Two Novel Mutations at the CD36 Gene Splicing Sites and Their Molecular Basis for the CD36 Deficiency / 中国实验血液学杂志

OBJECTIVE@#To study two novel CD36 gene mutations at the CD36 splicing sites found in Guangxi population, as well as the molecular basis and population incidence of them.@*METHODS@#DNA sequencing and cDNA clonal sequencing were used to detect CD36 exon sequence and the protein coding region sequence of CD36 mRNA for 2 CD36 deficient individuals (HHC and WGM) found in Guangxi population. Eukaryotic expression cell lines were established for the discovery of CD36 mRNA abnormal transcripts and Western blot assay was used to verify the effect of abnormal CD36 mRNA transcripts on CD36 expression. A DNA PCR-SSP genotyping method was established for the two CD36 novel mutations, and the population distribution was investigated among 110 CD36 deficient individuals in Guangxi region and 296 random individuals in Guangxi population.@*RESULTS@#Novel mutation of c.430 -1G>C was found at the CD36 splicing site in HHC and WGM individuals, and novel mutation of c.1006 +2T>G at the CD36 splicing site was also found in the WGM individual. CD36 cDNA clonal sequencing showed that CD36 c.430 -1G>C could lead to the production of the two CD36 mRNA transcript variants: c.429_430ins［430-17_430-2;C］(p.Ala144fsTer1) and c.430_609del(p.Ala144_Pro203del)(GenBank:HM 217023.1); and CD36 c.1006 +2T>G could lead to the production of CD36 mRNA transcript variant of c.819_1006 del (p.Ser274GlufsTer16) (GenBank: HM217025.1). It was verified that all the three transcript variants could lead to CD36 deficiency by establishment of eukaryotic expression cell lines and Western blot assay. A study of the population incidence of two novel CD36 splicing site mutations found showed that in 110 CD36 deficient individuals and in 296 random individuals in Guangxi region, the mutation rate of CD36 c.430 -1G>C was 10.91% (12/110) and 1.35% (4/296), respectively, while CD36 c.1006 +2T>G was 2.73% (3/110) and 0 (0/296), respectively.@*CONCLUSION@#This study identifies two novel CD36 mutations at CD36 splicing site, and preliminary clarified their molecular basis for the CD36 deficiency and the distribution characteristics in Guangxi population as well. It provides an experimental and theoretical basis for studying the molecular mechanism and characteristics of CD36 deficiency in Chinese population.

Anti-CD36 Mediated Platelet Transfusion Refractoriness and Related Cases After Stem Cell Transplantation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyse the cases of platelet transfusion refractoriness after received HLA-matched unrelated donor hematopoietic stem cell transplantation, to analyze and identify the phenotype and genotype of CD36 in both the patient and stem cell donor, as well as the characteristic of antibody induced platelet transfusion refractoriness, and to analyse the efficacy of matched CD36-deficiency platelets transfusions.</p><p><b>METHODS</b>The CD36 expression on platelet and monocyte was analyzed by flow cytometry (FCM) in both patient and donor. Polymerase chain reaction sequence-based typing (PCR-SBT) was used to analyze the exons sequence of CD36 and HPA. Fast monoclonal antibody-specific immobilization of platelet antigen (F-MAIPA) and FCM were used to identify platelet antibodies in the patient. Short tandem repeat polymerase chain reaction (STR-PCR) was applied to monitor engraftment evidence. The platelet level was monitored. CD36- deficiency donor's platelets were selected from CD36- deficiency donor blood bank.</p><p><b>RESULTS</b>The donor was CD36 positive and the patient was typed I CD36 deficiency. The anti-CD36 antibody was identified in patient's serum (after transplantation), while the HLA and HPA-related antibodies were excluded. Sequence analysis of CD36 exon in the patient showed Exon 6 -1G>C(Change in splicing site) homozygote, which was a novel CD36 mutation. STR, HPA and CD36 of the patient (complete chimerism) were conversed to that of donor gene types on day 18 after allo-HSCT. The positive CD36 expression on platelet and monocyte in the patient was observed on day 96 after allo-HSCT. The patient showed the platelet transfusion refractoriness which was significantly improved after platelets transfusions from CD36 deficiency donors.</p><p><b>CONCLUSION</b>Stem cell transplants resulted in anti-CD36 and caused platelet transfusion refractoriness, that was first reported in China. To ensure the efficacy of platelet transfusion, the CD36-deficiency patient should receive CD36 deficiency platelets for transfusion.</p>

