基因芯片技术在肾移植组织配型中的应用

目的比较基因芯片和特异性聚合酶链反应(PCRSSP)两种HLADR分型方法,探讨适用于肾移植供、受者分型的新方法。方法对60份肾移植供、受者的DNA样本同时采用基因芯片和PCRSSP进行HLADR分型,并进行分析比较。结果60例样本的两种分型方法结果完全一致56份,相同率达93%。结果不相同的样本共4份,经第三方验证,其中基因芯片分型漏1个位点2例、1个位点误判1例,PCRSSP分型漏1个位点1例。其中20份样本作了重复实验,其重复率达到96%。结论基因芯片用于HLA分型具有灵敏度高、效率高、标准化程度高的优点,是其它分型方法所无可比拟的,具有广阔的应用前景。     

哈尔滨某三甲医院丙型肝炎病毒基因分型研究

目的分析哈尔滨某三甲医院丙型病毒性肝炎患者的HCV基因分型情况,为地区性丙型肝炎的防治提供理论依据。方法收集50例抗-HCV阳性患者血清标本,提取HCV RNA进行核心(core)片段扩增和DNA测序。根据测序结果,采用进化树方法对所得基因序列进行基因分型,获得入组丙型肝炎患者的基因分型情况。结果本研究中中42例患者的血清样本成功扩增出HCV core基因部分片段(+328 nt~+726 nt),其中各种HCV基因分型分布如下:基因分型结果1b型12份(28.57%),2a型29份(69.05%),3a型1份(2.38%)。结论哈尔滨某三甲医院丙型肝炎患者HCV的基因型最常见为HCV 2a型,其次为HCV 1b型,偶见HCV 3a型。     

新疆南部维吾尔族人群尿石症与血管内皮生长因子基因多态性关系的研究

目的 探讨新疆南部维吾尔族人群血管内皮生长因子(VEGF)-460位点基因多态性与泌尿系结石的关系.方法 应用聚合酶链反应-限制性片段长度多态性分析(PCR-RFLP)、基因测序等方法检测200例尿石症患者的VEGF-460基因多态性,200例健康人群体检标本作为对照组,比较2组VEGF-460基因型和等位基因的分布频率.结果 2组VEGF-460位点CC、TT、CT基因型和等位基因比较差异均无统计学意义(P＞0.05),基因型分布频率分别为1.5%、29.0%、69.5%和0.5%、27.5%、72.0%,等位基因C、T分别为36.2%、63.7%和36.9%、63.1%,2组比较差异均无统计学意义(P＞0.05).结果 新疆南部地区维吾尔族人群VEGF-460基因多态性与尿石症无关,VEGF-460位点可能不是维吾尔族尿石症的易感基因.     

多聚酶链反应寡聚核苷酸探针反向杂交在HLAⅠ类抗原分型中的应用

目的比较多聚酶链反应寡聚核苷酸探针（PCR—SSOP）技术与血清学方法对HLAⅠ类抗原A、B分型的准确性及分型精度。方法选取25例曾行血清学HLA-A、B分型的肾移植受者血标本行PCR—SSOP反向杂交法HLA—A、B分型。结果25例PCR—SSOP法HLA—A抗原分型均成功，检出A位点等位基因总数46个，单一位点4例，杂合子21例；检出B位点等位基因总数47个，单一位点3例，杂合子22例。血清学方法检出A抗原单一位点7例，其中2例经PCR—SSOP证实存在第2个位点，血清学A位点总误差率为12％；B抗原单一位点6例，1例PCR-SSOP证实存在第2个位点血清学B位点总误差率为8％。结论PCR—SSOP反向杂交法能对HLA—Ⅰ类抗原行中等分辨度进行等位基因分型，操作简单，分辨度及灵敏度高，结果解读客观。     

肝细胞肝癌CDH1基因启动子C/A单核苷酸多态性与蛋白表达的关系

目的 探讨原发性肝细胞肝癌CDH1基因启动子-160位点的C/A单核苷酸多态性(SNP)与其蛋白表达的关系.方法 以34例肝癌手术病人为对象,DNA直接测序法检测其血液标本中CDH1基因启动子-160位点C/A SNP,免疫组化法检测组织标本中CDH1的蛋白产物--上皮钙黏素(E-cadherin)的表达情况,比较分析C/A SNP与E-cadherin表达的关系.结果 E-cadherin高表达组18例(52.9%),低表达组16例(47.1%),两组的基因型出现率CC与CA,AA比较差异均有统计学意义(P<0.05),CA与AA间差异无统计学意义(P>0.05),A、C等位基因频率在两组差异有统计学意义(P<0.05).结论 CDH1基因启动子-160位点的C/A SNP在肝癌E-cadherin表达中可能发挥重要作用,且A等位基因的出现与E-cadherin表达下调相关.     

