Establishment of a PCR-SSP method for the simultaneous amplification and identification of the presence of KIR genes / 中华医学遗传学杂志

OBJECTIVE@#To develop a polymerase chain reaction-sequence specific primer (PCR-SSP) method for simultaneous amplification and identification of the KIR genes among Chinese population.@*METHODS@#Peripheral blood samples from 132 healthy donors who had given blood at Shenzhen Blood Center from January 2015 to November 2015 were selected as the study subjects. Based on the polymorphism and single nucleotide polymorphism (SNP) information of high-resolution KIR alleles in the Chinese population and the IPD-KIR database, specific primers were designed to amplify all the 16 KIR genes and the 2DS4-Normal and 2DS4-Deleted subtypes. The specificity of each pair of PCR primers was verified by using samples with known KIR genotypes. During PCR amplification of the KIR gene, co-amplification the fragment of human growth hormone (HGH) gene by multiplex PCR was used as the internal control to prevent false negative results. A total of 132 samples with known KIR genotypes were randomly selected for blind inspection to verify the reliability of the developed method.@*RESULTS@#The designed primers can specifically amplify the corresponding KIR genes, with clear and bright bands for the internal control and KIR genes. The results of detection are fully consistent with the known results.@*CONCLUSION@#The KIR PCR-SSP method established in this study can yield accurate results for the identification of the presence of KIR genes.

Association of molecular genetic polymorphism of KIR-HLA systems with acute leukemia in Southern Chinese Han / 中华医学遗传学杂志

OBJECTIVE@#To investigate the association of molecular genetic polymorphism of KIR-HLA systems with acute lymphoblastic leukemia (ALL) and acute myelocytic leukemia (AML) in southern Chinese Han.@*METHODS@#A total number of 323 cases of adult ALL patients, 350 adult AML, and 745 random healthy controls were tested by KIR PCR-SSP and HLA-A, -B, -C sequence-based typing (PCR-SBT) methods. The molecular genetic polymorphisms of KIR genes and KIR gene profiles, classⅠ HLA ligands, and KIR receptor +HLA ligand combinations were compared between patient and healthy control groups.@*RESULTS@#A total number of 32 and 33 different kinds of KIR profiles were identified in the ALL and AML patient groups. Compared with the observed frequencies of KIR profiles in healthy controls, the observed frequencies of KIR profile AA1 were significantly lower in both the ALL and AML groups (ALL group: 45.79% vs. 55.30%, Pc=0.004; AML group: 48.27% vs. 55.30%, Pc=0.030). In the ALL group, the observed frequencies of 2DL2 gene and 2DL2+HLA-C1 combination, 2DS2 gene and 2DS2+HLA-C1 combination were significantly higher than those in healthy controls (P<0.05), whereas the frequencies of 2DL3 gene, HLA-A3/A11 ligand and 3DL2+HLA-A3/A11 combination were significantly lower than those in healthy controls. However, no significant differences remained after Bonferroni correction (Pc>0.05). In AML group, the observed frequencies of both 2DS1 and 2DL5 genes were significantly higher than that in healthy controls, whereas the frequencies of HLA-C2 ligand and 2DL1+HLA-C2 combination were significantly lower than that in healthy controls(P<0.05). However, no significant difference existed after Bonferroni correction (Pc>0.05).@*CONCLUSION@#This study revealed some potential susceptibility or protective factors related to acute leukemia in southern Chinese Han, especially the protective factor KIR profile AA1, which might provide new clues and theoretical basis for the pathogenesis of acute leukemia and individualized immunotherapy.

Association of human leukocyte antigen class Ⅰ, Ⅱ allelic and haplotypic polymorphisms with leukemia in Han nationality of southern China / 白血病·淋巴瘤

