Sequence-based Typing Method Research Articles

Discovery of the novel HLA‐DRB1*10:04 allele in a Taiwanese volunteer bone marrow donor and identification of the probable HLA‐A, ‐B, ‐C and ‐DRB1 haplotype in association with DRB1*10:04

We report here a novel variant of HLA-DRB1*10, DRB1*10:04, discovered in a Taiwanese volunteer bone marrow donor by a sequence-based typing (SBT) method. The DNA sequence of DRB1*10:04 differs from DRB1*10:01:01, in exon 2, at nucleotide positions 296 (G fi A) and 303 (T fi G). The nucleotide changes caused an amino acid substitution at amino acid residue 70 (R fi Q). We hypothesize that the formation of DRB1*10:04 was probably the result of a gene recombination event where DRB1*10:01:01 received a minimum length of DNA sequence from DRB1*04:05:01, as the sequence of DRB1*10:04 is identical to DRB1*10:01:01 in exon 2 except the sequence from nucleotide 296 to nucleotide 303, which is identical to DRB1*04:05:01. The plausible HLA-A, -B, -C and - DRB1 haplotypes in association with DRB1*10:04 was deduced as A*01:01-B*37:01-C*06:02-DRB1*10:04.

Association of HLA-DRB1 alleles and neuropsychological function in autism

Evidence suggests an association between autism and immune dysfunction. The associations between human lymphocyte antigen (HLA)-A2, B44, DRβ1*04 (DR4), C4B, and haplotype B44-SC30-DR4 and autism have been reported in western countries but there is a lack of such information in Asian population. This study aimed to assess the association between HLA-DRB1 allele frequencies and the clinical phenomenology of autism. The sample included 141 participants (male, 87.2%), who were diagnosed with autistic disorder based on clinical assessments and structured interviews using the Chinese version of the Autism Diagnostic Interview-Revised, and 156 healthy controls (male, 38.6%). The HLA-DRB1 alleles were determined by sequencing-based typing method. A subsample of patients (n=39) were assessed for intelligence and neuropsychological functions. The results showed that the pattern of DRB1 allele frequencies was significantly different between patients with autism and the controls (P=0.047). After adjusting for sex by haplotype regression, the frequencies of DR4, DR11, and DR14 were significantly different between patients with autism and healthy controls. In addition, patients with autism and DR4, DR11, or DR14 had different performance on intelligence and neuropsychology tests. Despite a relatively small sample size and a case-control association design, the findings suggest HLA-DRB1 gene might be associated with autism in Han Chinese. The true functional variants associated with autism in our samples remain to be further clarified. It warrants a replication study of a larger family sample and to validate the HLA genetic association with autism and its influence on neuropsychological function.

Identification of two novel HLA‐A*02 variants, A*02:319 and A*02:01:64, in two Taiwanese marrow stem cell donors by sequence‐based typing

We report here two novel variants of HLA-A*02 allele, A*02:319 and A*02:01:64, discovered in two Taiwanese unrelated volunteer bone marrow donors by sequence-based typing (SBT) method. The DNA sequence of A*02:319 is identical to A*02:07 in exons 2 and 3 but varies with one nucleotide at codon 9 (TTC->TCC). The variation caused one amino acid substitution at residue 9 (F->S). On the other hand, the DNA sequence of A*02:01:64 is identical to the sequence of A*02:01:01:01 in exons 2 and 3 except a silent mutation at codon 114 (CAC->CAT). The probable HLA-A, HLA-B and HLA-DRB1 haplotypes in association with A*02:319 and A*02:01:64 were deduced as A*02:319-B*46:01-DRB1*04 and A*02:01:64-B*38:02-DRB1*16:02, respectively.

Identification of BoLA‐DRB3.2 alleles in Korean native cattle (Hanwoo) and Holstein populations using a next generation sequencer

Bovine leucocyte antigen (encoded by BoLA) has been widely studied to identify the association with many traits related to immunity. Exon2 of BoLA-DRB3 is extremely polymorphic, and more than 100 alleles have been identified. We investigated polymorphisms of BoLA-DRB3.2 in Korean native cattle and Holstein populations using a next generation sequencer of the GS-FLX Titanium system. We found 38 alleles including 11 new alleles (BoLA-DRB3*1303, *4702, *7101, *7501, *7201, *7301, *7601, *1104, *7701, *7401 and *50021) in Hanwoo, and nine alleles including one new allele (BoLA-DRB3*7601) in Holstein. The 454 sequencing method is a promising alternative technology for high throughput genotyping of BoLA-DRB3.2 because of its technical advantages that allow it to overcome the disadvantages of sequence-based typing methods.

