A simplified PCR-SSP method for HLA-A2 subtype in a population of Wuhan, China

HLA-A2 is the most frequent HLA-A allele in all ethnic populations, and an important restriction element for peptide presentation to T cells in infectious disease and cancer. However, the HLA-A2 supertype consisting of up to 75 subtypes, mutation studies and analyses using cytotoxic T lymphocytes suggest the functional relevance of subtype-specific differences in HLA-A2 molecules for peptide binding and T-cell recognition. Therefore, it is necessary for T-cell response study to discriminate the HLA-A2 subtypes and to understand the profile of HLA-A2 allellc distribution in a given population. In this study, we developed a simple, robust approach based on the nested polymerase chain reaction using sequence-specific primers （PCR-SSP） to discriminate 17 HLA-A2 subtypes which cover the most HLA-A2 alleles （〉 99% allele frequency） reported in Chinese, using 15 combinations of 19 allelic specific primers. In the first round of PCR, 3 combinations of 5 primers were used to determine whether the tested sample was HLA-A2 positive, meanwhile the subtypes of HLA-A＊0209 and HLA-A＊0215N were determined for the variant position of these two subtypes is in exon 4 instead of exon 2, 3. Samples of HLA-A2 positive were subtyped in the second round of PCR, using PCR products of the first round as templates. This strategy was applied to test the samples of 78 random HLA-A2 positive individuals for their HLA-A2 subtypes. Those samples were screened for HLA-A2 positive by the first round PCR-SSP from 154 healthy blood donors in Wuhan, China. The subtyping results were verified by using flow cytometric analysis （FCM） with HLA-A2 specific monoclonal antibody BB7.2 and DNA sequencing. The typing results of the samples show 50.7% random individuals in the population carry HLA-A2, HLA-A＊0201 ranks the first （allele frequency = 15.5%）, followed by A＊0207 （5.8%）, A＊0206 （4.7%）, A＊0203 （2.6%）, A＊0210 （0.7%）, and these 5 alleles account for 99.0% HLA-A2 subtypes of allele frequency. Our study indicates that the developed typing method is simple and reliable for HLA-A2 subtyping in Chinese, and the profile of allelic distribution of HLA-A2 subtypes is revealed in the population of Wuhan, China.     

HLA—DR位点的PCR—SSP基因分型

目的：探讨适用于肾移植供体HLA-DR的快速基因分型方法。方法：自行设计合成引物建立顺序特异性引物聚合酶链反应技术,并对110例肾移植供体作HLA-DR基因分型。结果：DNA提取方法可以满足PCR-SSP分型方法对DNA模板的要求。全部操作耗时120min,110例标本均分型成功。基因频率范畴在0.0045-0.1227之间,以DR4、DR15和DR9占多数。结论：PCR-SSP法作HLA-DR分型具有准确,简便,快速等优点。适合在临床进行推广应用。     

高分辨分型方法分析福建地区HLA-B15组的基因多态性

目的研究福建地区HLA-B15组等位基因的分布特点,探讨其可能对临床供者选择的影响。方法用聚合酶链反应序列特异引物（PCR-SSP）方法,从已分型的300例供者中选出低分辨率结果为B＊15的样本进行高分辨率分型。结果共检出6种HLA-B15的等位基因,分属于4种血清学特异性。结论福建人群B＊15基因组中B＊1501占优势,其次为B＊1502。为选择最适供者,建议临床配型中采用高分辨率基因分型。     

孕妇外周血中单个胎儿有核红细胞产前诊断胎儿ABO血型

目的:探讨母血中单个胎儿有核红细胞(NRBCS)产前诊断胎儿ABO血型的可行性。方法:从154例孕妇外周血中分离单个NRBCS,应用引物延伸扩增(PEP)法扩增单个NRBCS的基因组,用PCR法检测扩增产物中的SRY基因以判断NBRCS的来源。再用特异性序列-聚合酶链反应(PCR-SSP)法测胎儿ABO血型基因型。抽取新生儿脐血提取淋巴细胞,应用PCR-SSP法测胎儿ABO血型基因型。用常规血清方法测新生儿血型。结果:用PCR-SSP法检测出154例中的152例血型,2例未测出,检出率98.7%。检测出的152例胎儿基因型与脐血淋巴细胞所测ABO血型基因型完全相符。常规血清学方法测新生儿ABO血型,检测率为85.7%。结论:用单个NRBCS结合PCR-SSP法检测胎儿ABO血型方法可靠。     

人类白细胞抗原DR基因与狼疮性肾炎相关性的探讨

目的 从基因水平探讨人类白细胞DR抗原(HLA-DR)基因与中国北方汉人狼疮性肾炎(LN)的相关性。方法 采用特异性引物-聚合酶链式反应(PCR-SSP)方法,检测89例系统性红斑狼疮(SLE)患者及106例健康者HLA-DRB1基因类型,进行临床相关性分析。结果 SLE患者的HLA-DR2及DR9基因频率明显高于对照组(0.36/0.20,RR=2.36,P<0.001;0.27/0.18,RR=1.69,P<0.05);DR2及DR9同时阳性的35例患者中,LN的发病率明显高于其它患者(RR=4.93,P<0.005),而且抗dsDNA抗体的阳性率也较高(RR=2.66,P<0.05)。结论 中国汉人HLA-DR2及DR9基因可能与SLE的遗传易感性有关,两者有相加作用,DR2及DR9同时阳性的SLE患者易患LN。     

Differential Genomics Output and Susceptibility of Iranian Patients with Unilocular Hydatidose

Background

The objective of this study was to investigate HLA-DRB1*and DQB1* allelic polymorphisms in Iranian patients with hydatidose. This is the first survey dealing with the correlation between HLA-DRB1* and DQB1* alleles and cystic echinococcosis in Iranian patients.

