Study of the distribution of KIR3DL2 alleles among ethnic Han Chinese from Zhejiang / 中华医学遗传学杂志

OBJECTIVE@#To study the distribution of KIR3DL2 alleles among ethnic Han Chinese from Zhejiang.@*METHODS@#Genomic DNA was extracted by using a magnetic bead method. The full sequence of the KIR3DL2 gene was amplified with four pairs by PCR primers. The coding regions of 208 unrelated ethnic Han Chinese blood donors were analyzed using a BigDye Terminator v3.1 Sequencing Kit. The genotypes were assigned based on the nucleotide polymorphism of the KIR3DL2 gene.@*RESULTS@#Among the 208 samples, 133 were KIR3DL2 heterozygotes and 75 were homozygotes. Forty six KIR3DL2 genotypes were detected. Respectively, 70, 33 and 23 individuals were found to have a KIR3DL2*00201/KIR3DL2*00201, KIR3DL2*00201/KIR3DL2*00701, and KIR3DL2*00201/KIR3DL2*01001 genotype. Twenty-two KIR3DL2 alleles were discovered, and the frequencies of KIR3DL2*00201, KIR3DL2*00701 and KIR3DL2*01001 were 57.45%, 13.46% and 9.13%, respectively.@*CONCLUSION@#The distribution of KIR3DL2 alleles among ethnic Han Chinese in Zhejiang has been determined and fits the criteria for genetic polymorphism.

Molecular characterization of a recombination allele of ABO blood group / 中华医学遗传学杂志

OBJECTIVE@#To analyze the molecular characteristics of a recombinant allele of the ABO blood group.@*METHODS@#The ABO phenotype was determined with the tube method. The coding regions of the ABO and FUT1 genes were analyzed by PCR-sequence based typing. The ABO alleles of the proband were determined by allele-specific primer sequencing. The full sequences of the ABO gene of the proband and her mother were determined through next generation sequencing.@*RESULTS@#The red blood cells of the proband did not agglutinate with anti-H, and the sequence of the FUT1 gene was homozygous for c.551_552delAG.The proband was thereby assigned as para-Bombay. Bi-directional sequencing also found that she was heterozygous for c.261G/del,467C>T,c.526C>G,c.657C>T,c.703G>A,c.796C>A,c.803G>C and c.930G>A of the coding regions of the ABO gene. Allele-specific primer sequencing also found her to carry a ABO*A1.02 allele and a recombinant allele from ABO*O.01.01 and ABO*B.01. The recombination site was located between nucleotide c.375-269 and c.526, and the allele was maternally derived.@*CONCLUSION@#An recombinant allele of the ABO gene has been identified, which has originated from recombination of ABO*O.01.01 with the ABO*B.01 allele.

Cloning and expression of MICB gene and its application for the detection of anti-MICB antibodies / 中华微生物学和免疫学杂志

Objective To construct a prokaryotic expression system for major histocompatibility complex class Ⅰ chain-related gene B ( MICB) and to establish an ELISA method for the detection of anti-MICB antibodies in patients with kidney transplantation. Methods The MICB cDNA fragments were ob-tained by RT-PCR with a pair of specific primers. The MICB cDNA and the prokaryotic expression vector pET-28a were digested by two restriction enzymes and ligated to construct the recombinant expression plas-mid pET-28a-MICB. The transformed E. coli BL21 DE3 strains carrying recombinant expression plasmid were induced by IPTG to express MICB protein. The expressed recombinant proteins were identified by Western blot assay and purified by Ni-NTA Spin column. The purified proteins were coupled to ELISA for the detection of anti-MICB antibodies in patients with kidney transplantation. Results Three common MICB fragments contained the exons 2 and 3 were obtained. The recombinant proteins were expressed in E. coli BL21 DE3 strains carrying pET-28a-MICB and successfully purified by the Ni-NTA Spin column. Results of the Western blot assay confirmed that the obtained proteins were the target proteins. The ELISA method was successfully established and used for the detection of anti-MICB antibodies in 24 patients with kidney trans-plantation. The absorbance values indicated that the sensitivities of three recombinant MICB proteins were different. Conclusion The expression system for MICB gene was successfully constructed. The established ELISA for the detection of anti-MICB antibodies would pave the way for further investigation on the correla-tions between MICB protein and transplantation immunity.