Objective:To investigate the association of human leukocyte antigen (HLA) class Ⅰ (A, B and C), class Ⅱ (DRB1, DQB1 and DPB1) allelic and haplotypic polymorphisms with acute lymphoblastic leukemia (ALL), acute myeloid leukemia(AML) and chronic myeloid leukemia (CML) in Han nationality of southern China.Methods:The peripheral blood samples of 845 leukemia patients (323 cases of ALL, 350 cases of AML and 172 cases of CML) and 745 healthy blood donors from Han nationality of southern China in Shenzhen Blood Center were collected. The HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 genes were genotyped by using polymerase chain reaction-reverse sequence specific oligonucleotide (PCR-rSSO) and polymerase chain reaction-sequence based typing (PCR-SBT) methods to identify the first 4 digits of HLA alleles. The Arlequin 3.5 software was used to analyze HLA haplotypes. The correlations between HLA allelic and haplotypic polymorphisms and three types of leukemia were statistically analyzed at HLA low-resolution level (the first 2 digits of alleles) and high-resolution level (the first 4 digits of alleles), respectively.Results:P-values were adjusted by Bonferroni correction. In ALL group, the frequencies of A*02 (36.22% vs. 28.26%, χ 2 = 13.41, PC < 0.01) and its haplotype A*02-B*46-C*01 (15.35% vs. 10.23%, χ 2 = 10.90, PC = 0.02), DRB1*12 (15.79% vs. 11.10%, χ 2 = 9.02, PC = 0.03), A*02:03 (9.75% vs. 5.32%, χ 2 = 14.25, PC = 0.002) and its haplotype A*02:03-B*38:02-C*07:02 (3.80% vs. 1.51%, χ 2 = 10.41, PC = 0.02) were higher than those in healthy controls, which implied that these factors could confer risk effect in ALL. In AML group, the frequency of A*11-B*15-C*08-DRB1*15-DQB1*06-DPB1*02 (1.34% vs. 0.07%, χ 2 = 12.54, PC = 0.003) was significantly higher than that in healthy controls, suggesting that the risk effect might be conferred by this haplotype. In CML group, the frequencies of A*02 (36.63% vs. 28.26%, χ 2 = 9.33, PC = 0.02) and its haplotype A*02-B*15-C*04 (2.17% vs. 0.29%, χ 2 = 11.74, PC = 0.02), and DRB1*03:01-DQB1*02:01-DPB1*02:01 (1.86% vs. 0.14%, χ 2 = 13.10, PC = 0.01) were higher than those in healthy controls, which implied that these factors could confer risk effect in CML, whereas the frequency of DRB1*13 (1.45% vs. 5.25%, χ 2 = 9.29, PC = 0.03) was lower than that in healthy controls, suggesting that it was a CML antagonistic gene. Conclusion:Leukemia susceptible or antagonistic HLA alleles and haplotypes are found at low-resolution and high-resolution levels of HLA, which might provide reference for investigating the pathogenesis of leukemia and guiding formulation of effective treatment strategies in Han nationality of southern China.

A protective effect conferred by KIR3DL1 and its cognate ligand against cervical cancer among ethnic Han Chinese population and its potential mechanism / 中华医学遗传学杂志

OBJECTIVE@#To explore the role of inhibitory KIR (iKIR) and its cognate HLA ligand in the occurrence and development of cervical cancer among ethnic Han Chinese and its potential mechanism.@*METHODS@#Peripheral blood samples from 265 Han Chinese patients with cervical intraepithelial neoplasia (CIN)/cervical cancer and 200 ethnically matched healthy controls were collected. The results of KIR PCR-SSP, HLA PCR-rSSO and KIR3DL1 PCR-SBT, together with cervical cancer data from the TCGA database, were used to assess the association of iKIR genes, receptor-ligand gene combinations, iKIR transcription level in the tumor tissue and the KIR3DL1 alleles with the occurrence and development of cervical cancer.@*RESULTS@#Among the four iKIR genes (KIR2DL1, 2DL2/3, 3DL1 and 3DL2), the frequencies of KIR3DL1 and KIR3DL1-HLA-Bw4 genes among controls were significantly higher than those of the cervical cancer group (96.5% vs. 87.0%, P = 0.018; 81.5% vs. 64.8%, P=0.009). The survival rate of cervical cancer patients with a high transcription level of KIR3DL1 in tumor tissues was significantly higher than those with a low/medium transcription level (P=0.028). The frequency of strong-inhibitory and high-expression KIR3DL1*01502 allele among the healthy population was significantly higher than that of the cervical cancer group (76.0% vs. 59.3%, P =0.015).@*CONCLUSION@#Combined KIR3DL1 and KIR3DL1-HLA-Bw4 can confer a protective effect against the development of cervical cancer, which may be attributed to the strong-inhibitory and high-expression allele of KIR3DL1*01502.

A protective effect conferred by KIR3DL1 and its cognate ligand against cervical cancer among ethnic Han Chinese population and its potential mechanism / 中华医学遗传学杂志