补体裂解片断C4d在移植肾急性排斥反应中的临床意义

目的探讨肾小管周围毛细血管补体裂解片断(C4d)沉积在移植肾急性排斥反应中的临床意义。方法对肾移植后发生急性排斥反应的78例受者进行移植肾活体穿刺检查,共获取移植肾活检穿刺标本86份。根据Banff97病理分型将86份活检标本分为BanffⅠ型32份,Ⅱ型51份,Ⅲ型3份。应用免疫组织化学法检测出86份标本中有30份出现肾小管周围毛细血管C4d沉积,阳性率为34.9%。分析C4d阳性其与Banff97分型、术前一般情况、抗排斥治疗、移植肾功能及移植肾预后的关系。结果BanffⅠ和Ⅱ型受者移植肾中C4d阳性率分别为21.9%和39.2%,两者相比差异无统计学意义(P=0.101)。有妊娠史、术前群体反应性抗体(PRA)>30%和再次移植的受者C4d阳性率较高。C4d阳性的受者发生排斥反应时血肌酐较阴性受者高,分别为(312.56±196.26)μmol/L和(210.97±136.59)μmol/L,两组差异有统计学意义(P=0.0115)。C4d阳性受者对激素和ATG冲击治疗与阴性受者比较,敏感率明显降低。C4d阳性的受者移植肾1年生存率较阴性受者低,分别为64.3%和90.0%,两组间差异有统计学意义(P=0.006)。结论移植肾C4d阳性的受者发生排斥反应时,对常规的激素冲击和ATG抗排斥治疗不敏感,血肌酐明显升高,移植肾1年存活率下降,受者预后较差。     

含缬酪肽蛋白基因单核苷酸多态性在肝细胞癌中的研究

目的:探讨含缬酪肽蛋白(valosin containing protein,VCP)基因不同位点单核苷酸多态性(single nucleotide polymorphism,SNP)与肝细胞癌(hepatocellular carcinoma,HCC)的关系。方法:采用病例组与对照组研究,收集122例HCC(HCC组)和120例非HCC(对照组)外周血标本,对VCP基因的4个标签SNPs采用直接测序法进行基因测定分型。采用卡方检验比较基因型及等位基因在HCC组与对照组之间分布的差异,采用非条件Logistic回归分析多态基因型与HCC的关系。结果:rs546982位点等位基因及基因型在HCC组与对照组中的分布具有显著性差异(P<0.05)。携带rs546982位点AA基因型者比GG基因型者发生HCC风险低,而携带AA基因型的HCC病人比携带GG基因型的HCC病人发生脉管癌栓及淋巴结转移的风险高。位点rs2074549、rs607671、rs10972300的基因型在HCC组和对照组之间差异无统计学意义。结论:VCP基因多态性与HCC存在关联,rs546982位点AA基因型可降低HCC发生率,但可增加HCC病人的淋巴结转移及脉管癌栓的风险。     

PCR-ST Amp法应用于HLA-DRB1测序分型的研究

目的采用单管PCR扩增(ST Amp)方法建立高效的人类白细胞抗原-DRB1(HLA—DRB1)基因测序分型(SBT)技术并探讨其应用价值。方法合成7个5’端特异性扩增引物和1个3’端共用引物，将上述引物混合在一起组成单个PCR反应用于HLA—DRB1的DNA测序分型分析。结果所有样本都能成功分型．分型结果准确可靠，重复性好。结论STAmp法改进了测序分型技术，具有高分辨率、高特异性和高效率的特点．值得推广应用。     

