-risk class II HLA haplotypes in families with the disease. However, sample size limits the differences that can be detected in this study.

Is HLA the cause of the high incidence of type 1 diabetes in the Canary Islands? Results from the Type 1 Diabetes Genetics Consortium (T1DGC)

IntroductionIncidence of childhood-onset type 1 diabetes mellitus in the Canary Islands is the highest reported so far in Spain, and among the highest worldwide. The HLA region accounts for approximately half the genetic risk of type 1 diabetes. Our aim was to assess distribution of high-risk and protective HLA haplotypes in the Canarian families included in the T1DGC, as compared to the rest of Spain. MethodsThe T1DGC study, an international project to study the genetics and pathogenesis of type 1 diabetes, enrolled more than 3000 families with type 1 diabetes worldwide. Spain provided 149 of these families, of whom 42 were from Tenerife and Gran Canaria. HLA was genotyped centrally using a PCR-based, sequence-specific oligonucleotide probe system. Haplotypes were reconstructed using the deterministic algorithm alleHap in the R programming environment. Based on prior T1DGC results in Caucasian population, haplotypes DRB1*0405-DQA1*0301-DQB1*0302, DRB1*0401-DQA1*0301-DQB1*0302, DRB1*0301-DQA1*0501-DQB1*0201, DRB1*0402-DQA1*0301-DQB1*0302 and DRB1*0404-DQA1*0301-DQB1*0302 were considered high-risk. DRB1*0701-DQA1*0201-DQB1*0303, DRB1*1401-DQA1*0101-DQB1*0503, DRB1*1501-DQA1*0102-DQB1*0602, DRB1*1101-DQA1*0501-DQB1*0301, DRB1*1104-DQA1*0501-DQB1*0301, DRB1*1303-DQA1*0501-DQB1*0301, DRB1*1301-DQA1*0103-DQB1*0603 and DRB1*0403-DQA1*0301-DQB1*0302 were considered protective. The distribution of protective, high-risk, and other haplotypes in the (first two) affected siblings and unaffected parents from Canarian and non-Canarian Spanish families was compared (Chi-square test). ResultsNo significant differences were found between the regions in distribution of the HLA haplotypes in the affected siblings or in the non-affected parents. ConclusionsThe high incidence of childhood-onset type 1 diabetes in the Canarian population does not appear to be explained by a greater prevalence of high-risk class II HLA haplotypes in families with the disease. However, sample size limits the differences that can be detected in this study.

The incidence of HLA-DQ2/DQ8 in Turkish children with celiac disease and a comparison of the geographical distribution of HLA-DQ.

IntroductionCeliac disease (CD) is an auto-immune enteropathy that occurs in genetically pre-disposed people as a result of the consumption of gluten-containing foods.AimTo identify the incidence of HLA-DQ2 and HLA-DQ8 observed in children with CD.Material and methodsIn this study, we focused on children ranging in age from 2 to 18 years and diagnosed with celiac disease. In our patients diagnosed with CD, in addition to tissue transglutaminase antibodies (anti-tTG), we also evaluated HLA-DQ2 B1 and HLA-DQ8 B1 alleles using the method of polymerase chain reaction (PCR)/sequence-specific oligonucleotide probes (Luminex®). The detection of 0201/0202 for HLA-DQ2 allele and 0302 for HLA-DQ8 allele was accepted as a positive result.ResultsThe mean age of our patients with celiac disease was 7.42 ±3.18 years, and the female/male ratio was 1.5/1. Seventy-six percent of our patients were HLA-DQ2 and/or HLA-DQ8 positive, 67% were HLA-DQ2 positive, and 25% were HLA-DQ8 positive. Nevertheless, 24% of them were HLA-DQ2 and HLA-DQ8 negative. The incidence of HLA-DQ2 in the control group was 18.8% with a significant difference compared to the HLA-DQ2 incidence in the patient group (67%) (p < 0.05). Similarly the HLA-DQ8 incidence in the control group (5.7%) was significantly lower than the incidence in the patient group (25%) (p < 0.05).ConclusionsThe incidence of the patients diagnosed with CD, who are HLA-DQ2 and HLA-DQ8 negative, varies among different populations.

Association of human leukocyte antigen DRB1 with anti-cyclic citrullinated peptide autoantibodies in Saudi patients with rheumatoid arthritis.

