A new HLA-DRB1 genotyping method using single nucleotide polymorphism (SNP) analysis with multiplex primer extension reactions and its application to mixed samples

We have improved on conventional methods for HLA-DRB1 genotyping and devised a new method that is simple, cost-effective, and adequately applicable to routine forensic practice. This method consists of group-specific polymerase chain reaction (PCR) of the exon 2 region of the HLA-DRB1 gene and simultaneous detection of single nucleotide polymorphisms (SNPs) at multiple sites using multiplex primer extension reactions. With this method, we successfully detected HLA-DRB1 genotypes from the following materials: the peripheral blood of 142 donors, 6 aged saliva stains of known DRB1 genotype stored for 5-10 years at room temperature, 10 aged bloodstains of unknown DRB1 genotype stored for 29 years at room temperature, and minimal bloodstains and saliva stains from 3 donors of known DRB1 genotypes. Furthermore, we were able to type DRB1 alleles of the minor component in mixed samples at a proportion of 1/1,000 or 1/10,000. In a criminal case, DRB1 alleles detected from mixed bloodstains on a sword found at the scene enabled us to explain the case. This method is expected to be useful for forensic medicine.     

Detection of HLA-DRB1 microchimerism using nested polymerase chain reaction and single-strand conformation polymorphism analysis

For the detection of microchimerism, molecular methods detecting donor-specific HLA-DRB1 alleles in the recipient are most commonly used. Nested polymerase chain reaction sequence specific primer (nested PCR-SSP) methods widely used to increase the sensitivity of detection have been reported to give frequent false-positive reactions. We have developed a new method combining nested PCR with single-strand conformation polymorphism analysis (nested PCR-SSCP) and tested the 1 to 0.00001% level of microchimerism for 27 different HLA-DRB1 alleles. For most (26/27) of the HLA-DRB1 alleles tested, this method could detect 0.01 to 0.001% of microchimerism and its sensitivity was equal to or better than that of nested PCR-SSP tested in parallel. Its specificity was verified by visualizing particular DRB1-specific SSCP bands under test. Nested PCR-SSP indicated frequent false-positive reactions, mainly caused by nonspecific amplification of DRB3/B4/B5 alleles present in the major (recipient) DNAs. We have compared a real-time quantitative PCR for non-human leukocyte antigen (HLA) target (insertion/deletion marker) using a commercial kit (AlleleSEQR Chimerism assay), and its microchimerism detection sensitivity (around 0.1%) was 1 step (10 times) lower than that of nested PCR-SSP or -SSCP methods for HLA-DRB1 alleles. We validated that the newly designed nested PCR-SSCP affords good sensitivity and specificity and may be useful for studying microchimerism in clinical settings.     

High-resolution typing for HLA-DRB1*15 amd -DRB1*16 by fluorescence-marked sequence-specific priming (TaqMan assay)

Sequence-specific primed polymerase chain reaction (PCR-SSP) is widely used in HLA laboratories. The TaqMan method, which is described here for high-resolution typing of HLA-DRB1*15 and -DRB1*16, does not require elaborate and time-consuming post-PCR detection steps. In this one-tube assay, conventional PCR-SSP and fluorescence detection of the amplicon with a doubly labeled fluorescent probe are combined: a fluorogenic hybridization probe (FHP) labeled with a spectral resolvable fluorescent reporter dye (FAM or TET) at its 5' terminus and a common quencher dye (TAMRA) at its 3' terminus is cleaved by the 5' nuclease activity of Taq DNA polymerase only if the target sequence is amplified. An increase of fluorescence intensity indicates a successful amplification. For high-resolution typing of HLA-DRB1*15 and -DRB1*16 alleles we designed two FHPs and 14 specific primer mixes (7 for DR15 and 7 for DR16). Amplification of the specific sequence was detected by a FAM-labeled FHP, whereas amplification of the internal control was detected by a TET-labeled FHP. We were able to type all heterozygous DRB1*15/DRB1*16 subtype combinations. For evaluation, 60 HLA-DRB1*15-positive and 40 HLA-DRB1*16-positive individuals were typed and the results were compared with conventional PCR-SSP DR15/16 subtyping. There were no discrepancies between the two methods. The TaqMan method is an alternative to conventional PCR-SSP typing which is suitable for routine use in HLA laboratories.     

