Residues Met76 and Gln79 in HLA-G alpha1 domain involve in KIR2DL4 recognition.

Human leukocyte antigen-G (HLA-G) has long been speculated as a beneficial factor for a successful pregnancy for its restricted expression on fetal-maternal extravillous cytotrophoblasts and its capability of modulating uterine natural killer cell (uNK) function such as cytotoxicity and cytokine production through NK cell receptors. HLA class I alpha1 domain is an important killer cell Ig-like receptor (KIR) recognition site and the Met76 and Gln79 are unique to HLA-G in this region. NK cell receptor KIR2DL4 is a specific receptor for HLA-G, yet the recognition site on HLA-G remains unknown. In this study, retroviral transduction was applied to express the wild type HLA-G (HLA-wtG), mutant HLA-G (HLA-mG) on the chronic myelogenous leukemia cell line K562 cells and KIR2DL4 molecule on NK-92 cells, respectively. KIR2DL4-IgG Fc fusion protein was generated to determine the binding specificity between KIR2DL4 and HLA-G. Our results showed that residue Met76, Gln79 mutated to Ala76,79 in the alpha1 domain of HLA-G protein could affect the binding affinity between KIR2DL4 and HLA-G, meanwhile, the KIR2DL4 transfected NK-92 cells (NK-92-2DL4) showed a considerably different cytolysis ability against the HLA-wtG and HLA-mG transfected K562 targets. Taken together, our data indicated that residue Met76 and Gln79 in HLA-G alpha1 domain plays a critical role in the recognition of KIR2DL4, which could be an explanation for the isoforms of HLA-G, all containing the a1 domain, with the potential to regulate NK functions.

[The relationship between haplotypes of multilocus markers and ankylosing spondylitis].

OBJECTIVE: To investigate the relationship between haplotypes of multilocus markers and ankylosing spondylitis (AS). METHODS: Five families with AS were recruited from Shanghai area. Eleven microsatellite markers around D6S276 were analyzed by Linkage package and by Cyrillic package. RESULTS: Fine linkage analysis showed the significant Lod score values with D6S276 was 3.8821, Lod score values with D6S1691 and D6S1618 near D6S276 were larger than 1.5. The crossover value in 5 pedigrees was 14%. The haplotype analysis showed that the regions between D6S1691 and D6S1618 were associated with AS. CONCLUSION: The regions of D6S1691-D6S276-D6S1618 may harbor a susceptible gene of AS. The specific haplotypes of different pedigrees may play an important role in the presymptomatic diagnosis for AS.

[A genomewide scan for the susceptibility gene loci to ankylosing spondylitis in Chinese Han population].

Ankylosing spondylitis (AS) is a common inflammatory arthritis, with a prevalence of 1@1000-3@1000 in Caucasian and 2@1000 in Chinese population. The recognition of the association of HLA-B27 with AS confirmed the importance of heritable factors in the disease. Whole-genome scans showed that some affected-sibling-pair families with AS not only demonstrated strong linkage to the MHC locus, but also identified other regions with suggestive or stronger linkage on chromosomes 1p, 2q, 9q, 10q, 16q and 19q. In order to localize the regions containing genes that determine susceptibility to AS in Chinese Han population, a genomewide screen was performed in nine Chinese families with AS, including 29 affected individuals. LINKAGE and GENEHUNTER packages were used for parametric (LOD) and non-parametric (NPL) analysis. The significant two-point LOD score value with D6S276 (at 44.9 cM from the 6p terminal) was 3.8821 in parametric analysis. Fine mapping showed LOD scores of D6S1691 (at 42.7 cM) and D6S1618 (at 47.6 cM) around D6S276 were 1.5717 and 2.0056, respectively. Single point NPL score of D6S276, D6S1691 and D6S1618 were 2.6053, 2.7490, 2.0202, respectively, and minimum P value were 0.0072, 0.0047, and 0.0265, respectively. Using multipoint NPL, the maximum LOD score values, NPL score and minimum P value obtained near D6S276 were 5.0623, 3,7561, and 0.000233, respectively. As a result, the strong linkage of the D6S276 with AS was found, the region of D6S1691-D6S276-D6S1618 existed a susceptibility gene of AS. In addition, the LOD scores of D3S1292, D4S1535 and D18S64 were larger than 1.0, so they might be some suggestive linkage markers with AS.

[Analysis on HLA-E polymorphism in Guangdong Han population].

To investigate the distribution of HLA-E alleles and linkage between HLA-E and HLA-A or -B loci in Chinese Han in Guangdong area, HLA-E alleles were detected by using PCR-SSP in 150 unrelated healthy individuals from Guangzhou area; HLA-A, -B antigens typing in 106 individuals was carried out with NIH standard microlymphocytoxic method. Analysis of linkage was performed between HLA-E and HLA-A, -B. The results showed that three alleles of HLA-E could be detected in this population. They are E * 0101, E * 01031, E * 01032, with the frequency of 45.33%, 32.33%, 22.33% respectively. No E * 0102 and E * 0104 could be detected in all of these individuals. The analysis of linkage on two loci between HLA-E and HLA-A or -B showed that no significant difference could be found between expected frequencies and observed frequencies except B15/E * 01032 and A2/E * 01032. In conclusion, the high conservative polymorphism of HLA-E suggests that it's biological characteristic is different from that of classical HLA class Ia molecules.

[Killer Ig-like receptor gene content diversity and haplotype analysis in Chinese Han population in Shanghai].