BACKGROUNDThe genetic association between human leukocyte antigen (HLA)-DRB1 alleles and the risk of development of autoantibodies has been investigated, but there are few studies from the Gulf region.OBJECTIVESTo investigate the association between the HLA-DRB1 shared epitope and the risk for development of autoantibodies in rheumatoid arthritis (RA) patients in a Saudi population.DESIGNAnalytical cross-sectional study.SETTINGTertiary care hospital in Riyadh, Saudi Arabia.PATIENTS AND METHODSWe enrolled consecutive Saudi RA patients attending the rheumatology clinic between January and April 2015. Previously published data on HLA typing on unmatched healthy controls were used for comparison. HLA typing was performed using sequence-specific oligonucleotide probes (SSOP). Rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) antibodies, and antinuclear antibodies (ANA) were also measured. Logistic regression analysis was used to study the autoantibodies as possible explanatory variables for the presence of the HLA-DRB1 shared epitope.MAIN OUTCOME MEASURE(S)The association between the presence of the shared epitope and the risk of developing anti-CCP antibodies, ANA, and RF.RESULTSIn 76 patients with RA, carrying the shared epitope was associated with a significantly higher risk of having RA [OR=2.65, 95% CI (1.42–4.94), P=.0009]. However, only HLA-DRB1*04:05 was significantly associated with RA [OR=3.73, 95% CI (1.61–8.96), Pc=.016]. In the logistic regression analysis, only anti-CCP was significantly associated with the shared epitope [OR=14.51, 95% CI (1.53–137.49), P=.02].CONCLUSIONSOur analysis indicates that the presence of the HLA-DRB1 shared epitope is strongly associated with the development of anti-CCP antibodies in Saudi patients with RA.LIMITATIONSA larger sample size is needed to confirm our finding.

HLA-B60 and HLA-DR4 alleles in Javanese children with steroid-sensitive primary nephrotic syndrome

Background Steroid-sensitive nephrotic syndrome (SSNS) ofchildren is associated with several human leucocyte antigen (HLA)class I and class II.Objective To investigate the association between HLA-B60 andHLA-DR4 alleles and primary nephrotic syndrome (PNS) inJavanese children.Methods A case control study was conducted on 47 Javanesechildren with PNS who were typed for HLA-B60 and HLA-DR4 al-leles, using DNA sequence specific oligonucleotide probe (SSOP)as control sample, 47 healthy children were also typed for thoseHLA antigens using the same technique.Results Compared with control group, children with PNS had higherfrequency of both HLA-B60 (23.32% vs 4.3%; OR=6.85 [CI=1.32-35.65]; P&lt;0.01) and HLA-DR4 (40.0% vs 2.1%; OR=30.67 [CI:3.71-253.33]; P&lt;0.0002). There was association between HLA and PNSwith SSNS in children.Conclusion The strong association between PNS and HLA anti-gen support the immunogenetic background of the disease, whichseems to be stronger in young children with SSNS.

Analysis of HPA1-16 and HLA-A, B gene polymorphisms among ethnic Han population from Shandong

To study the polymorphisms of human platelet antigen (HPA) 1-16 and human leukocyte antigen (HLA)-A and -B loci among ethnic Han population from Shandong. A total of 588 samples from platelet donors were genotyped for the above loci with sequence-specific primer PCR and sequence-specific oligonucleotide probe PCR. The frequencies of HPA-la, -1b, HPA-2a, -2b, HPA-3a, -3b, HPA-4a, -4b, HPA-5a, -5b, HPA-6a, -6b, HPA-15a, -15b were 0.9974, 0.0026, 0.9456, 0.0544, 0.5417, 0.4583, 0.9983, 0.0017, 0.9889, 0.0111, 0.9903, 0.0097, 0.5434 and 0.4583, respectively. The HPA-7-14 and HPA-16 showed no heterozygosity as the b allele was not detected in such loci. The most common genotypic combination for HPA was HPA-(1,4,7-14,16,17) aa-2aa-3ab-5aa -6aa-15ab (0.1820). HLA-A2 (0.3070) and HLA-B13 (0.1361) demonstrated the highest frequencies at their respective loci. The HPA and HLA loci are highly polymorphic among ethnic Hans from Shandong. The distribution of HPA polymorphisms also shows a great ethnic and territorial difference. It is important to construct regional database for the genotypes of HPA and HLA loci for platelet donors.

The Association of HLA-class I Genes and the Extent of Atherosclerotic Plaques in Patients with Psoriatic Disease.