移植肾活检组织检测供者HLA四分位基因分型的临床应用

目的明确移植肾活检组织检测的供者HLA四分位基因分型结果的准确性及临床应用。方法回顾性分析2019—2022年在西安交通大学第一附属医院进行肾移植后随访的受者资料, 对可疑排斥反应但缺乏供肾HLA四分位基因分型的38例肾移植受者进行移植肾穿刺, 提取移植肾穿刺组织DNA, 采用LABType SSO法, 进行HLA四分位基因分型检测, 与受者已知HLA基因型进行比对, 预测供者HLA基因型。采用Labscreen Single试剂盒进行供者特异性抗体(DSA)检测, 对38例肾移植的供、受者的临床基线资料、HLA分型资料、受者DSA抗体数据、移植肾病理学指标等进行统计分析。结果 38例受者中, 12例(31.58%)HLA-A、B、C、DR、DQ位点全部检测出;14例(36.84%)检出4个位点;10例(26.32%)检出3个位点;2例(5.26%)检出2个位点, 检出位点和移植时间存在负相关关系(rs=-0.707, P=0.001)。HLA位点检出率分别为A:78.94%(30例);B:65.78%(25例);C:84.21%(32例);DR:57.89%(22例);DQ:100%...     

高通量测序分析肝移植急性排斥反应相关基因单核苷酸多态性位点的突变

目的利用高通量测序分析肝移植术后急性排斥反应相关度最高的基因单核苷酸多态性(SNP)位点的突变情况。方法收集同种异体原位肝移植术的68例受体外周血样本,根据有否发生急性排斥反应分为急性排斥组(13例)和非排斥组(55例)。通过查阅文献,最终确定与排斥反应发生相关的44个SNP突变位点。以44个SNP位点作为检测靶点,用高通量测序分析对两组受体外周血样本进行检测,经生物信息学分析出与急性排斥反应发生相关基因SNP位点的突变率。结果急性排斥组中的白细胞介素(IL)-10 TT基因型、T等位基因、AA基因型、A等位基因的SNP位点突变率明显高于非排斥组;急性排斥组中的细胞趋化因子受体(CCR)5AG基因型的SNP位点突变率明显低于非排斥组,CCR5 GG基因型的SNP位点突变率明显高于非排斥组;急性排斥组中的IL-4 CT基因型的SNP位点突变率明显高于非排斥组,IL-4 TT基因型的SNP位点突变率明显低于非排斥组;急性排斥组中核因子-κB抑制因子α(NF-κBIA)C等位基因的SNP位点突变率明显高于非排斥组;急性排斥组中维生素D受体(VDR)CC基因型和C等位基因的SNP位点突变率明显低于非排斥组,差异均有统计学意义(均为P0.05)。结论高通量测序分析发现肝移植术后急性排斥反应相关基因中,其SNP位点突变率较高的包括IL-10 TT基因型、T等位基因、AA基因型、A等位基因,CCR5 GG基因型、AG基因型,IL-4 CT基因型、TT基因型,NF-κBIA C等位基因,VDR CC基因型和C等位基因。     

基因芯片快速HLA-DR52组分型

目的 用基因芯片对HLA-DR52组快速分型。方法 根据中国汉族人群常见的HLA-DRB位点及其基因多态性的独特序列，设计特异性的寡核苷酸分型探针，制成寡核苷酸芯片。通过组间特异引物扩增基因组DNA，扩增中用荧光标记，扩增标记后的产物与芯片的探针杂交，通过杂交产生的荧光信号确定样品的基因亚型。83份样本分别用基因分型芯片和序列特异性引物聚合酶链反应技术(PCR-SSP)对HLA-DR52组分型。结果PCR-SSP分型DR52组57个位点中，经芯片分型有2个无DR52组位点，3个PCR-SSP分型为DR52组纯合子，芯片为DR52组杂合子，1个PCR-SSP分型为非DR52组纯合子，芯片分型为含有1个DR52组位点的杂合子。结论 基因芯片能对HLA-DR52组快速、准确的分型，比PCR-SSP能更多的检出DR52组位点，特别是能将纯合子进一步分型，适合临床应用。     