Identification of A Novel HLA-B*13:92 Allele and Analysis of Its 3D Model Structure / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To identify a novel human leukocyte antigen (HLA) allele HLA-B*13:92 and analyze 3D model of HLA molecule.</p><p><b>METHODS</b>Polymerase chain reaction sequencing-based (PCR-SBT) was used in routine HLA typing, the B locus typing results of one sample was one base mismatch with B*13:01:01, B*58:01:01 at locus 189, The Group Specific Sequencing Products (GSSP) which target at B*13 and B*58 were used to confirm difference between the new allele and highest homologous allele, then the new allele was modeled by Swiss-model to its 3D structure.</p><p><b>RESULTS</b>The sequencing results showed that the new allele with highest homologous allele B*13:01:01 was the difference in the second exon at position 189 C>A (codon 39 GAC>GAA), 39 Asp (D) was changed to Glu (E). The amino acid substitution at residue 39 of the HLA polypeptide was located in α-helices of antigenic peptide-biding region.</p><p><b>CONCLUSION</b>This allele is a new HLA-B allele found in Chinese Guangxi Zhang population and has been designated as HLA-B*13:92 by the World Health Organization (WHO) HLA Nomenclature Committee.</p>

Detection, diagnosis and analysis of the first case of neonatal alloimmune thrombocytopenia purpura associated with anti-HPA-5b in China / 中国实验血液学杂志

This study was aimed to investigate the detection and diagnosis of the neonatal alloimmune thrombocytopenia purpura (NAITP) caused by anti-HPA-5b antibody. The platelet count and clinical manifestation in the newborn were examined. The HPA-1-21bw genotypes of the newborn and her parents were detected by multiple-PCR and DNA sequencing. The HPA-specific antibody in the sera of newborn and her mother were detected and identified by flow cytometry (FCM) and monoclonal antibody-specific immobilization of platelet antigens (MAIPA). The results indicated that the clinical manifestations of the newborn were lighter. The HPA genotyping showed that the genotype of the newborn was HPA-5ab, while that of her mother and father were HPA-5aa and HPA-5ab, respectively. The antibody against the platelet of newborn's father existed in the newborn's mother sera. The HPA antibody of the mother was identified as anti-HPA-5b. It is concluded that the newborn with neonatal alloimmune thrombocytopenia purpura was caused by the antibody against HPA-5b.

A case of neonatal alloimmune thrombocytopenia purpura caused by anti HPA-3a antibody and literature review / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the diagnosis and treatment of a case of neonatal alloimmune thrombocytopenia purpura (NAITP) caused by anti HPA-3a antibody.</p><p><b>METHODS</b>The platelet counts and purpuric symptom in the newborn were clinical examined. The HPA-1-21bw genotypes of the newborn and his parents were detected by multiple DNA-PCR, gene sequencing and genotyping. The HPA specificity antibody in the sera of newborn and his mother were detected by flow cytometry (FCM), and the HPA specificity antibody was identified by monoclonal antibody-specific immobilization of platelet antigens (MAIPA).</p><p><b>RESULTS</b>The newborn had the typical symptom of NAITP, multiple subcutaneous petechia, hematuria and coffee-like vomitus. The HPA genotype of the newborn was HPA-3ab, while that of his mother and his father were HPA-3bb and HPA-3aa, respectively. The sera of newborn and his mother existed antibody against the platelet of newborn's father. The HPA antibody of the newborn and his mother were identified as anti HPA-3a. The newborn was approved a patient of NAITP caused by anti HPA-3a antibody.</p><p><b>CONCLUSION</b>The diagnosis and treatment for NAITP newborn caused by anti HPA-3a antibody in this study was the first domestic report. It could provide successful experiences and references for the similar cases.</p>

Ambiguous allele combinations in the HLA high resolution genotyping--review / 中国实验血液学杂志

Human lymphocyte antigen (HLA) is the most complicated human dominant polymorphic genetic system. Accurate HLA genotyping is clinically important for hematopoietic stem cell (HSC) transplantation, also important for research on many human diseases. Polymerase chain reaction-sequence based typing (PCR-SBT) provides the highest resolution level and defines new alleles, so it is widely used for HLA typing. One great disadvantage of PCR-SBT method is the fact that it cannot resolve sequences of heterozygous samples in diploid genomes, leading to ambiguous typing results which make much trouble to the accurate definition of HLA genotype. This article reviewed the occurring reasons and solution method of ambiguous allele combinations in the HLA high resolution genotyping as well as the research prospect in this field.