To investigate the association between HLA susceptibility and disease severity markers and the extent of atherosclerosis in patients with psoriatic disease. White patients with psoriatic arthritis (PsA) and psoriasis without PsA (PsC) were recruited. An ultrasound of the carotid arteries was performed and the size of each atherosclerotic plaque was measured. The resulting score, the total plaque area (TPA), represented the extent of atherosclerosis. HLA genotyping was performed using sequence-specific oligonucleotide probes. The association between 10 HLA susceptibility and severity markers of PsC and PsA and the severity of atherosclerosis was assessed by ordinal logistic regression models adjusted for age, sex, and cardiovascular (CV) risk factors. The study involved 411 patients (273 PsA, 138 PsC). Of them, 61.8% had at least 1 atherosclerotic plaque. HLA-B*13:02 and HLA-C*06:02 were associated with more severe atherosclerosis (age- and sex-adjusted OR 2.31, 95% CI 1.23-4.32 and OR 1.68, 95% CI 1.12-2.52, respectively). HLA-B*38:01 was associated with less severe atherosclerosis (OR 0.49, 95% CI 0.28-0.86). These associations remained statistically significant after adjusting for CV risk factors. Higher levels of erythrocyte sedimentation rate (ESR) were associated with more severe atherosclerosis (age- and sex-adjusted OR 1.33, p = 0.02). HLA-B*13:02-positive (p = 0.01) as well as HLA-C*06:02-positive (p = 0.008) patients had higher levels of ESR over time. HLA-C*06:02 and B*13:02 alleles are associated with a higher burden of atherosclerosis in patients with psoriatic disease. This association may be mediated by a higher level of systemic inflammation.

P031 Laboratory algorithm for the first cardiac transplant in Panama

Aim The first heart transplant in Panama, made in March 2016, involved two hospitals, the Metropolitan Panama Complex and Punta Pacifica Hospital. The National Transplant Laboratory algorithm included all aspects of immunological compatibility in order to minimize one of the most common complications of transplantation: rejection such as acute heart donor. This paper presents the algorithm developed in the Laboratory in this first heart transplant. Methods The patient is female, 52 years old, blood type “A”. Molecular HLA typing use amplified DNA with specific HLA primer and hybridized with a panel of sequence specific oligonucleotide probes (SSOP) (LIFECODES HLA-SSO-Typing kits) The results of HLA recipient are: A ∗ 02, A ∗ 24, B ∗ 51, B ∗ 58, DRB1 ∗ 04, DRB1 ∗ 13, C ∗ 05, Cw ∗ 15, DQB1 ∗ 03(08), DQB1 ∗ 03(07), DPB1 ∗ 04, DPB1 ∗ 05. The studies to establish the presence of anti-HLA preformed antibodies included LIFECODES Antibody Screen, identification of anti-HLA Class I, II using LIFECODES Class I, II ID, and LIFECODES LSA Class I, II. The patient has in his serum anti-A ∗ 36,anti-B ∗ 14(B64) and anti DQB1 ∗ 02 with PRA Class I and Class II, 4%, 30%. The deceased donor was male, 24 years old, blood “A”, and HLA: A ∗ 02, A ∗ 24, B ∗ 58, B ∗ 63, DRB1 ∗ 13, DRB1 ∗ 13, Cw ∗ 07, C w ∗ 07, DQB1 ∗ 06, DQB ∗ 01. DPB1 01, DPB1 ∗ 02. The virtual Crossmatch was negative and actual crossmatching Test (DSA) was negative (LIFE CODES Donor Specific antibodies). In the Bank Laboratory we keep historical sera of the recipient and the pre-transplant serum and preserve HLA glycoproteins of the deceased donor for future humoral antibody-mediated rejection. Conclusion The time used by the laboratory to complete the algorithm was 4 h and the patient was successfully transplanted. Subsequent studies at 15, 30, 45 days have shown the absence of donor specific antibodies. The Laboratory has developed an algorithm that includes all high-tech procedures that avoid the risk of acute antibody-mediated rejection and guarantee further studies to monitor AMR presence. Final del formulario. Download high-res image (95KB) Download full-size image