Sequence-based typing of 82 strains of serotype I Legionella pneumophila isolated from 9 provinces in China

To analyze the characteristics of Sequence-based Typing (SBT) of the Serotype 1 Legionella pneumophila (Lp1) isolated from environmental water in China, and then create a preliminary database. A total of 82 strains of Lp1 isolated from environmental water in 9 provinces of China between 2005 and 2008 were genotyped by SBT method and Pulsed-field Gel Electrophoresis (PFGE) method. The results of the two different typing methods were then compared by cluster analysis, adopting BioNumerics version 5.1 software. By SBT method, the 82 strains of Lp1 were divided into 22 ST types, of which 17 new types and one new allele was discovered. The dominant type was ST-1 type, found in 8 provinces, accounting for 46.3% (38/82). ST-1, ST-150, ST-154, ST-159, ST-160 and ST-630 types were found in more than 2 isolated-sites; while more than 2 different ST types were found in 5 isolated-sites, as site B4, B5, B6, S3 and S8. In cluster analysis, 15 ST types were grouped into three complexes (ST-1 complex, ST-154 complex and ST-149 complex); and the other 7 ST types were not assigned complex. By PFGE method, 46 banding patterns were observed. As a result of the combination of the two methods, the 82 isolates strains could be divided into 54 molecular types, which showed a reliable accordance in the cluster analysis between the two methods. The SBT of the Lp1 in environmental water in China was unique. From the study, a preliminary SBT database was set up.

Association of MICA and MICB alleles with symptomatic dengue infection

Dengue viruses (DV) are one of the most important arthropod-borne viral diseases in the developing world. DV can cause syndromes that are either self-limiting or severe. Allelic variants of human leukocyte antigen (HLA) genes have been demonstrated to be associated with disease susceptibility. Here we report the association of nonclassical HLA class I MICA–MICB genes with disease outcome during DV infection. A sequencing-based typing method and genotyping of MICA and MICB in a well-characterized group of Cuban individuals with dengue hemorrhagic fever (DHF), dengue fever (DF), or asymptomatic dengue infection (ADI) was performed. Statistical analysis revealed a tendency for MICA*008 and MICB*008 to associate with susceptibility to illness when symptomatic versus asymptomatic cases (odds ratio [OR] = 2.1, p v = 0.03, and OR = 10.4, p = 0.0096, respectively) were compared. Surprisingly, a stronger association of both allelic forms was observed for the DF patients compared with the ADI group (MICA*008, OR = 5.2, p = 0.0001; and MICB*008, OR = 13.2, p = 0.0025) rather than the severe cases. Major histocompatibility class I-related gene-related natural killer cells and/or γδ and αβ T-cell activation might regulate the development of symptomatic DF and DHF.

Comparison of molecular diagnostic methods for the detection of Acanthamoeba spp. from clinical specimens submitted for keratitis

Acanthamoeba spp. are responsible for a significant annual number of keratitis (AK) cases leading to vision-threatening disease worldwide. Current methods rely on direct examination of specimens by microscopy and/or culture. The former lacks sensitivity and the latter suffers from a poor turnaround time. We undertook a comparison of all published molecular methods, evaluating performance characteristics such as analytical sensitivity, specificity, limit of detection (LOD), reproducibility, accuracy, and cost of test. The study population comprised 128 patients. Eligible specimens were tested prospectively between April 2007 and May 2010 by microscopy and/or culture. Eleven different specimen types were used including corneal scrapings (51.5%), corneal swab (17.9%), and contact lens material (10.9%). Results of 2 published gel-based polymerase chain reaction (PCR) and 2 published real-time quantitative (Q) PCR methods were compared in a blinded manner to direct microscopic examination and/or culture for the detection of Acanthamoeba in clinical specimens. QPCR (Riviere method) had the highest sensitivity at 89.3%, excellent accuracy using ROC analysis (AUC ∼0.90), lowest LOD down to 0.1 organism per microliter, and superior linear correlation with parasite density (R2 = 0.9965) when compared with microscopy, culture, and other molecular methods. Phylogenetic analysis using a sequence-based typing method revealed that clinical isolates in this population with AK were genetically distinct from granulomatous amebic encephalitis or environmental isolates. The QPCR method was more expensive ($14.80) than traditional methods such as culture ($2.50) or microscopy ($2.50). However, 13 culture- and microscopy-negative specimens were positive by QPCR during the study period, suggesting that detection using QPCR may result in reduced complications and health care costs associated with misdiagnosed AK.