HLA-DRB1基因与子痫前期相关性研究

目的探讨HLA-DRB1基因与子痫前期相关性。方法采用序列特异性引物技术（PCR—SSP）对119例子痫前期患者、117例正常孕妇及其新生儿进行HLA-DRB1等位基因分型，比较其基因频率。结果共检出13种等位基因，两组孕母或两组新生儿间，其HLA-DRB1等位基因频率差异无统计学意义（Pc〉0．05）；子痫前期组和正常晚孕组母婴所携带HLA-DRB1＊014、-DRB1＊10、-DRB1＊14等位基因配伍差异有统计学意义（P〈0．05）。结论母婴所携带某些HLA-DRB1基因配伍与子痫前期易感性或抗性相关；父源性HLA-DRB1＊014基因与子痫前期易感性相关。     

Nested polymerase chain reaction-based HLA class II typing for the unique identification of formalin-fixed and paraffin-embedded tissue

Human leukocyte antigen (HLA) genotyping is routinely performed prior to organ transplantation using peripheral blood leukocyte-derived DNA. In addition, polymerase chain reaction (PCR)-based methods have permitted HLA genotyping using DNA extracted from formalin-fixed and paraffin-embedded tissue, with proven applications in HLA–disease association studies and surgical biopsy identification. The utility of current techniques may be limited by the poor yield of intact DNA from such paraffin biopsies. This paper describes a new nested PCR-based HLA class II genotyping method which reliably detects HLA DRB alleles within DNA extracted from even extremely small paraffin biopsies. This method comprises initial PCR amplification of exon II sequences of the HLA DRB1, 3, 4, and 5 genes using generic PCR primers. Identification of the HLA DRB1 alleles and detection of the DRB3, 4, and 5 genes is then performed using a series of separate individual second-round PCR reactions, each of which contains PCR primer pairs detecting a single HLA DRB allele or group of alleles (PCR-SSP). The ability of this method to detect 19 individual HLA DRB1 alleles or groups of alleles, covering all common DRB1 specificities, was confirmed via concordant results when compared with ‘direct’ (single amplification step) PCR-SSP analysis of one cell line-derived and nine peripheral blood DNA samples, and with five DNA samples extracted from paraffin biopsies. The technique was then successfully applied to 11 further paraffin biopsy-derived DNA samples, of which ten were untypable by ‘direct’ PCR-SSP analysis, from five cases in which doubt existed as to the individual origin of the tissues. © 1997 John Wiley & Sons, Ltd.     

HLA class II typing of whole genome amplified mouth swab DNA

Postal collection of mouth swabs provides a cheap and convenient means of DNA sampling but hitherto has not provided sufficient genetic material for HLA typing by polymerase chain reaction using sequence-specific primers (PCR-SSP). This study examined the feasibility of collecting mouth swabs from a test population by post, amplifying the DNA by whole genome amplification and genotyping for selected HLA class II alleles. We optimised a strategy for whole genome amplification or primer extension preamplification using a random 15 base pair primer which resulted in a 1,000-fold increase in DNA template. The amplified DNA was of sufficient quality for analysis of selected HLA Class II alleles by PCR-SSP and PCR using sequence-specific oligonucleotide probes. To test the reliability of our data, blood DNA from 30 individuals in 10 families, previously tested for all DRB1 alleles in a routine diagnostic laboratory, was then tested in our laboratory for DRB1 *03 and *04 following whole genome amplification. Further whole genome amplified product from another 10 families was tested for DRB1 *03, *04 in our laboratory and then tested for all DRB1 alleles in a routine diagnostic laboratory. One repeat typing was required to achieve 100% concordance between laboratories. Amplification of whole genome amplified DNA by PCR-SSP was then extended successfully to low-resolution HLA DRB1, DQA1, DQB1 and DPB1 typing. Mouth swab collection by post, followed by whole genome amplification of DNA provides an effective strategy for genetic analysis of large cohorts. We have optimised conditions for HLA class II typing on whole genome amplified DNA collected by mouth swab, but this method could potentially be applied to low concentrations of DNA from other sources.     

Rapid HLA-DQB typing by eight polymerase chain reaction amplifications with sequence-specific primers (PCR-SSP)

Molecular genotyping of HLA class II genes using group-specific DNA amplification by the PCR followed by probing with (PCR-SSO) probes is too time consuming for the typing of cadaveric organ donors. Recently, amplification of DNA using PCR-SSP has proved a reliable and rapid method for typing HLA-DRB1 genes. PCR-SSP takes 2 hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers that in eight PCR reactions will positively identify the HLA-DQB1 alleles corresponding o the serologically defined series HLA-DQ2, DQ4, DQ5, DQ6, DQ7, DQ8, and DQ9. Presently, 30 homozygous cell lines and 138 individuals have been typed by the DQB1 PCR-SSP technique and compared with a combination of serology and RFLP with 100% concordance. No false-negative or false-positive amplifications were recorded. All combinations of DQB1 can be readily identified. DQB1 PCR-SSP can take as little as 130 minutes from start to finish, including DNA preparation.     