P029 Algorithm for haploidentical hematopoietic cell transplant in Panama

Haploidentical hematopoietic stem cell transplantation (HSCT) provides an opportunity for patients to benefit when HLA genotypically matched sibling is not available. Virtually all patients have at least one HLA partially matched family member, parent, sibling or child, who is immediately available to serve as a donor. In Panama, between 2007 and 2014, the chance of finding genotypically identical sibling donor was only 36.7%, for these reasons we started in November 2013 HSCT, according to the protocol development in our Laboratory. Methods HLA phenotyping was performed in all the studies, using 2 different molecular methods. DNA samples were amplified with HLA locus-specific primers, then hybridized to panels of sequence specific oligonucleotide probes (LIFECODES HLA-SSO- Typing kits). Potential family members initially typed at the HLA-A, -B, Cw, DRB1, DQB1, DPB1 loci at intermediate resolution. The family members with IPA/NIMA haplotypes, or IMA/NIPA were then typed at the at high-resolution with ABI 3500 Genetic Analyzer additionally perform KIR genotyping to the donor (LIFECODES KIR Typing Kits), HLA antibody screen (LIFECODES Class I, II ID, LIFECODES LSA Class I, II), LIFECODES Donor Specific antibodies (DSA) and chimerism post-transplant using Promega Powerplex 21. We use HLA Matchmaker for determine epitopes mismatch we keep at −86 °C the serum of the recipient, DNA and HLA glycoproteins of the donor. Results The first HSCT was performed in November 2013, and a total of five another HSCT had been performed. Conclusions Haploidentical provides an opportunity for patients to benefit from HCT when and HLA genotypically sibling is not available. Our protocol presents a better logistic to matched donor and it include the necessary procedure for better selection of the haploidentical. We establish advantages for the haploidentical transplant: 1. Shorter wait time for a stem cell transplant. 2. The stem cell transplant is done in about 2–3 weeks, as opposed to several months with other methods. Download : Download high-res image (94KB) Download : Download full-size image R. Aguilar: Grant/Research Support; Company/Organization; No. 2. Consultant; Company/Organization; No. 3. Speaker’s Bureau; Company/Organization; No. 4. Scientific/Medical Advisor; Company/Organization; No. 5. Employee; Company/Organization; No. 6. Stock Shareholder; Company/Organization; No. 7. Other (Identify); Company/Organization; NO.

An in-house multilocus SNP genotyping assay for evaluation of complex genetic diseases

Background: With an increase in the discovery of newer genetic loci/polymorphisms in complex multifactorial diseases, there is also an increased need for methods that can simultaneously genotype multiple loci in a cost-effective manner. Using coronary artery disease (CAD) as a model, the study aimed to develop an in-house multilocus assay for simultaneous detection of 17 genetic variants in 11 genes implicated in CAD.Methods: A multiplex polymerase chain reaction (PCR)-based reverse line blot hybridization (MPCR-RLBH) approach was used, where each DNA sample was amplified using two separate MPCRs, and the alleles were genotyped using covalently immobilized, amino-linked sequence-specific oligonucleotide probes using an enhanced chemiluminescence system. The assay performance was tested on 75 healthy controls and 75 angiographically proven CAD cases. Validation was done by automated Sanger sequencing.Results: The assay could successfully discriminate both the alleles at CETP (I405V), LPL (D9N), NOS3 (T-786G and E298D), LIPC (C-514T), FGB (G-455A), ITGB3 (L33P), AGT (M235T), and MTR (A2756G) loci. Certain mutations included in this assay such as ins242G, ins397G, E387K, L393K in the LDLR; N291S in the LPL; D442G in the CETP; and T833C in the CBS genes were found to be absent. The genotype results obtained using this assay showed 100% concordance with sequencing.Conclusion: The study demonstrated development and validation of a multiplex SNP genotyping assay that can be used to assess genetic risk factors in CAD. The assay provides a cost-effective alternative to expensive high throughput genotyping systems in common molecular research laboratories.

Killer-cell immunoglobulin-like receptors genotyping of 127 individuals from Terceira Island, Azores, Portugal

One hundred and twenty-seven unrelated Azorean individuals were randomly selected to study the gene frequencies of Killer-cell immunoglobulin-like receptors (KIR) in the Azorean (Terceira) population. KIR genotyping was performed by polymerase chain reaction using commercial sequence-specific oligonucleotide probe kits. All loci were in HWE, showing no locus-level deviations. The genotype data is available in the Allele Frequencies Net Database under the population name “Azores Terceira Island KIR”.

HLA-B (*) 58:01 for Allopurinol-Induced Cutaneous Adverse Drug Reactions: Implication for Clinical Interpretation in Thailand.