OBJECTIVE: To detect the diversity of killer Ig-like receptor(KIR) gene content and the combination of haplotypes in Chinese Han population in Shanghai area. METHODS: DNA samples from 87 randomly unrelated healthy individuals in Shanghai Han population were genotyped with SSP/PCR method. RESULTS: (1) Frequencies of KIR genes: All of 18 known KIRs genes, such as 2DL1-5, 2DS1-5, 3DL1-3, 3DS1, KIR1D and the pseudogenes X, Xv and Z(KIR2DP1) were observed in Shanghai Hans. All individuals contain 3DL3, 2DL4, 3DL2 and 3DL1; the most common genes were 2DL3, Z, 2DL1 and X; the following were 2DS4, 1D, 2DL5, 2DS1, 3DS1 and 2DS5; the next were 2DS2, 2DL2, 2DS3 and Xv. (2) Frequencies of KIR gene haplotypes; there were 13 haplotypes detected in 87 Han individuals, among them, the most frequent one was type 2 (haplotypeA-2DS4). (3) Frequencies of KIR genotypes: 18 kinds of the combinations of the haplotypes were observed; the most frequent ones were AJ(2,2), AF (1,2). Also, In this study were identified five new genotypes FZ1 2 9 , FZ2 1 16 , FZ3 6 17 , FZ4 4 13 and FZ5 2 6 ,which had not been observed in Caucasians so far. CONCLUSION: These findings suggest that there are distinctive frequencies of KIR gene content, haplotype as well as genotype in Chinese Han population in Shanghai area.

[Genome-wide scanning for susceptibility genes in researches on six HLA-associated diseases].

Six human leucocytic antigen(HLA)-associated diseases, including ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, type 1 diabetes mellitus and psoriasis, were selected as objects of this review. The characteristics of these diseases in whole-genome scans on susceptibility genes or loci undertaken to date were analyzed and compared. Meanwhile, the potential proposals for dealing with the existing problems were put forward.

Frequency of cytochrome P450 2C9 allelic variants in the Chinese and French populations.

Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme responsible for the metabolism of different drugs with low therapeutic index such as oral anticoagulants. CYP2C9*2 and CYP2C9*3 are two single nucleotide polymorphic allelic variants. The frequency of these alleles in different ethnic populations is extremely variable. In this study, we compared the frequencies of CYP2C9 allelic variants among 394 Chinese living in Shanghai to 151 French Caucasians living in Paris. The allelic frequencies of CYP2C9 variants of the Chinese and the French subjects were 0.963, 0.001, 0.036 and 0.77, 0.15, 0.08 for CYP2C9*1, CYP2C9*2, CYP2C9*3, respectively. Chinese CYP2C9*3 allelic frequency was twice as lower as the French subjects, but three times higher than Korean (0.036 vs. 0.011). The CYP2C9*2 allele could be detected in only one Chinese subject, whereas it represented the major allelic variant in French Caucasians. The low frequency of the CYP2C9*2 and CYP2C9*3 allelic variants in Chinese subjects does not justify their detection in clinical practice, unlike French Caucasians.

[Expression of human KIR2DL1-Ig fusion protein in COS-7 cells].

AIM: To express, purify and identify the KIR2DL1-IgG Fc fusion protein. METHODS: Extracellular region of KIR2DL1 cDNA was amplified by RT-PCR from peripheral blood mononuclear cells, and cloned this fragment into fusion expression vector CD5lnegl. The recombinant vector CD5lnegl-KIR2DL1 was obtained after restriction endonuclease digestion and sequencing. COS-7 cells were transfected with this eukaryotic expression vector CD5lnegl-KIR2DL1 constructed in our Laboratory through DEAE-dextran/chloroquine method. The expressed KIR2DL1-IgGFc fusion protein in COS-7 cell culture surpernatant was identified by ELISA with mAb EB6 and HRP-anti-hIgFc mAb and Western blot. RESULTS: Restriction endonuclease digestion and sequencing indicated that the CD5lnegl-KIR2DL1 had been constructed successfully. The fusion protein could be detected by ELISA in COS-7 cell culture surpernatant. Western blot analysis also showed that the fusion protein could react to both EB6 and anti-hIgFc mAb. There was only one specific band at the position of the relative molecular mass (M(r)) 73 000, and it was equivalent to the expected value. CONCLUSION: KIR2DL1-IgG Fc fusion protein expressed in COS-7 cells successfully and the protein can mimic the natural KIR2DL1 protein and is used as a potential tool in the recognition of its ligands.

[Analysis on polymorphism in exons 2,3 and 4 of the MICA gene in three different Chinese populations].

OBJECTIVE: To detect genetic polymorphism in the exons 2 to 4 of the MICA gene in Chinese Han population in Shanghai, Dai population in Yunnan province and Uygur population in Xinjiang province respectively. METHODS: DNA samples from 183 random healthy individuals in Han population, 41 in Dai population and 66 in Uygur population were genotyped by using the polymerase chain reaction (PCR) and sequence-specific oligonucleotide probing (SSOP) method. RESULTS: In total, 10, 7 and 9 alleles of MICA were observed in Han, Dai and Uygur population respectively. MICA*008 was the most common allele in the population of both Han and Uygur, whereas MICA*010 was the most popular one in Dai population. Han and Dai had a bit similar distribution pattern (Chi-square=12.809 P=0.046), in contrast with Han to Uygur (Chi-square=58.499 P=0) and Dai to Uygur (Chi-square=49.273 P=0). CONCLUSION: MICA gene displayed different allele distributions in different populations.