Ambiguity results distribution and its solutions of HLA-A, B and DRB1 sequence-based typing / 重庆医学

Objective To investigate the ambiguity results distribution of HLA-A,B and DRB1 gene sequence-base typing in Guangxi population and to propose the way to resolve.Methods HLA-A,B and DRB1 genes of 1 000 donors in the Guangxi branch bank of China'bone marrow bank were genotyped by PCR-SBT,and then the ambiguity results distribution of the three loci was analyzed.The typing ambiguities resultswere resolved by high-resolution polymerase chain reaction-sequence-specific primers(PCR-SSP) and group specific sequencing primer(GSSP) methods,respectively.Results Among 1 000 samples,at least 1 locus in HLA-A,B and DRB1 genes in 96.7％ samples appeared the ambiguity results,in which the proportions of HLA-A,B and DRB1 loci appearing ambiguity results were 65.7 ％,58.8 ％ and 77.2 ％ respectively.For the samples of detected ambiguity results,single using the GSSP method could resolve the ambiguity typing results of 87.37％ HLA-A,93.54％ HLA-B and 60.49％ HLA-DRB1,using high-resolution PCR-SSP could resolve the ambiguity typing results of 12.63 ％ HLA-A,4.76 ％ HLA-B and 15.29 ％ HLA-DRB1,and the rest 1.70 ％ HLA-B and 24.22 ％ HLA-DRB1 ambiguity results were resolved by both GSSP and high-resolution PCR-SSPs method.Conclusion GSSP and high-resolution PCR-SSPs methods have high abilities to solve HLA ambiguity results both locate inside and outside the sequencing region,respectively.GSSP and high-resolution PCR-SSPs methods are supplement for each other,which can effectively resolve the problem of ambiguity results in high resolution HLA typing.

Observation and analysis of STR loci mutation among 1 786 cases of paternity test in Guangxi area / 重庆医学

Objective To observe and analyze the mutation characteristics of 17 STR loci among the paternity test cases in Guangxi area .Methods Among 1 786 cases of non—exclusion parentage ,1 430 cases were parental triplet and 356 cases were uniparental diad ,1 001 persons were Han people ,2 102 persons were Zhuang people and 113 persons were other ethnic group in the parents .The genome DNA was extracted by Chelex-100 method .17 short tandem repeat (STR) loci were detected by Power Plex ? 18D System Kit .The paternity testing containing mutant STR loci were screened out from 1786 cases .The locus-specific ,specificity of paternal and maternal ,and allele-specific mutation rates were observed and analyzed ,respectively .The characteristics of the muta-tions were studied .Results In total ,75 mutations events were observed at 16 of the 17 loci .Among them ,73 (97 .34% ) times were one step mutation ,onece(1 .33% ) was two—step mutation ,and once(1 .33% ) was three—step mutation ,no mutation was found at the TPOX locus .The mutation rates ranged 0 .031 1% —0 .404 2% ,and the mean mutation rate was 0 .145 8% .The proportion of the paternal mutations and the maternal mutations was 5 .4:1 .0 ,the difference had statistical significance(P0 .05) .Conclusion STR loci mutation is common phenomenon in paternity test .The data of STR loci mutations should be constantly accumulated for selecting the genetic characteristics in line with the Guangxi population and the genetic markers of STR loci with high identification ability to ensure ac-curate and reliable identification results .

A novel CD36 mutation T538C (Trp180Arg) results in CD36 deficiency and establishment of a genotyping method for the novel mutation based on sequence-specific primer PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the molecular basis for a CD36 deficiency individual and distribution of CD36 gene mutation in Guangxi population.</p><p><b>METHODS</b>A female individual was studied. CD36 phenotype was detected by monoclonal antibody immobilization of platelet antigens assay (MAIPA) and flow cytometry (FCM). The coding regions of the CD36 gene were sequenced. A DNA-based polymerase chain reaction-sequence specific primer (PCR-SSP) assay was used to verify the identified mutation. Cell lines expressing the mutant and wild-type CD36[CD36(MT) and CD36(WT)] were established, with the expression of CD36 determined by Western blotting. The distribution of CD36 gene mutation was investigated among 1010 unrelated individuals with the PCR-SSP assay.</p><p><b>RESULTS</b>Both MAIPA and FCM assays showed that the patient had type II CD36 deficiency. DNA sequencing showed that she has carried a heterozygous mutation T538C (Trp180Arg) in the exon 6 of CD36. Sequencing of cDNA clone confirmed that there was a nucleotide substitution at position 538 (538T>C). Western blotting also confirmed that the CD36 did not express on the CD36(MT) cell line that expressed the 538C mutant, but did express on the CD36(WT) cell line. The novel CD36 mutation T538C was further verified with 100% concordance of genotyping results by DNA-based PCR-SSP assay and 1010 unrelated individuals. No CD36 538C allele was detected among the 1010 individuals.</p><p><b>CONCLUSION</b>This study has identified a novel CD36 mutation T538C(Trp180Arg)(GenBank: HM217022.1), and established a genotyping method for the novel sequence-specific primer PCR. The novel mutation is rare in Guangxi and can cause type II CD36 deficiency.</p>