Background: The aim of this study was to investigate the predisposition to different types of allopurinol-induced cutaneous adverse drug reactions (CADR), including Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN; SJS-TEN, n = 13), drug reaction with eosinophilia and systemic symptoms (DRESS, n = 10) and Maculopapular eruption (MPE; n = 7), conferred by HLA-B*58:01 in a Thai population.Methods: This case-control association study compares 30 patients with allopurinol-induced CADR, allopurinol-tolerant control patients (n = 100), and a Thai general population (n = 1095). Patients' human leukocyte antigen type B (HLA-B) alleles were genotyped by using a two-stage sequence-specific oligonucleotide probe system.Results: Of a total 30 patients with CADR due to allopurinol, 29 (96.7%) patients were found to be at least heterozygous for HLA-B*58:01, compared to only 4.0% in allopurinol-tolerant patients (p < 0.001). Odds ratio (OR) for the association of HLA-B*58:01 with allopurinol-induced CADR in this population was 696.0 (95% CI: 74.8–6475.0). The HLA-B*58:01 allele was present in all patients with allopurinol-induced SJS-TEN (OR = 579.0, 95%CI: 29.5–11362.7, p < 0.001) and DRESS (OR 430.3, 95%CI: 22.6–8958.9, p < 0.001). Additionally, OR of HLA-B*58:01 was highly significant in the allopurinol-induced MPE patients (OR 144.0, 95%CI: 13.9–1497.0, p < 0.001).Conclusion: In this study we confirmed the association between HLAB*58:01 and allopurinol-induced SJS-TEN in a Thai population. In addition, we identified an association between HLA-B*58:01 and allopurinol-induced DRESS and MPE in this population. Therefore, HLA-B*58:01 can be used as a pharmacogenetic marker for allopurinol-induced CADR including SJS-TEN, DRESS and MPE. These results suggest that screening for HLA-B*58:01 alleles in patients who will be treated with allopurinol would be clinically helpful in preventing the risk of developing CARD in a Thai patients.Summary Regardless of phenotype, this is the first pharmacogenetic study of allopurinol-induced CADR in patients of Thai ancestry.In this study we confirmed the association between HLA-B*58:01 and allopurinol-induced SJS-TEN, DRESS, and MPE in Thai population.Regarding to our findings, the pharmacogenetic interpretation could be generalized to drug hypersensitivity including DRESS, SJS-TEN, and MPE.

HLA-A, -B, -C, -DRB1 and -DQB1 alleles and haplotypes in 951 Southeast Asia Malays from Peninsular Malaysia

A total of 951 Southeast Asia Malays from Peninsular Malaysia were genotyped for HLA-A, -B, -C -DRB1, and -DQB1 loci using polymerase chain reaction sequence-specific oligonucleotide probe hybridization methods. In this report, there were significant deviation from Hardy-Weinberg proportions for the HLA-A (p<0.0001), -B (p<0.0001), -DRB1 (p<0.0001) and -DQB1 (p<0.01) loci. Minor deviations from HWEP were detected for HLA-C (p=0.01). This genotype data was available in Allele Frequencies Network Database (AFND) Gonzalez-Galarza et al. (2015).

HLA-A, -B, -DRB1 allele and haplotype frequencies of 920 cord blood units from Central Chile

We present human leukocyte antigen (HLA) haplotype and allele/antigenic group frequencies derived from a data set of 920 umbilical cord blood units collected in Central Chile. HLA-A and -B genotypes were typed using sequence specific oligonucleotide probe methods while HLA-DRB1 genotypes were obtained from sequencing-based typing. The most frequent haplotype is A*29~B*44~DRB1*07:01 with an estimated frequency of 2.1%.

HLA-C*18:01: A Rare Allele in the European Caucasian Population Coinciding with Difficult-to-Treat Plaque Psoriasis

Advances in knowledge about the metabolic pathways involved in the pathogenesis of psoriasis and related diseases have led to a search for new therapeutic targets and the development of new biological drugs. Several studies have focused on HLA-C*06 and investigated correlations between the genetic risk factors of psoriasis and clinical parameters such as the severity of the disease and the response to treatment. Our objective is to share experience from our institution in the observation of two patients with severe chronic plaque psoriasis who were unresponsive to any anti-TNF-α treatment and only partially responsive to ustekinumab. The patients are carriers of a rare allele of HLA-C that occurs in Caucasians. The patients with moderate-to-severe chronic plaque psoriasis, and candidates for biological therapy were typed for the HLA-C locus at high resolution via polymerase chain reaction sequence-specific oligonucleotide probes (PCR-SSOP) using a commercial kit (LAB(®)Type, One Lambda Inc., Canoga Park, CA, USA). The socio-demographic variables and clinical data were collected. In our cohort of 134 patients, only two showed the presence of the allele HLA-C*18:01. To our knowledge, a coincidence between HLA-C*18 and severe psoriasis in Caucasian patients has not previously been described. The fact that both of these patients showed the same clinical history (unresponsive to any anti-TNF-α treatment and partially responsive to ustekinumab) cannot be attributed to a random observation because HLA-C*18 is extremely rare in Europe. As a consequence, at this latitude, it probably indicates a severe disease for which the proper therapy has still not been identified.