A new method for typing bovine major histocompatibility complex class II DRB3 alleles by combining two established PCR sequence‐based techniques

Recently, two polymerase chain reaction sequence-based typing (PCR-SBT) methods were reported for the genotyping of the bovine leukocyte antigen (BoLA)-DRB3. One technique is a single PCR-SBT (sPCR-SBT) method that generates heterozygous sequences that are subsequently analyzed by the haplofinder program, while the other technique is a nested PCR-SBT (nPCR-SBT) method that allows the analysis of heterozygous sequences using the assign 400ATF software. In this study, these techniques were compared and then integrated to produce an improved genotyping method. The primer set used for sPCR-SBT was more accurate than those used for nPCR-SBT. Combining sPCR-SBT with the assign 400ATF software previously reported for nPCR-SBT enables rapid and accurate genotyping of a large number of DNA samples.

Molecular typing methods for outbreak detection and surveillance of invasive disease caused by Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, a review.

Invasive disease caused by the encapsulated bacteria Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae remains an important cause of morbidity and mortality worldwide, despite the introduction of successful conjugate polysaccharide vaccines that target disease-associated strains. In addition, resistance, or more accurately reduced susceptibility, to therapeutic antibiotics is spreading in populations of these organisms. There is therefore a continuing requirement for the surveillance of vaccine and non-vaccine antigens and antibiotic susceptibilities among isolates from invasive disease, which is only partially met by conventional methods. This need can be met with molecular and especially nucleotide sequence-based typing methods, which are fully developed in the case of N. meningitidis and which could be more widely deployed in clinical laboratories for S. pneumoniae and H. influenzae.

Identification of two novel HLA-B*40 alleles, B*40:137 and B*40:158, in Taiwanese individuals

Using sequence-based typing method we discovered two new HLA-B*40 variants, B*40:137 and B*40:158, in Taiwanese individuals. The sequence of B*40:137 has three nucleotide (nt) changes from B*40:21 at nt 353 (C→T), nt 355 (C→A) and nt 369 (C→T) resulting two coding changes at residue 94 (T→I) and residue 95 (L→I), whereas the sequence of B*40:158 differs from B*40:01:01 with five nt substitutes at nt 463 (C→A), nt 477 (C→G), nt 499 (T→A), nt 512 (T→G) and nt 527 (T→A) causing five amino acid exchanges at codons 140 (Y→S), 155 (R→S), 168 (S→T), 171 (L→W) and 179 (V→E). Our hypotheses on the generation of the two novel alleles are presented.

A novel sequence-based coa genotyping method to discriminate nosocomial methicillin-resistant Staphylococcus aureus isolates

To evaluate a novel sequence-based coa typing method in classification for nosocomial methicillin-resistant Staphylococcus aureus. D1 and D2 regions of coa gene were used to discriminate methicillin-resistant Staphylococcus aureus and compared with other classical sequence-based methods with discriminate power. All MRSA isolates were divided into 11 types and about 20 subtypes with coa typing method here. The discriminate power of this coa typing method was better than MLST or spa method. It was demonstrated that this coa method is a simple, rapid, and effective technique applicable for outbreak or local nosocomial MRSA investigations in the future.