Methods

The study was carried out on 56 patients with confirmed cystic echinococcosis and 30 apparently healthy individuals living in Arak- Markazi Province by HLA-DRB1 and DQB1 typing through PCR-SSP method. The first step was to identify the patients and blood sampling. DNA was prepared from whole blood and PCR-SSP with 31 primer mixes for per sample was used. PCR reaction mixtures were loaded in agarose gels and bands were observed under UV illumination and gel document after electrophoresis. Analysis of results was carried out with specific softwares and frequency and interpretation tables for calculation of P-value in χ² test were provided via Fisher’s exact test. Significant samples were analyzed by logistic regression and odds-ratios were calculated.

Results

A statistically significant positive association was found between HLA-DQB1*03 and the resistance to cystic echinococcosis (P < 0.02) (odds-ratio = 2.87).

Conclusion

Immunogenetic susceptibility to unilocular hydatidose varies according to the HLA antigens in Arak, Markazi Province, and DQB1*03 molecules are associated with the level of immune response to parasite antigens.     

Typing of HLA-B*15 alleles using sequence-specific primers

Abstract: We have developed a DNA based typing method to detect 38 known B*15 alleles using sequence-specific primers (PCR-SSP). This method involves 38 primers and 39 PCR-SSP reactions with results that can be obtained in 3 hours. The method is easy, fast and suitable for clinical typing for bone marrow and organ transplantation. We have typed 106 HLA-B15 samples using this method. For homozygous HLA-B15 samples, some B*15 allele combinations need to be resolved by additional PCR reactions not included in this article. The method allows the detection of potential new alleles requiring sequencing for confirmation, and it is useful to resolve unusual serological reaction patterns for different HLA-B15 serological specificities. In addition, it could be used to resolve ambiguous PCR-SSOP typing results and for recognition of mismatches in serologically matched unrelated individuals.     

Simultaneous genotyping of HPA-17w to -21w by PCR-SSP in Chinese Cantonese

Studies have reported the polymorphism of human platelet antigen (HPA)-17w, -18w, -19w, -20w, and -21w. However, the distribution of these five antigens in Chinese Cantonese is still unknown. In this study, we designed new sequence-specific primers for HPA-19w to -21w and used published primers for HPA-17w and -18w to develop a polymerase chain reaction with the sequence-specific primers (PCR-SSP) method for simultaneously genotyping HPA-17w to -21w. A total of 820 unrelated Cantonese apheresis platelet donors in Guangzhou were involved in this study. Among the five HPAs, complete a/a homozygosity was observed for HPA-17w to -20w with an allele frequency of 1.0000. For HPA-21w, nine individuals (9/820, 1.10%) were found to be HPA-21a/bw heterozygous and the allele frequencies of HPA-21a and HPA-21bw were 0.9945 (1631/1640) and 0.0055 (9/1640), respectively. The reliability of the PCR-SSP method was determined by comparing with the genotyping results by DNA sequencing, and no inconsistencies were observed between the two methods. This study provides a reliable PCR-SSP method for simultaneously genotyping HPA-17w to -21w and could improve HPA-matched platelet transfusion in Chinese Cantonese.     

老年人MBL基因ExonⅠ点突变频率及其血浆含量变化的研究

目的探讨老年人甘露糖结合凝集素(MBL)基因ExonⅠ52、54和57位密码子点突变频率和血浆MBL含量变化。方法采用PCR-SSP法检测MBL基因ExonⅠ52位密码子点突变,PCR-RFLP法检测54和57位密码子点突变;ELISA法测定血浆MBL含量。结果老年组和中青年组52、54和57位密码子基因突变频率分别为0.6%和0.8%(确切概率P=0.801)(52位)、20.9%和16.4%(P=0.336)(54位)、0%和0%(57位)。血浆MBL含量老年组和中青年组分别为(2017±1804)μg/L和(2523±1955)μg/L(P=0.109)(野生型+突变型组);(2956±1701)μg/L和(3432±1687)μg/L(P=0.1775)(野生型组);(530±360)μg/L和(709±416)μg/L(P=0.3448)(突变型组)。结论MBL基因ExonⅠ52、54、57位密码子点突变频率和MBL血浆含量老年组和中青年组差别无统计学意义。     

Allelic polymorphisms of human platelets-specific alloantigens in South Tunisian population

Abstract

Objectives

Human platelet-specific alloantigens (HPA) are polymorphic epitopes which vary among ethnic groups.

Background

In Tunisia, HPA frequencies were determined in North and centre; however, the pattern of HPA in South Tunisian population is not been studied yet. The aim of this work was to determine allelic frequencies of HPA-1, -3, and -5 systems in south Tunisian population, in order to estimate the risk of anti-platelet allo-immunization and to create a register of HPA-typed blood donors.

Methods

Our study concerned 212 unrelated healthy, regular blood donors from southern Tunisia. Allelic polymorphisms of each system were determined using a polymerase chain reaction with sequence-specific primers.

Results

Genotype frequencies a/a, a/b, and b/b were, respectively, 0.670, 0.288, and 0.042 for HPA-1 system, 0.430, 0.462, and 0.108 for HPA-3 system, and 0.750, 0.241, and 0.009 for HPA-5 system. The allele frequencies were 0.814 and 0.186 for HPA-1a and -1b alleles; 0.660 and 0.340 for HPA-3a and -3b alleles and 0.870, and 0.130 for HPA-5a and -5b alleles.

Discussion

The reported frequencies are more similar to those of Caucasians than those of north Tunisian population.