基因芯片与血清学方法用于120份供、受者 HLA-A分型的比较

目的 比较基因芯片和血清学方法用于汉族的移植供、受者白细胞抗原Ⅰ类A抗原（HLA—A）分型的准确性和临床实用性。方法 采集供、受者的外周血共120份,再将每份血样分为2部分,分别采用血清学和基因芯片方法检测HLA—A抗原分型,对两种方法分型结果不同的样本,采用序列特异引物聚合酶链反应技术（PCR-SSP）检验。通过比较分型结果和操作方法,评价两种方法的准确性和临床实用性。结果 血清学分型总耗时3h,基因芯片分型总耗时4．5～5h,共有112份样本分型成功,其中两种方法结果一致的有91份,不一致的有21份。经验证,基因芯片分型错误2份,总误差率为2％;血清学分型错误19份,其中5份抗原误差,14份空白误差,总误差率为17％。结论 基因芯片分型准确性明显优于血清学,随着芯片性能的不断完善,将来可能完全取代血清学方法,并具有广阔的应用前景。     

采用顺序特异引物聚合酶链反应技术行快速HLA－DR53组基因配型

采用顺序特异引物聚合酶链反应技术（ＰＣＲ－ＳＳＰ）对６５例肾移植供受者的ＨＬＡ－ＤＲ５３组（ＤＲ４、ＤＲ７、ＤＲ９）进行基因水平分型。结果本组所有位点均分型成功，无一例假阳性和假阴性；每位点重复１０次，重复性１００％；总耗时５小时。分型结果经标准细胞株、限制性内切酶分析和寡核苷酸探针杂交予以确证，特异性１００％。显示本方法用于ＨＬＡ－ＤＲ５３组基因分型具有高分辨率、高特异性和简捷快速的特点，适合于临床应用。     

HLA-Ⅰ、Ⅱ类PCR-SSP基因分型在肾移植配型及亲子鉴定中的应用

目的：建立快速、准确的HLA基因分型方法,满足临床移植配型的需要。方法：对21对已进行HLA抗原血清学分型的肾移植的供、受者,12例亲子鉴定应用聚合酶链反应－序列特异引物（PCR－SSP）进行HLA－I、Ⅱ类基因A、B、DRB、DQB1位点扩增,琼脂糖凝胶电泳分析PCR产物并确定其基因型。结果：基因分型结果与血清学分型一致者有18对,基因分型能明确判断而血清学分型无法判断者有3对;排除亲子关系的有两例。结论：PCR－SSP方法具有操作简单、快速、结果可靠的特点,不仅适用于移植配型、法医学亲子鉴定和个人识别,也可用于相关疾病及人类遗传学的研究。     

HLA class I A and B typing in the clinical laboratory using DNA-based techniques

Abstract  The potential for clinical HLA class IA and B typing utilizing the polymerase chain reaction com bined with sequence-specific oligo-nucleotide probes (PCR-SSOP) was investigated. Two hundred and ten clinical samples for the HLA-B lo cus and 100 clinical samples for the HLA-A locus were typed by DNA-based methods and serology. For the HLA-B locus an improved SSOP typing system was developed which involved using HLA-B specific 5' primers and two 3' primers, in sepa rate reactions. Using a panel of 30 digoxigenin-labelled SSOPs, HLA-B types were assigned for all 210 in dividuals with an improvement in resolution over previously described DNA-based systems and confirming serologically assigned types in all cases except one. In addition, using a single primer pair and a panel of 16 SSOPs, 100 samples were suc cessfully HLA-A typed by PCR-SSOP resolving ambiguous serolog-ical types, including HLA-AL9 sub types and A2 homozygosity. In 25 samples, the assigned types were also confirmed by the amplification refractory mutation system (ARMS-PCR). These results indicate that non-urgent clinical HLA-A and -B typing may be performed by PCR-SSOP with a resolution at least equal to that of serology.     

HLA-C polymorphisms in two cohorts of donors for bone marrow transplantation

The typing for HLA-C in transplantation was rather neglected in the past. However, several recent studies have emphasized its role in transplantation and its association with the outcome. Serological typing of HLA-C could identify only a limited number of HLA-C antigens, resulting in a number of HLA-C blanks. This was mainly due to the low expression of surface HLA-C and the small number of available specific anti-sera. Performing molecular methods has identified new HLA-C alleles and filled the blank of most serological typed antigens. In this study, we compared serological and molecular typing of HLA-C in two cohorts of healthy Saudis. Our serological typing method identified HLA-C1-7 with different frequencies, 23.5% of the alleles were not identified and thus defined as blank. Using the SSP molecular method, all samples were typed and all alleles were defined. Both methods showed that C?07 and C?06 have the highest frequency in the Saudi population. Our study emphasizes the importance of molecular methods in identifying all possible HLA-C alleles.     