Establishment of platelet antigen panel cells and application in platelet alloantibody detection / 重庆医学

Objective To establish the platelet antigen panel cells and to apply them in the detection and identification of platelet alloantibody.Methods Human platelet antigen(HPA)1-16 genotyping from 1 500 un-related blood donors in Nanning area were performed by the polymerase chain reaction-sequence specific primers(PCR-SSP)technique,platelet antigen cells with O blood type were chosen to establish the panel cells of platelet antigen.The phenotype of the panel cells were verified by the reference sera from the 14th platelet immunology workshop of the International Society of Blood Transfusion(ISBT).And then these panel cells were used in clinic to detect the platelet alloantibody and the samples from the 14th and 15th platelet immunology workshop of ISBT.Re-sults Six platelet cells with consistent phenotype and genotypes and covering the HPA 1-5 and 1 5 systems were selected to estab-lish the platelet panel cells and successfully applied them in the clinical and scientific sample detection and identification.Conclusion Platelet antigen panel cells are established successfully,which provides the experimental basis for the diagnosis and research of platelet allogenic abnormal immunity diseases.

Proficiency evaluation of platelet alloantiboby analysis in 42 platelet Immunology laboratories around the world:the 14t~h ISBT Platelet Immunology Workshop and Cooperative Research Project / 中国输血杂志

Objective To organize the 14th ISBT Platelet Immunology Workshop and Cooperative Research Project,and proficiency evaluation on the techniques and quality level of platelet alloantiboby analysis will be taken effect for the 42 platelet immunology laboratories around the world. Methods Organized and confirmed by Nanning Institute of Transfusion Medicine,9 quality control samples contained the Human Platelet Antigen (HPA) specificity antibody have been supplied to all the participated laboratories in this project. The participants could use the In-House technology or/and market kits to test these quality control samples. The forms and sheets have been provided for recording of results. Results There were 36 laboratories from 23 countries participated in this Cooperative Research Project,and 35 laboratories have reported their results. The appraised consistent rate of the 9 quality control samples ranges from 20% to 97.14%,the HPA antibody specificities have been showed as anti HPA-1a,anti HPA-1b,anti HPA-3a,anti HPA-3a,anti HPA-3b,anti HPA-5b,anti HPA-5a,anti GPIV and anti HPA-5b+15b,respectively. Among the appraised consistent rate,the anti HPA-3b were the lowest and the anti HPA-5b and anti HPA-5a was the highest. Over 10 technologies of platelet alloantiboby analysis were used by the laboratories. Conclusion This international cooperative research project has successfully made the proficiency evaluation and report on the techniques and quality level of platelet alloantiboby analysis for the 35 international platelet immunology laboratories.