Influence of different products of platelet membrane glycoprotein monoclonal antibodies used internationally on tests for monoclonal antibody-specific immobilization of platelet antigens / 中国实验血液学杂志

This study was aimed to investigate the influence of different platelet membrane glycoprotein monoclonal antibodies (McAb) which are common used in laboratories on the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technique according to the request of 14th International Society of Blood Transfusion Platelet Immunology Workshop. 30 participant laboratories were provided with 10 known human platelet antigen (HPA) antibodies, 1 normal serum, 9 different McAbs (against GPIIb/IIIa, GPIa/IIa, GPIb/IX and GPIV respectively), and the same protocol. Each participant laboratory carried out the test as the protocol to compare the results of different McAbs against the same glycoprotein and submitted the data to organizer. The results indicated that in McAbs against GPIIb/IIIa, AP2, Gi-5 and PL2-73 showed higher mean S/CO than that of others; in GPIa/IIa, MBC202.2 and 143.1 showed higher mean S/CO than that of others; in GPIb/IX, 142.11 and CLB-MB45 (CD42b) showed higher mean S/CO than that of others; as to GPIV, 131.4 showed higher mean S/CO. In conclusion, capture effects of various McAbs are different, so that different products of McAbs exert influences on the sensitivity of MAIPA. To use a panel of McAbs against the same glycoprotein may avoid the false negative results.

Polymorphism of D2S1338 and D19S433 loci in Southern Chinese Han nationality population and its application / 中国实验血液学杂志

This study was aimed to investigate the polymorphism of D2S1338 and D19S433 loci in Southern Chinese Han nationality population, and to evaluate its forensic application. The DNA was extracted by chromatography on che-lex-100. Fifteen STR loci (D2S1338, D19S433 etc.) were amplified by Identifiler kit and analyzed by 3100 genetic analyzer. The softwares of analysis were GeneScan 3.0 and Genetype 3.7. The results indicated that the allele frequencies of H, DP, PIC, PE of D2S1338 and D19S433 loci were obtained. The allele frequency of samples were in Hardy-Weinberg equilibrium by chi2 test. No mutation was found on D2S1338 and D19S433 loci in 370 cases of meiosis. It is concluded that the D2S1338 and D19S433 loci are high polymorphic and their mutation rates in Southern Chinese Han nationality population are low. Their good distribution is suitable for forensic individual identification and paternity testing.

Study on molecular genetic structure of Ael blood subgroup / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the ABO allele molecular characteristics of Ael blood subgroup.</p><p><b>METHODS</b>Five individuals of diagnosed as Ael blood subgroup were subjected to PCR amplify ABO alleles using four pairs of sequence-specific primers. Exon 6 and exon 7 at ABO locus of all samples were sequenced. An individual with AelB phenotype was chosen for further analysis of transcript structure of ABO gene.</p><p><b>RESULTS</b>Sequence analysis indicated one Ael phenotype sample with reported Ael01 allele, one Ael phenotype sample with an Ael05 allele, and two AelB and one Ael individuals did not contain referred A allele, but contain O01 or O02 allele with 261G deletion.</p><p><b>CONCLUSION</b>Molecular bases for the Ael have highly polymorphism. The mechanism responsible for the express weak A antigen of O allele with 261G deletion awaits to be elucidated.</p>