HLA alleles and haplotypes in the east Black Sea Turkish population

HLA class I and class II alleles were studied for the first time in 234 unrelated individuals inhabiting the East Black Sea region in Turkey. This region is on the historic silk road and close to Georgia. HLA class I (A* and B*) and class II DRB1* typing was done by the PCR-SSP method. A total of 17 HLA-A* alleles, 34 HLA-B* alleles, and 15 HLA-DRB1* alleles were detected. HLA-A*-B*, A*-DRB1*, and B*-DRB1* two-loci linkage disequilibrium data show that two specific combinations (A2-B35, A2-DRB1*11, and B35-DRB1*13) had the highest frequency (more than three or four times) compared with the other two-loci combinations, possibly reflecting an ancient founder effect. A*24 B*18 DRB1*13 and A*32 B*27 DRB1*11 were the most common haplotypes in the east Black sea Turkish population. HLA-B* showed the highest heterozygosity (94%) among the samples. The observed diversity in the HLA-A* and HLA-DRB1* loci was quite similar, ranging from 79% to 84%. We suggest that the east Black Sea Turkish population is characterized by the features of the Turkish anthropological type with some influence of other groups, such as Caucasians, Asians, and Mediterraneans.     

Variation in HLA-B27 gene subtypes and susceptibility of ankylosing spondylitis in the Croatian population

The differences in amino acid residues of HLA-B27 subtypes are minor, but may play role in pathogenesis of ankylosing spondylitis (AS). Aim of this study was to investigate of frequency of B27 subtypes in Croatian AS patients and B27 positive healthy controls. Group of 50 AS patients and 38 B27 positive controls were typed for B27 subtypes by PCR-SSP method. In the group of AS patients we found four subtypes: B*2705 (83.0%), B*2702 (13.2%), while remaining two alleles B*2701 and B*2704 had one individual each. In the group of B27+ controls we also observed B*2705 (76.3%) as most frequent allele while frequency of B*2702 was 21.1%. No significant evidence for association between AS and a particular HLA-B27 subtype in the Croatian population were found.     

The probability of HLA-C matching between patient and unrelated donor at the molecular level: estimations based on the linkage disequilibrium between DNA typed HLA-B and HLA-C alleles.

BACKGROUND: Recent evidence suggests a more significant role of HLA-C as a target of alloreactions after bone marrow transplantation than previously suspected. Although linkage disequilibrium (LD) between HLA-B and -C serogroups is well documented, the level of LD at the allelic level is not known. In this study, we determine the LD between HLA-B and -C alleles and estimate the probability of molecular HLA-C matching between unrelated individuals who match for both HLA-B alleles. METHODS: The study included 727 haplotypes from 849 individuals who were HLA-A, -B, -C and -DRB1 typed by high-resolution PCR-SSOP technique. Zelterman's statistic was used to test for global LD between HLA loci. LD between specific HLA-B and -C allelic combinations was calculated from their observed and expected frequencies in the study haplotypes. The probability of HLA-C matching for specific HLA-B allele was estimated from contingency table generated from the HLA-B and -C haplotypes. RESULTS: HLA-C was found to exist in LD with HLA-A and -B, as well as -DRB1, loci; however, it was strongest between HLA-B and -C loci. A marked variability in the level of LD between specific HLA-B and -C alleles was noticed. A strong LD was seen in some allele pairs like B*0702-C*w0702, B*3501-Cw*0401, and B*0801-Cw*0701. The overall estimated probability of HLA-C matching between unrelated individuals that match for both HLA-B alleles is 42.25%. For 237 (72.9%) of 325 combinations involving the 25 commonest HLA-B alleles, the estimated probability that the HLA-B-matched unrelated individuals will match for both HLA-C alleles is less than 50%. In addition, a 100% probability of matching for both HLA-C alleles is expected only if both individuals bear either B*0801/ B*0801 or B*4901/B*4901 or B*0801/B*4901. Probability tables for common alleles are presented. CONCLUSIONS: We conclude that, despite matching for both HLA-B alleles by high resolution DNA typing and the presence of a strong LD between HLA-B and HLA-C loci, unrelated individuals are more likely to mismatch rather than match for one or both HLA-C alleles.