Short-form HLA-DP typing with 48 primer mixes using the polymerase chain reaction and sequence-specific primers.

We have developed a short-form SSP-based HLA-DP typing system for routine use adapted from a comprehensive HLA-DP typing method described by Gilchrist et at. (1998). Our short-form system detects 93 alleles, including the 18 most common HLA-DPB1 alleles and eight HLA-DPA1 alleles. The primer mixes described were tested using the PCR-SSP Manager (Bunce et al., 1998) database to confirm the specificity of selected primers, and to detect potentially ambiguous amplifications. This short-form HLA-DP typing system was validated using 50 fully typed samples obtained through the UCLA International DNA Exchange. All samples gave 100% concordance with the consensus type. Our laboratory routinely uses a PCR-SSP based system of 48 primer mixes for HLA-DRB and HLA-DQB typing. The advantage of the short-form HLA-DP typing system described here is that the additional 48 HLA-DP primer mixes required can be included on the second half of a 96-well format tray. This method now enables a full HLA class II typing at the level of allele group resolution in 2 1/2 h.     

异基因骨髓移植中PCR-SSP和RFLP对HLA-DRB、DPB1分型的应用

目的:探讨序列特异性引物聚合酶链反应（PCR-SSP）和聚合酶链反应限制性片段长度多态性分析（PCR-RFLP）对HLA-DRB、DPB1分型在异基因骨髓移植中应用的可行性。方法:应用19对不同的DRB抗原所对应等位基因序列特异性的引物,PCR扩增供受者各20个样本DRB,产物直接琼脂糖凝胶电泳分析;同时用另一对引物PCR扩增DPB1第二外显子,产物10种限制性内切酶酶切分析。结果:各DRB带清晰可辨,直接确定为该型;DPB1的多态性片段编码转换后确定其基因型;4例找到供者。结论:它们是异基因骨髓植前快速、精确和可靠的基因分型手段。     

Rapid HLA-DR fluorotyping based on melting curve analysis

Real-time polymerase chain reaction (PCR) has been used in the study of human leukocyte antigen (HLA) genotyping as a potential alternative for routinely used molecular methods such as PCR-sequence specific primers (PCR-SSP) and PCR-sequence specific oligonucleotide probes (PCR-SSO). Combined with fluorescent dye like SYBR GREEN I, it has more advantages such as low cost and consistent background. The aim of this study was to optimize the fluorescent dye-based method and introduce it into the fluorotyping for HLA-DR lotus. 24 pairs of allele-specific primers and 1 pair of internal control were optimized to discriminate HLA-DRB1, -DRB3/B4/B5 alleles. Additionally, conditions of real-time PCR amplifying and melting curve recording had been improved for convenient and clear readout. Forty-two clinical samples previously typed by conventional PCR-SSP or sequence based typing (SBT) were tested and all got identical results. With this technique, 15 DNA samples can be assayed in parallel within 2 hours on the Real-time PCR instrument. These data strongly suggest a rapid HLA-DR fluorotyping method based on melting curve analysis, which could be a more economic and automatic alternative for clinical HLA-DR typing.     

Rapid HLA-DR Fluorotyping Based on Melting Curve Analysis

Real-time polymerase chain reaction (PCR) has been used in the study of human leukocyte antigen (HLA) genotyping as a potential alternative for routinely used molecular methods such as PCR-sequence specific primers (PCR-SSP) and PCR-sequence specific oligonucleotide probes (PCR-SSO). Combined with fluorescent dye like SYBR GREEN I, it has more advantages such as low cost and consistent background. The aim of this study was to optimize the fluorescent dye-based method and introduce it into the fluorotyping for HLA-DR lotus. 24 pairs of allele-specific primers and 1 pair of internal control were optimized to discriminate HLA-DRB1, -DRB3/B4/B5 alleles. Additionally, conditions of real-time PCR amplifying and melting curve recording had been improved for convenient and clear readout. Forty-two clinical samples previously typed by conventional PCR-SSP or sequence based typing (SBT) were tested and all got identical results. With this technique, 15 DNA samples can be assayed in parallel within 2 hours on the Real-time PCR instrument. These data strongly suggest a rapid HLA-DR fluorotyping method based on melting curve analysis, which could be a more economic and automatic alternative for clinical HLA-DR typing.     