Rare HLA alleles and their predicted haplotypes in Tzu Chi Taiwanese marrow donor registry

The marrow donor registry of Buddhist Tzu Chi Stem Cells Centre (BTCSCC) maintained an HLA database of 291677 bone marrow volunteer donors from 1993 to 2008. From the 291677 donors we identified 28 rare alleles and discovered six novel alleles in Taiwanese. Our criterion of a rare allele was according to the definition, set by the National Marrow Donor Program (NMDP), one having a frequency of 0.002% (1/50,000) or less. The authenticity of the rare alleles was confirmed by sequence specific primer (SSP) typing protocol and/or sequence-based typing (SBT) method. This study reports the number of times the alleles were observed between 1993 and 2008, and the ethnicity of the donors carrying the rare or novel alleles.

An HLA-A*02:01-B*13:01-DRB1*14:01:03 haplotype conserved in Taiwanese and a possible close relationship between DRB1*14:01:03 and DRB1*14:54

We here report sequence confirmation and analysis of the variant HLA-DRB1*14:01:03 on three voluntary bone marrow donors and the conserved haplotype carrying DRB1*14:01:03 allele in Taiwanese population. In exon 2, the DNA sequence of DRB1*14:01:03 is identical to HLA-DRB1*14:01:01 except a silent nucleotide substitution at position 192. However, sequence specific primer (SSP) reaction pattern of DRB1*14:01:03 matched with the pattern of DRB1*14:54 instead of DRB1*14:01:01, 14:01:02 or 14:01:03. In exon 3, at position 421, DRB1*14:01:03 has an identical nucleotide as DRB1*14:54 but differs from DRB1*14:01:01. We think the discrepancy of the allele assignment by SSP typing protocol and by sequence-specific oligonucleotide probe (SSO) and sequence-based typing methods should be addressed. We assume DRB1*14:54 is probably the parental allele for DRB1*14:01:03.

Development of new HLA-B*3505 genotyping method using Invader assay

Several pharmacogenetic studies have revealed strong associations between specific human leukocyte antigen (HLA) alleles and the susceptibility to drug hypersensitivity. Recently, we reported HLA-B*3505 as a strong genetic biomarker for the nevirapine-induced skin rash in Thai population. Here, we developed a new HLA-B*3505 genotyping method by a combination of the Universal Invader assay and sequence-specific primer PCR. From the sequence alignment of 68 HLA-B alleles in the Thai population, we selected the two most discriminative SNPs (rs1140412 and rs4997052) as target SNP sites. When we carried out the assay using 324 Thai individuals, fluorescence intensities of HLA-B*3505-positive and HLA-B*3505-negative samples were apparently discriminated at the endpoint of the reaction. Our results were 100% concordant with those obtained by a sequence-based typing method. As our assay is simple and rapid, we believe our method will be a useful tool for pharmacogenetic testing of the nevirapine-induced skin rash.

Association between carbamazepine-induced cutaneous adverse drug reactions and the HLA-B*1502 allele among patients in central China

The aim of this study was to investigate the association between carbamazepine (CBZ)-induced cutaneous adverse drug reactions (cADRs) and the HLA-B*1502 allele among patients from central China. Eight patients with Stevens–Johnson syndrome and toxic epidermal necrolysis (SJS/TEN), 28 with mild maculopapular eruptions (MPEs), 50 CBZ-tolerant controls, and 71 healthy volunteers were recruited. HLA genotyping was performed using the polymerase chain reaction sequence-based typing (SBT) method. As a result, the HLA-B*1502 allele was observed at the following rates: (1) 100% (8/8) among those with CBZ-induced SJS/TEN, (2) 10.7% (3/28) among those with CBZ-induced MPEs; (3) 8.0% (4/50) among CBZ-tolerant controls; (4) 8.5% (6/71) among healthy volunteers. The eight patients with SJS/TEN positive for the HLA-B*1502 allele had an odds ratio (OR) of 184 compared with CBZ-tolerant controls. There was no significant difference in frequency between patients with MPEs and CBZ-tolerant controls ( P > 0.05). Thus, CBZ-induced SJS/TEN, but not MPEs, is strongly associated with HLA-B*1502. Testing for HLA-B*1502 should be recommended for patients from central China prior to initial CBZ treatment.