Effects of methylated CpG islands in promoter region on the expression of KIR3DL1 protein / 中华微生物学和免疫学杂志

Objective To investigate the effects of methylated CpG islands in the promoter region on the expression of killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1).Methods Three voluntary unpaid blood donors carrying high expression allele KIR 3DL1*01502 and three donors carrying low expres-sion allele KIR3DL1*005 were recruited in this study .The nucleotide sequences and the methylated CpG islands in the promoter regions of KIR 3DL1*01502 allele and KIR3DL1*005 allele were analyzed .The NK cells expressing KIR3DL1*01502 and KIR3DL1*005 were respectively treated with 5-aza for the dem-ethylation of CpG islands within the promoters .The expression of KIR3DL1 protein on the surface of NK cells was measured with flow cytometer .Results Two differences at nucleotide sites -65 and -269 were detected within the promoter regions of KIR3DL1*01502 and KIR3DL1*005, resulting in two distinct CpG islands.The CpG islands within the promoter of KIR 3DL1*01502 allele were highly methylated .The ex-pression of KIR3DL1 protein on NK cells which carried KIR 3DL1*01502 allele was significantly increased after the demethylation of CpG islands .However , the treatment of demethylation had no significant effects on the expression KIR3DL1 protein on NK cells harboring KIR3DL1*005 allele.Conclusion The methylated CpG islands within the promoter of KIR 3DL1*01502 allele affected the antigen expression on the surface of NK cells.Different KIR3DL1 alleles might show different mechanisms in regulating antigen expression .

Establishment of a polymerase chain reaction sequencing based typing method for HLA-DPB1 exons 2 and 3 and investigation of their polymorphisms / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a polymerase chain reaction sequencing-based typing (PCR SBT) method for HLA-DPB1 exons 2 and 3, and to analyze their polymorphisms.</p><p><b>METHODS</b>Based on the sequences of HLA-DPB1 loci, locus-specific primers were designed and applied to amplify the target sequences encompassing the entire exons 2 and 3 of HLA-DPB1. The amplification products were digested by enzymes and directly sequenced in both directions. The genotype was assigned by Assign 3.5+ SBT software.</p><p><b>RESULTS</b>Specific target fragment was obtained with the PCR amplification, and good quality electropherogram was derived by direct sequencing. Among 242 individuals from Zhejiang Han population, 18 HLA-DPB1 alleles were detected. Alleles with a frequency of > 0.05 have included DPB1*05:01:01/135:01 (0.4112), DPB1*02:01:02 (0.1901), DPB1*04:01:01 (0.1136) and DPB1*02:02 (0.0620). A novel HLA-DPB1*168:01 allele has also been identified. Nine polymorphism sites were founded in the exon 3 region, which included a new SNP site 517 A>T.</p><p><b>CONCLUSION</b>The PCR-SBT method for exons 2 and 3 of HLA-DPB1 is reliable, which allowed detection of polymorphisms in exon 3 of the HLA-DPB1 gene.</p>

Development of a method for the separation of HLA-A, -B and -C haploid using biotinylated probe and streptavidin magnetic beads / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a method for separating the human leukocyte antigen (HLA)-A, -B and -C haploid using biotinylated probes and streptavidin magnetic beads in order to solve ambiguous HLA genotyping results.</p><p><b>METHODS</b>Based on sequence information of HLA alleles from the IMGT/HLA database, the 5-biotinylated probes were designed. The probe was mixed and extended with corresponding genomic DNA, and incubated with streptavidin magnetic beads, which could form a streptavidin magnetic beads-biotin-probe DNA complex. The unique DNA haploid binding to corresponding probe was isolated after washes and elution. The separated haploid genomic DNA was used as template for HLA-A, -B and -C loci amplification and sequencing analysis.</p><p><b>RESULTS</b>Among the 12 HLA-A probes, 19 HLA-B probes and 13 HLA-C probes, DNA sequencing has confirmed that 9 HLA-A probes, 9 HLA-B probes and 5 HLA-C probes could successfully separate the haploid from genomic DNA samples.</p><p><b>CONCLUSION</b>The developed method for HLA-A, -B and -C haploid separation is reliable, which can solve certain ambiguity and improve the accuracy of HLA genotyping.</p>