HLA class II polymorphism in Thai patients with non-Hodgkin's lymphoma.

The distribution of HLA-DRB1 alleles and DQB1 alleles in 100 Thai patients with non-Hodgkin's lymphoma (NHL) was analysed using the polymerase chain reaction with sequence-specific primer (PCR-SSP) method, and the association between the disease and the presence of certain HLA class II alleles was investigated. The frequencies of HLA-DRB1*1502 and DRB1*09012 were increased while those of DRB1*0404, DRB1*0803 and DRB1*1106 were decreased. On the other hand, the incidence of HLA-DQB1 alleles was similar to that in the normal population. Interestingly, only HLA-DRB1*1502 showed a significant positive association with NHL, especially in patients < or / = 45 years and in male patients. It is concluded that the DRB1*1502 allele may contribute to NHL susceptibility in the Thai population. However, further studies on the functional roles of the HLA class II alleles are necessary to elucidate NHL susceptibility.     

Short-form HLA-DP typing with 48 primer mixes using the polymerase chain reaction and sequence-specific primers

We have developed a short-form SSP-based HLA-DP typing system for routine use adapted from a comprehensive HLA-DP typing method described by Gilchrist et al. (1998). Our short-form system detects 93 alleles, including the 18 most common HLA-DPB1 alleles and eight HLA-DPA1 alleles. The primer mixes described were tested using the PCR-SSP Manager (Bunce et al., 1998) database to confirm the specificity of selected primers, and to detect potentially ambiguous amplifications. This short-form HLA-DP typing system was validated using 50 fully typed samples obtained through the UCLA International DNA Exchange. All samples gave 100% concordance with the consensus type. Our laboratory routinely uses a PCR-SSP based system of 48 primer mixes for HLA-DRB and HLA-DQB typing. The advantage of the short-form HLA-DP typing system described here is that the additional 48 HLA-DP primer mixes required can be included on the second half of a 96-well format tray. This method now enables a full HLA class II typing at the level of allele group resolution in 2¹/2 h.     

High-resolution HLA-DRB typing using denaturing gradient gel electrophoresis and direct sequencing

High-resolution HLA-DRB typing is required for bone marrow transplantation between unrelated donors and recipients and also for identification of novel HLA-DRB alleles. Here we describe a method for the unambiguous identification of HLA-DRB alleles using the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The highly variable second exon of all HLA-DRB1, -DRB3, -DRB4, -DRB5, -DRB6 and -DRB7 alleles was amplified using a single pair of generic DRB -specific primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and the sequences of these alleles were determined using fluorescent-based sequencing and allele-assignment software. The validity of this typing procedure was confirmed by identification of HLA-DRB alleles for 17 individuals previously characterized by PCR-SSP and/or cloning and sequencing techniques. We identified 34 different HLA-DRB alleles in these 17 unrelated individuals. Importantly, our analysis revealed HLA-DRB1 alleles which had not been identified using the PCR-SSP typing technique. Additionally, alleles from the HLA-DRB3, -DRB4 and -DRB5 loci were identified. Whereas traditional HLA-DRB typing methods provide limited information or require the use of multiple oligonucleotide primers or probes, our technique provides a reliable, specific and relatively rapid way of identifying all HLA-DRB alleles for high-resolution tissue typing.     

Multiplex genotyping of human minor histocompatibility antigens

Minor histocompatibility antigens (mHAg) induce major histocompatibility complex-restricted, T cell-mediated immune responses that may contribute to increased risk of graft-versus-host disease and graft-versus-leukemia effects. Unlike human leukocyte antigen genes, mHAg are encoded by genetically and functionally unrelated genes located throughout the chromosome. The role of mHAg in stem cell transplantation and the population frequencies of mHAg alleles remain unknown due in part to the lack of suitable high throughput methods for genotyping these diverse genes. Here we describe the development and utility of a multiplexed Luminex assay for genotyping human mHAg, including HA-1, HA-2, HA-3, HA-8, HB-1, CD31(125), and CD31(563). The assay uses a multiplexed, allele-independent, gated amplification of mHAg genes followed by differential detection of allele-specific primer extension products using the MultiCode PLx system (EraGen Biosciences, Madison, WI). The alleles are interrogated using a multiplex allele-specific primer extension reaction using primers tagged with EraCodes. The products are hybridized to Luminex beads and the hybridization duplexes are detected using streptavidin-phycoerythrin. The assay resolved the mHAg genotypes of 259 Caucasian donors and provided population estimates of mHAg gene and phenotypic frequencies. All mHAg alleles evaluated in this study exhibited Hardy-Weinberg equilibrium, although some mHAg phenotypes were present in large majority of individuals tested (HA-2, HB-1). This assay will provide a valuable tool for determining mHAg frequencies in other ethnic populations, as well as for establishing the clinical importance of mHAg disparities in stem cell transplantation.     