The genetic polymorphisms of nine Y-STR loci with short fragment size alleles in southern Chinese Han population and its application in forensic science / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the genetic polymorphisms of 9 Y-chromosome specific STR loci that the allele size is less than 180 bp in length in the southern Chinese Han population, and to utilize the studied result to forensic science.</p><p><b>METHODS</b>Nine Y-STR loci were amplified by single multiplex PCR, and the PCR products were sequenced by using ABI Prism 3100 DNA Sequencer. The allele and haplotype frequencies at 9 Y-STR loci were determined in a total of 213 unrelated males from southern Chinese Han population. Eighty-four father/son pairs with demonstrated paternity and thirty-six non-paternity father/son pairs were detected by using our Y-STR multiplex system.</p><p><b>RESULTS</b>Three Y-STR alleles for DYS426, five alleles for DYS393, DYS460, DYS391 and DYS389 I respectively, six alleles for DYS456, seven alleles for both H4 and DYS388, and eight alleles for DYS458 loci were detected in 213 unrelated male individuals. Except for the DYS426 locus with a low GD value of 0.1489, the GD values for other 8 Y-STR loci ranged from 0.5064 to 0.9133. A total of 178 haplotypes were found at 9 Y-STR loci, of which 154 haplotypes were observed only once, and the haplotype diversity was 0.9983. None of Y-STR allele mutation was observed in the 84 father/son pairs with demonstrated paternity. Among the 36 non-paternity father/son pairs, two cases could get the paternity exclusions at 2 Y-STR loci; and the paternity of 33 cases could be ruled out by 3 or more Y-STR loci; only one case was found no exclusion of paternity regardless of detecting 9 Y-STR loci.</p><p><b>CONCLUSION</b>This result indicates that the 9 Y-STR loci with short fragment size alleles are highly polymorphic. The fluorescent multiplex amplification system that we developed is suitable for personal identification and paternity testing.</p>

Molecular genetic analysis for the A3 alleles / 中国实验血液学杂志

To study four A(3) subgroup samples identified by serologic tests, among which two belong to a family, three were A(3) subgroup, one was A(3)B subgroup. All four samples were genotyped by PCR-SSP method, and the nucleotide sequences of Exon 6, Exon 7 and part introns at the ABO locus for these samples were detected by ABI Prism 3100 DNA sequencer. Comparison with the consensus of A101 was performed. The results showed that haplotypes of two A(3) subgroups were common A102 allele and O1-2 allele, and haplotypes of one A(3) subgroup were common A102 allele and rare O(1v)-4 allele. Unexpectedly, a synonymous substitution 838C-->T had been found in A allele of the A(3)B subgroup sample, which predict a Leu280Phe alteration. The results suggested that molecular genetic background of the A(3) phenotypes is polymorphic. Possibly, the missense mutation 838C-->T is the molecular genetic basis of A(3)B subgroup that lead to low activity of the glycosyltransferases.

Study on the correlation between acute lymphoblastic leukemia and HLA genes in southern China Han population / 中国实验血液学杂志

To study the correlation between acute lymphoblastic leukemia (ALL) and HLA-A, B and DRB1 gene in southern Chinese Han population and to investigate the susceptible HLA gene to ALL, a total of 4707 healthy volunteer bone marrow donors from southern Chinese Han population were used as a control group, 201 patients diagnosed as patient group from southern Han individuals were genotyped at HLA-A, B and DRB1 loci by PCR-SSP, PCR-SSOP and SBT. HLA allele frequency and its distribution of ALL patient group were compared with the control group by using chi(2) test, and calculated the statistic value of relative risk (RR), pathogenicity score (EF) and preventive score (PF). The results showed that in comparison with the control group, the gene frequence of HLA-A26, B56 and DR9 increased significantly, but the gene frequence of HLA-A30, A33 and B58 allele frequency decreased significantly for patients with ALL. It is concluded that HLA-A26, B56 and DR9 gene have a high correlation with ALL and seem to contribute the genetic susceptibility to ALL in southern Chinese Han populations. However, HLA-A30, A33 and B58 gene seem to have protective role for southern Han individuals suffered from ALL.