Distribution of Diego blood group alleles and identification of four novel mutations on exon 19 of SLC4A1 gene in the Chinese Han population by polymerase chain reaction sequence-based typing

The Diego blood group system plays an important role in transfusion medicine. Genotyping of DI1 and DI2 alleles is helpful for the investigation into haemolytic disease of the newborn (HDN) and for the development of rare blood group databases. Here, we set up a polymerase chain reaction sequence-based typing (PCR-SBT) method for genotyping of Diego blood group alleles. Specific primers for exon 19 of the solute carrier family 4, anion exchanger, member1 (SLC4A1) gene were designed, and our PCR-SBT method was established and optimized for Diego genotyping. A total of 1053 samples from the Chinese Han population and the family members of a rare proband with DI1/DI1 genotype were investigated by the PCR-SBT method. An allele-specific primer PCR (PCR-ASP) was used to verify the reliability of the PCR-SBT method. The frequencies of DI1 and DI2 alleles in the Chinese Han population were 0.0247 and 0.9753, respectively. Six new single nucleotide polymorphisms (SNPs) were found in the sequenced regions of the SLC4A1 gene, and four novel SNPs located in the exon 19, in which one SNP could cause an amino acid alteration of Ala858Ser on erythrocyte anion exchanger protein 1. The genotypes for Diego blood group were identical among 41 selected samples with PCR-ASP and PCR-SBT. The PCR-SBT method can be used in Diego genotyping as a substitute of serological technique when the antisera is lacking and was suitable for screening large numbers of donors in rare blood group databases.

Anthropological analysis of Koreans using HLA class II diversity among East Asians

Human leukocyte antigens (HLAs) are useful markers for anthropological investigations because the allele and haplotype distributions at these loci vary widely among ethnic groups. HLA frequencies in Koreans, however, have not previously been analyzed on a phylogenetic basis. We determined the allele frequencies of four HLA class II (HLA-DRB1, -DQA1, -DQB1, and -DPB1) loci in 149 unrelated Korean individuals using a sequence-based typing method. A total of 29 HLA-DRB1, 17 HLA-DQA1, 16 HLA-DQB1, and 15 HLA-DPB1 alleles were identified. The most common allele at each locus was DRB1*0901, DQA1*0102, DQB1*0301, and DPB1*0501, respectively. Four-locus allelic association analysis showed the existence of 25 DRB1-DQA1-DQB1-DPB1 haplotypes with a frequency greater than 0.010. A dataset comprising ethnicity-specific information from published literature and the dbMHC database, as well as the allele frequencies determined in this study, was subjected to phylogenetic analysis. The analysis showed that Koreans are most closely related to Japanese and Han Chinese from Shandong province. Correspondence analyses showed that the current Korean population is located in a position intermediate between the northern and southern East Asian groups, supporting the theory of a bi- and/or multidirectional route of migration of early Korean settlers. This report can be used for anthropological studies, and may also be of use in the International Hematopoietic Stem Cell Sharing program.

Identification of the novel allele HLA-B*51:84 by sequence-based typing method in a Taiwanese individual

A new human leukocyte antigen-B allele, B*51:84 (B*5184), has been found in Taiwan. It differs from B*51:01:01(B*510101) by one nucleotide substitution at codon 145 (CGC →GGC), resulting to one amino acid change (Arginine to Glycine).

Identification of the novel allele HLA-B*13:01:03 by sequence-based typing method in a Taiwanese individual

A new human leukocyte antigen (HLA)-B allele, B*13:01:03 (B*130103), has been found in a Taiwanese individual. It differs from B*13:01:01 (B*130101) by a synonymous substitution at codon 113 (CAT->CAC).

Identification of a novel HLA‐DQB1 allele, DQB1*0326, in a College of American Pathologists 2009 survey specimen

We identified a novel DQB1*0326 allele from a proficiency test sample provided by the College of American Pathologists (CAP) medical society. This novel DQB1 allele was unexpectedly discovered by sequence-based typing method in an attempt to resolve a discrepant typing result between the CAP survey report and our laboratory report. This novel DQB1 allele is most similar to DQB1*030302 and DQB1*0311. DQB1*0326 has a nucleotide substitution resulting an amino acid change when compared with DQB1*030302 (M to L) and it differs from DQB1*0311 by one nucleotide variation causing an amino acid replacement (A to D).