Genotyping of ABO blood group in Chinese Han population / 中国组织工程研究

BACKGROUND: ABO is the most important blood group system for blood transfusion. Though widely used in determining ABO blood group for its simplicity and rapidity, serological technology has its inherent limitation, for which ABO genotyping provides a valuable alternative.OBJECTIVE: To study ABO gene polymorphism in Chinese Han population and apply ABO genotyping technique to solve serological problems in clinical practice of blood transfusion.DESIGN: Comparison of ABO genotyping results of random selected samples with those of routine serological phenotyping.SETTING: An institute of transfusion medicine in a municipal blood center.PARTICIPANTS: Totally 260 unrelated healthy Chinese blood donors of Han nationality were randomly selected in Shenzhen Blood Center from March to December in 2002, including 110 male and 150 female subjects aged between 18 and 50 years. A sample with discrepancy in serological ABO phenotype was from our blood center, and the donor' s family was investigated. Six samples suspected to be A2 phenotype by serological test were from four hospitals in Shenzhen including the Second People' s Hospital of Shenzhen.METHODS: The DNA was extracted from the peripheral blood by rapid salt fractionation, and subjected to polymerase chain reaction(PCR) with sequence-specific primers (PCR-SSP) to amplify the ABO gene for ABO genotyping. The alleles of the blood type difficult to determine were amplified with PCR-SSP on the basis of serologic tests including absorption and elution test and agglutination inhibition assay of salivary blood-group substances.MAIN OUTCOME MEASURES: Genotypes and phenotypes of the blood samples from 260 individuals and of the samples with serological ABO discrepancy.RESULTS: In the 260 Chinese Han individuals, in accordance with Hardy-Weinberg equilibrium, the gene frequencies of O1, B, A1O1(A467c), A1O2/1O3(A467T) alleles were 0. 582 7, 0. 184 6, 0. 009 6, and 0. 2231, respectively. Two of the six individuals with difficulty of blood type determination and suspected to have A2 phenotype by serological tests proved to have A2O1O1 genotype, and the rest were all of A1O2/A1O3O1. Three children of a family with difficult identification were para-Bombay types, and their ABO types were A102B, A102B and A102O1, respectively.CONCLUSION: ABO PCR-SSP genotyping is simple, rapid and accurate and can be a valuable complement to serological identification.

Identification of a novel B variant allele in a Chinese Han individual with B subgroup / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the molecular genetic background of B subtype in Chinese Han population and identify novel allele at the ABO locus.</p><p><b>METHODS</b>Ten samples from randomly selected blood donors of normal B phenotype used as control, and six samples from individuals diagnosed as B subgroup by serological tests were genotyped by sequence specific primer polymerase chain reaction and direct DNA sequencing at the exons 6 and 7 of ABO gene. The exons 6 and 7 and the intervening intron 6 of the B allele from each B subgroup sample were analyzed by cloning and haplotype sequencing.</p><p><b>RESULTS</b>A novel B variant allele was identified in two individuals whose blood samples were diagnosed as belonging to Bx subgroup and Bw subgroup respectively, the novel B allele being different from the allele B101 by single 695T>C missense mutation in exon 7. A family with the individual possessed Bx subgroup was studied. Among 22 family members tested, seven family members were found to carry the novel B variant allele. No novel point mutation at the exons 6 and 7 of ABO gene were detected in the ten control samples and the other four samples with B subgroup.</p><p><b>CONCLUSION</b>The present authors define this allele as a novel B variant allele. The mutation of this novel allele, in which the nucleotide alters from T to C at position of 695 in exon 7 and hence results in an amino acid change from Leu to Pro, is expected to diminish the enzyme's activity. It indicates that the alteration of amino acid at the position of 232 is critical to the activity of glycosyltransferases.</p>

A novel O~1 variant allele at the ABO blood group locus / 中华检验医学杂志

Objective To identify novel ABO allele in Chinese population. Methods The ABO blood group was tested by serological method, and then genotyped by sequence-specific primer (PCR-SSP) , gene cloning and sequence analysis. Results A healthy blood dornor who was diagnosed as having A2 subgroup and A2O1genotype was subjected to ABO gene cloning and sequence analysis. The haplotype-specific sequence analysis indicate that two single-base deletions, where G-deletion at nucleotide position 261 and A-deletion at nucleotide position 496 were determined in the O1 allele. The nucleotide sequence of the novelO1 allele were identical to ABO 0101 allele except for A-deletion at nucleotide position 496 in exon7 of ABO locus. Conclusion We defined this 0 allele as a novel O1 variant allele, and its registered number by GenBank is AY374123.

Study on the FUT2 gene structure of Xinjiang Uighur people of China / 中国输血杂志

Objective To study the Secretor gene (FUT2) molecular structure of Uighur population in Xinjiang area,China. Methods DNA was extracted from 40 Uygur unrelated donors' blood and sequence analysis of FUT2 genes was performed. Results Four mutations in the FUT2 genes of Uighur donors have been identified. The frequencies of mutations were 71.25% for 357T, 28.75% for 357C,77.50% for 385A,22.50% for 385T,70% for 428G,30% for 428A,72.50% for 739G and 27.50% for 739A. Conclusion Based on the characteristics of FUT2 gene structure of Xinjiang Uighur,it cauld be thought that there are some relationships between Xinjiang Uighur, Taiwanese of China and Caucasiany.