Effect of α -1,2 fucosyltransferase gene 682A> G and 547_552delAG mutations on the activity of fucosyltransferase / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the effect of α -1,2 fucosyltransferase (FUT1) gene 682A> G and 547_552delAG mutations on the expression of FUT1 mRNA and activity of α -1,2 fucosyltransferase.</p><p><b>METHODS</b>Recombinant expression vectors of FUT1 682A> G and FUT1 547_552delAG were constructed and transfected into COS-7 cells for stable expression screening. Expression of FUT1 mRNA was determined using real-time quantitative PCR. The activity of FUT1 was measured with high-performance liquid chromatography.</p><p><b>RESULTS</b>Stably transfected COS-7 cells with wild type FUT1, FUT1 682A> G and FUT1 547_552delAG were respectively obtained. The FUT1 mRNA level of transfected cells with 682A> G and 547_552delAG recombination vectors have measured 101.69% and 102.79% compared with that of wild type FUT1 transfected cells. A specific protein band with about 46 kD was confirmed in the 682A> G transfected cell lysates by SDS-PAGE electrophoresis and Western blotting with 6× His Tag antibody. Similar protein was not identified in the 547_552delAG cells lysates. Enzymes activity of FUT1 682A> G has measured 61.01% compared with wild type FUT1 protein, whilst the activity of FUT1 547_557delAG was completely abolished.</p><p><b>CONCLUSION</b>FUT1 682A> G and 547_552delAG mutations do not affect the transcript efficiency, although various mutations have different impact on the enzyme's activity.</p>

Sequence analysis of a novel HLA-B * 9534 allele and establishment of group specific primers polymerase chain reaction method / 中华微生物学和免疫学杂志

Objective To analyze the molecular genetic basis of novel allele HLA-B * 9534 and establish the allele group specific primer PCR method. Methods Genomic DNA was extracted from whole blood by commercial DNA extraction kit. The HLA-B exons 1 to 8 coding sequences of the proband were am-plified by PCR and the amplification product was purified with double enzymes digestion and both strands of exons 2, 3 and 4 were sequenced. The exon 2-4 amplification of the HLA-B * 9534 was performed with al-lele group specific primers PCR and the PCR product was directly sequenced for exon 2 to 4. Results The proband has two HLA-B alleles. The result was assigned for HLA-B * 1518 and B * 4601 combination with a mismatch in 593A/G heterozygote by DNA sequencing of exon 2 to 4 with loci primers. After separating the two alleles of the proband with allele group specific primers polymerase chain reaction method, HLA-B * 4601 and HLA-B * 9534 alleles were identified after sequencing. The HLA-B * 9534 is identical to HLA-B * 1518 except for one nucleotide substitutions in exon 3 at position 593 A→G, this results in amino acid substitution at cedon 174 from Asn to Ser. The sequences of the novel allele have been submitted to GenBank (EU046491) and the allele has been officially nominated by the WHO Nomenclature Committee. Conclusion Identification of a novel HLA-B * 9534 allele and allele group specific primer PCR for HLA-B * 9534 was re-liable.

Sequence analysis and identification of two novel HLA alleles HLA-B*9536 and HLA-B*4612 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify two novel HLA alleles HLA-B*9536 and B*4612, in an individual.</p><p><b>METHODS</b>DNA was extracted from whole blood by Invitrogen DNA extraction kit. The amplification for HLA-B exons 2-4 of the proband was performed separately with allele group specific primers and the PCR products were directly sequenced for exons 2-4 in both direction.</p><p><b>RESULTS</b>There were two novel HLA-B alleles in the proband. The sequences of the two alleles have been submitted to GenBank (EU081878 and EU081879). The two alleles have been officially named as B*9536 and B*4612 by the WHO Nomenclature Committee. The sequence of exons 2-4 of HLA-B*9536 showed one nucleotide difference in exon 3 at position 544 (G to A) comparing with the closest allele B*1505, which resulted in an amino acid change from Ala to Thr at codon 158. In the HLA-B*4612 allele, there was one nucleotide change in exon 3 at position 363 (G to A), when compared to the closest allele B*4601, which lead to an amino acid change from Arg to Ser at codon 97.</p><p><b>CONCLUSION</b>Two novel HLA-B alleles were identified in one individual and have been officially named by the WHO Nomenclature Committee.</p>