A rapid HLA-DRB1*04 subtyping method using PCR and DNA heteroduplex generators

We describe here a rapid polymerase chain reaction (PCR)-based method for the identification of HLA-DRB1*0401-*0412 alleles. The method is based on the generation of specific DNA heteroduplex patterns between PCR products derived from selective group-specific amplification of the various DRB1*04 alleles and PCR products derived from two synthetic DNA heteroduplex generator (DHG) molecules following non-denaturing polyacrylamide minigel electrophoresis. One DHG was designed to detect DRB1*0401, *0405, *0407, *0408, and *0409 alleles, whilst the other was designed to detect DRB1*0402, *0403, *0404, *0406, *0410, *0411, and *0412 alleles. Characteristic heteroduplex patterns were obtained for all DRB1*04 alleles tested both in homozygous and heterozygous situations. Both DHG and PCR-SSP (sequence-specific primer) typing were performed on 41 DRB1*04 positive DNAs from Singaporean Chinese blood donors and complete concordance in results was obtained. HLA-DRB1*0403, *0405, and *0406 were found to account for 95% of the DRB1*04 alleles in the population studied. The DHG technique described is technically simple and rapid since it essentially involves only two PCR amplifications per individual subtyping. The technique is particularly useful for resolving DRB1*04 combinations which are indistinguishable by PCR-SSO (sequence-specific oligonucleotide) or PCR-SSP subtyping.     

HLA class II sequence-based typing in normal Saudi individuals

We have adopted a system that combines low resolution PCR-SSP followed by sequence-based typing (SBT) to analyze HLA-DRB1, -DPB1 and -DQB1 alleles in the Saudi population. The SBT method was used to identify HLA class II alleles in Saudis for the first time. Nineteen HLA-DRB1 alleles in currently recognized subtypes of the DRB locus were detected. DR1 and DR9 were not encountered. SBT did not detect diversity within the DR7 and DR10 alleles. Sixteen HLA-DQB1 and 10 HLA-DPB1 alleles were identified. This study represents the first molecular report on the HLA class II allele frequency in the population of Saudi Arabia.     

New HLA-DRB1*07 allele identified from microlymphocytotoxicity/SSP discrepancies

A HLA-DRB1*07 variant allele has been identified in a cadaver kidney donor. Serological typing using monoclonal antibodies detected HLA-DR4 and HLA-DR7. HLA class II DNA typing using sequence-specific primer (PCR-SSP) polymerase chain reaction only detected DRB1*04, while sequence-specific oligonucleotide (PCR-SSO) polymerase chain reaction confirmed the presence of both DRB1*04 and DRB1*07 alleles, although two extra reactions were also found. Exon 2 of the HLA-DRB1*07 was isolated using allele-specific PCR, then cloned and sequenced. Four mutations, at positions 170 (T --> C), 171 (C --> T), 174 (C --> G), and 179 (C --> A), were observed. These mutations changed codons 57 and 60 (V --> A; S --> Y, respectively). This amino acid sequence at position 56-61 is only found in DRB1*0811.     

辽宁汉族人群HLA-DRB1基因多态性分布

目的调查HLA-DRB1基因位点遗传多态性在辽宁汉族人群中的分布。方法应用聚合酶链反应．序列特异性引物方法和反向聚合酶链反应．序列特异性寡核苷酸探针杂交的方法对13265名辽宁汉族人进行中低分辨率HLA-DRB1基因分型。结果共检出HLA-DRB1位点的13种等位基因，其中以HLA-DRB1＊15频率最高（17．49％），其次为HLA-DRB1＊09、＊12和HLA-DRB1＊07，基因频率分别为13．40％、11．87％和11．8l％。HLA-DRB1＊03（18）和HLA-DRB1＊14（8）等位基因未检出。对观察值和期望值进行X^2检验，符合Hardy-Weinberg平衡（X^2=73．34，af=78，P〉0．5）。该人群与南北方汉族人群、日本人、白人和黑人分别进行X^2值检验差异有统计学意义，X^2值分别为112．053、8．514、692．141、70．558和121．755。结论辽宁汉族人群HLA-DRB1基因分布有自身特点。