Prepae B-lymphoblastoid cell lines of HLA novel allele B ~*5610 in a family / 中国输血杂志

Objective To prepare B-lymphoblastoid cell lines of HLA novel allele B*5610 in a family for further study and identification . Method Isolate mononuclear cells under aseptic conditions from the peripheral blood. After infection with Epstein-Barr virus, the cells were cultured in 20% FBS, 2?g/ml CsA RPMI 1640. Results Immortalized B-lymphoblastoid cell lines of five B *5610 carriers in a family were achieved, and the new genes were inherited stably. Conclusion Our work is important for storing and breeding the precious material of biomedicine because the B *5610 genes in the immortalized B-lymphoblastoid cell lines were inherited stably.

Non-invasive detection of prenatal fetal ABO and/Rh(D)blood groups by flow cytometry(FCM) / 中国输血杂志

Objective To establish the FCM method of non invasive detection of fetal ABO and Rh(D) blood groups in maternal blood.Methods Using absorption and elution method, we obtained the IgG anti A and anti B from human sera. The IgG anti A, B, D were used as the first antibody to react with RBCs in maternal peripheral blood.The goat anti human IgG F(ab')2 FITC was used as the second antibody to conjugate anti A, B, D antibodies, Meanwhile anti i PE was used to mark fetal RBCs in maternal peripheral blood.The fluorescence dot plot diagrams of maternal and fetal cells acquired by FCM were used to detect fetal ABO and Rh(D) blood groups.Results Peripheral blood from 69 pregnant women between 8 and 39 weeks of gestation were studied.Fetal cells could not be found in 13 samples.Of the remaining 56 samples,fetal red cells were identified successfully with ABO/Rh(D) blood types identical to those tested after the birth of the baby.Conclusion In women with fetomaternal hemorrhage(FMH) during pregnancy,the FCM method established by the author can accurately and non invasively detect the blood groups of fetuses.This method can possibly be used for diagnosis of hemolytic disease of the newborn.

Analysis of the relative factors of the onset of repeated ectopic pregnancy / 中华妇产科杂志

Objective To determine the relative factors of the onset of repeated ectopic pregnancy. Methods The clinical data of the first time ectopic pregnancy of 28 cases with repeated ectopic pregnancy were analysed, and compared with those of 56 cases of non-repeated ectopic pregnancy onsetting at the same period. The factors measured included: age at onset of disease, age at first coitus, gravidity, parity, methods of contraception, duration of amenorrhea,duration of vaginal bleeding, serum ?-human chorionic gonadotropin level, volume of intraperitoneal bleeding, types of ectopic pregnancy, methods of therapy and inflammation evidence of fallopian tube. Logistic regression analysis was performed to determine the relative factors for onset of repeated pregnancy. Results The risk factors and its odds ratio (OR) from the multivariate analysis were as follow: anastomosis of the tube(62.74, P=0.043), positive evidence of inflammation of the tube (54.85, P=0.000), no contraception (11.29, P=0.002), contraception by condom occasionally (4.75, P=0.046); the protective factors and its OR were as follow: therapy being salpingectomy and sterilization of the opposite tube(0.06, P=0.049), oral contraception (0.10, P=0.050) and pharmacotherapy (0.33, P=0.002). Conclusions The risk factors of onset of repeated ectopic pregnancy include: anastomosis of the tube, positive evidence of inflammation of the tube, no contraception and contraception by condom occasionally; the protective factors include: therapy being salpingectomy and sterilization of the opposite tube, oral contraception and pharmacotherapy.

Application of Solid-Phase Platelet Immunoserological Assay for the Platelet Transfusion Therapy:Ⅱ.The Technical Study on the Treatment of Refractory Idiopathic Thrombocytopenic Purpura with Vinca-Alkaloids-Bin-ding Platelets / 中国输血杂志

A technique of solid-phase platelet im-munoserological assay was devised to selectsuitable platelet donors for patients withrefractory idiopathic thrombocytopenic pur- pura(RITP).The suitable platelets act-ed as carriers to bind with vinca-alkalo-ids,and then were gived to the patientswith RITP.The technique showed clini-cally significant.

Identification of a novel Bx allele in a Chinese Han individual with B subgroup / 中国输血杂志

T missense mutation in exon 7. No novel point mutation at exons 6 and 7 of ABO gene was detected in the other four samples with B subgroup. Conclusion We define this allele as a novel B allele in Chinese Han individuals. The mutation of this novel allele in which the nucleotide changes from C to T at position 721 in exon 7, resulting in an amino acid change from Arg to Trp, results in the decrease of the enzyme activity. It indicates that the alteration of amino acid at position of 241 is critical to the activity of glycosyltransferases.