Polymorphism of HLA-B* 40 gene family in Chinese Han population / 中国实验血液学杂志

To investigate the allele distribution of HLA-B* 40 gene family in Chinese Han population and to study its influence on the selection of clinical transplantation donor, the HLA-B genetypes of 381 individuals randomly selected from Chinese National Marrow Donor Project were identified by PCR-SSO, and then all the HLA-B* 40 positive samples from the above population and the B* 40 homozygote samples received from another 1 270 registered donors were analyzed by PCR-SBT and PCR-SSP at high resolution. The results showed that the population of 381 registered donors was examined at HLA-B locus by using Hardy-Weinberg equilibrium, the gene frequency of HLA-B* 40 was 0.1692. Four different HLA-B* 40 alleles (B* 4001, B* 4002, B* 4003, B* 4006) were identified, and the serological specificity was B60 and B61 respectively. The relative frequency of each allele was 0.1192 for B* 4001, 0.0154 for B* 4002, 0.0038 for B* 4003, 0.0308 for B* 4006. The distribution of B* 40 homozygote revealed a certain regularity at high-resolution, B* 40XX (B* 4001 group), at low-resolution; B* 4001 at high resolution; B* 40XX (B* 4002 group), at low-resolution; B* 4002 or B* 4006 or heterozygote of both at high-resolution. It is concluded that in Chinese Han population, predominant allele in HLA-B* 40 gene family is B* 4001, the high-resolution typing may be recommended to use for the selection of clinical transplantation donor.

Study of HLA polymorphism in the 6965 Han bone marrow registry donors / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze human leukocyte antigen (HLA) polymorphism and search for new alleles in Chinese Han population bone marrow registry donors.</p><p><b>METHODS</b>DNA-based HLA genotyping methods were used including PCR-SSP, BST and molecular cloning.</p><p><b>RESULTS</b>A total of 6965 unrelated donors, 4707 from South China origin and 2258 from north, were typed for HLA-A, B, and DRB1 loci. Seventy-two specificities of HLA alleles were identified. The HLA-A25, A34, A74, B41, B42, B53, B73 and B81 that were rarely reported in previously Chinese population studies were identified in this study. Estimation of gene frequency indicated that the blank gene frequency was less than 0.2% for HLA-A, 0.25% for HLA-B and 0.70% for HLA-DRB1 loci. Three novel alleles were identified and officially assigned by the World Health Organization (WHO) Nomenclature Committee as A*0253N, A*1114 and B*5610.</p><p><b>CONCLUSION</b>Large-scale DNA-based HLA genotyping used in bone marrow registry donors is highly accurate and reliable for estimating gene frequency and searching for new alleles. The discrepancy of HLA gene distribution between South and North China Han population showed the necessity of setting the more regions in South and North China to screen the bone marrow registry donors for bone marrow transplant.</p>

Experimental study on quantitative monitoring engraftment of an adult with mixed umbilical cord blood transplantation / 中国实验血液学杂志

The purpose of this research was to monitor quantitatively and study the dynamic changes and development rules of engraftment, chimera types, as well as relative amount of donor cells after allogeneic transplantation of mixed umbilical-cord blood from two units. An adult patient with acute myeloid leukemia received two units HLA one locus mismatched unrelated umbilical cord blood transplantation (2.5 x 10(7)/kg karyocytes in umbilical cord blood unit 1, and 1.53 x 10(7)/kg karyocytes in umbilical cord blood unit 2). Nine STR loci of the blood sample before and after transplantation were determined by quantitative detecting technique with fluorescence labeling polymerase chain reaction, while the engraftment and chimera types were qualitatively evaluated by comparing differential loci between the recipient and the donors. Then the relative proportion of chimera from two units of umbilical-cord blood in the patient after transplantation was calculated according to the differential gene peak areas of two donors on 377XL DNA sequencer after fluorescence scanning, and the engraftment level and the development rules of donor cells were analyzed. In addition, the results were also compared with that of HLA loci distinct analysis for engraftment. The results showed that two umbilical cord blood units at 15 days after transplantation were engrafted simultaneously and revealed a complete chimerism of the two. The relative amounts of chimera from unit 1 vs that of unit 2 were 51.3% vs 48.7%; subsequently relative amounts of chimera from unit 1 went up to 70.0% at 30 days, and that from unit 2 declined to 30.0%. However, at 52 days, only the genotype of umbilical cord blood unit 1 was detected, so that the engraftment turned to a complete chimerism of a single donor type. The one with fewer karyocytes was rejected and the one with more karyocytes finally engrafted in long-term. It is concluded that quantitatively detecting STR chimera with fluorescence labeling polymerase chain reaction can depict precisely the engraftment level and the change course of two umbilical cord blood units. It provides an accurate and reliable experimental basis for clinical umbilical cord blood application and donor selection, and is proved to be feasible for adult transplantation by using dual unit of umbilical-cord blood with HLA one locus mismatched at the same time.