Mutational analysis of FUT1 and FUT2 genes in para-Bombay individuals / 中国输血杂志

Objective To study the mutation of FUT1 and FUT2 genes in para-Bombay individual.Methods Direct DNA sequencing of FUT1 and FUT2 gene coding region were analyzed in two individuals with para-Bombay phenotype.Results One individual lost one of the three AG repeats located at nucleotides 547~552 of the FUT1 gene, whereas two of the three T repeats located at nucleotides 880~882 were deleted in the other.Conclusion Two frame-shift mutations of FUT1 gene are responsible for the H antigen deficiency

A study of FUT2 gene point mutation in the Chinese Han Population / 中国输血杂志

Objective To study the point mutation of FUT2 gene in Chinese Han population.Methods Using direct sequencing,molecular cloning techniques and the comparing with the gene sequence reportedby Kelly, the FUT2 gene structures of 41Chinese Han individuals have were studied.Results The G428A mutations of FUT2 gene was not found,but the A385T and C357T mutations were found in the 41 Chinese Han individuals.Among the 41 individuals,24 had A385T mutation and 17 had no A385T mutation.The neutral mutation C357T was found in all 41individuals.Conclusion The G428A point mutation of FUT2 which is commonly found in non secretor of Africans and Caucasian was not found in Chinese population.There are A385T and C357T point mutations which were found in 41 Chinese Han individuals.The present stady shows the difference between Chinese and Caucasian,and other non secretor mutations will be revealed by further investigation

Application of Y-chromosome specific short tandem repeat loci in noninvasive prenatal diagnosis / 中国输血杂志

Objective To establish a noninvasive method for prenatal genetic analysis by using maternal serum and apply the method in fetal sex determination,paternity testing. Methods Samples of maternal serum from 53 pregnant women (11 to 36 weeks of gestation) were collected. The DNA extracted from each sample was amplified by using"Y-PLEX 6" amplification kit .which enabled the simultaneous analysis of six Y-STR loci including DYS393.DYS19.DYS389 II, DYS390, DYS391 and DYS385. The PCR products were detected by using ABI PrismTM 377 Sequencer and genotyped by related analysis software. Results (1) Y-STR specific alleles were detected in the maternal sera of all 29 mothers bearing male babies. Among the six Y-STR loci,specific alleles were detected in 29/29 at DYS393 locus,in 18/29 at DYS19 locus and in 10/29 at DYS390 locus. (2) Y-STR specific alleles were not detected in maternal sera of 24 pregnant women bearing female babies. (3) According to the presence of specific alleles at DYS393 locus and the value of allelic peak height and peak area, the accuracy of fetal sex determination was 100% . (4)The observed Y-STR alleles of each prenatal specimen from pregnant women with male fetuses were the same as the results of their husbands. Conclusion The assay of highly polymorphic Y-STR genotyping system developed by the authors provided a sensitive, accurate and non-invasive method to prenatal diagnosis. Our results demonstrate that fetal sex can be accurately determined and imply that paternity testing could be performed for pregnant women carrying male fetuses.

Mutations observed at 13 CODIS STR loci in cases of paternity testing / 中国法医学杂志

Objective Mutations of 13 CODIS (Combined DNA Index System) STR core loci in 532 cases of paternity testing were observed in confirming paternity, the mutation rate and the mutation type were studied. Methods 587 cases of paternity testing were routinely carried out using AmpFe STR Profiler Plus and Cofiler PCR Amplification Kits. When one or two STR exclusions were found, then HLA system and other blood groups were tested by molecular typing, and sixteen STR loci were genotyped by using PowerPlexl6 PCR Amplification Kit. If necessary, the genotyping of Y chromosome specific STR and HLA allelic sequencing were added. Results 1052 meiosis were observed among the 532 cases in confirmed paternity, 18 mutation events were found in 17 paternity cases. Single-locus mutation was observed in 16 cases, and mutation at two STR loci was observed in one case. The observed mutational loci include: D5S818, D3S1358, D16S539, CSFIPO, D21S11, D13S317, D7S820, vWA, D18S51 and FGA. The mutation rates for D18S51 and FGA loci were both 0.29% , which were the highest among the ten mutational loci. 11 events of paternal source mutations, 5 events of maternal source mutations and two events of indistinguishable mutations were observed in 18 STR mutational events. Conclusion When one or two STR exclusions were found in paternity testing, other more genetic markers must be detected as complement before making final conclusions.