Objective@#To explore the role of inhibitory KIR (iKIR) and its cognate HLA ligand in the occurrence and development of cervical cancer among ethnic Han Chinese and its potential mechanism.@*Methods@#Peripheral blood samples from 265 Han Chinese patients with cervical intraepithelial neoplasia (CIN)/cervical cancer and 200 ethnically matched healthy controls were collected. The results of KIR PCR-SSP, HLA PCR-rSSO and KIR3DL1 PCR-SBT, together with cervical cancer data from the TCGA database, were used to assess the association of iKIR genes, receptor-ligand gene combinations, iKIR transcription level in the tumor tissue and the KIR3DL1 alleles with the occurrence and development of cervical cancer.@*Results@#Among the four iKIR genes (KIR2DL1, 2DL2/3, 3DL1 and 3DL2), the frequencies of KIR3DL1 and KIR3DL1-HLA-Bw4 genes among controls were significantly higher than those of the cervical cancer group (96.5% vs. 87.0%, P = 0.018; 81.5% vs. 64.8%, P=0.009). The survival rate of cervical cancer patients with a high transcription level of KIR3DL1 in tumor tissues was significantly higher than those with a low/medium transcription level (P = 0.028). The frequency of strong-inhibitory and high-expression KIR3DL1*01502 allele among the healthy population was significantly higher than that of the cervical cancer group (76.0% vs. 59.3%, P = 0.015).@*Conclusion@#Combined KIR3DL1 and KIR3DL1-HLA-Bw4 can confer a protective effect against the development of cervical cancer, which may be attributed to the strong-inhibitory and high-expression allele of KIR3DL1*01502.

Genetic polymorphisms of KIR2DS4 gene among ethnic Hans from southern China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study genetic polymorphisms of the KIR2DS4 gene among ethnic Hans from southern China.</p><p><b>METHODS</b>Genomic DNA was isolated from 306 unrelated individuals and amplified with KIR2DS4-specific PCR primers. KIR2DS4-positive samples were genotyped for the entire coding sequence by sequencing-based typing (SBT). Assignment of allelic genotypes was accomplished by using Assign 3.5 software. For samples with inconclusive SBT results, RT-PCR products covering the entire coding sequence of the KIR2DS4 gene were subjected to cloning and haplotype sequencing.</p><p><b>RESULTS</b>Among all tested samples, 297 were demonstrated to have carried the KIR2DS4 framework gene. For KIR2DS4-positive samples subjected to SBT for the entire coding sequences, no background was observed with the obtained sequences. Three of the seven identified alleles were of novel types, which were officially named by the KIR subcommittee of the World Health Organization Nomenclature Committee for Factors of HLA System. The observed frequencies for the 7 alleles were KIR2DS4*00101 (78.8%), *003 (10.5%), *004 (16.0%), *010 (23.2%), *017 (0.3%), *00105 (0.3%) and *018 (0.7%), respectively. Allele KIR2DS4*007 was not found. The overall frequency for normal cell-surface expression KIR2DS4 alleles including 2DS4*00101, *017 and *00105 was 79.4%, and that for non cell-surface expression alleles including 2DS4*003, *004, *010 and *018 was 50.4%. The ratio between the two was 1.6:1.</p><p><b>CONCLUSION</b>The present study has elucidated the allelic diversity of KIR2DS4 among ethnic Hans from southern China, which may provide valuable data for transplantation as well as studies on KIR-associated disease and evolution.</p>

Association of genetic polymorphisms of KIR-HLA system with chronic myeloid leukemia among ethnic Hans from southern China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the association of KIR-HLA gene polymorphism with chronic myeloid leukemia (CML) among ethnic Hans from southern China.</p><p><b>METHODS</b>A total of 172 adult CML patients and 480 unrelated healthy controls were screened for the presence of KIR with sequence-specific primers-PCR (PCR-SSP) and sequence-based typing (SBT) of HLA-A, -B and -C loci. Polymorphisms of the KIR-HLA system were analyzed at 4 levels, and the frequencies of KIR framework genes and KIR profiles, classⅠHLA ligands, matched KIR+HLA pairs and KIR-HLA compound profile were compared between the two groups. P values were calculated using SPSS 13.0 software.</p><p><b>RESULTS</b>For the CML group, the frequencies of HLA-C2 ligand, 2DL1+HLA-C2 pair and HLA-B Bw4-80I were significantly lower than those of the control group, suggesting a protective effect against CML (HLA-C2: OR=0.386, 95%CI:0.240-0.620, P<0.01; 2DL1+HLA-C2: OR=0.316, 95%CI:0.191-0.525, P<0.01; HLA-B Bw4-80I: OR=0.576, 95%CI:0.384-0.862, P<0.01). The frequencies of KIR2DL1 ligand (HLA-C2) and KIR3DL1 ligand (HLA-B Bw4-80I) in the CML group were significantly lower than that of the control group, suggesting that the HLA-C2 and HLA-B Bw4-80I expression is probably decreased in the CML patient group, which led to reduced inhibitory signal and enhanced activating signal of KIR2DL1and/or KIR3DL1NK cells. Notably, the frequency of KIR-HLA compound profiles ID2 (KIR AA1-HLA-C1/C1-Bw6/Bw6-A3/11) in CML patients significantly increased in the CML patient group compared with the control group, suggesting that the KIR-HLA compound profiles ID2 may be a risk factor for CML (OR=2.163, 95%CI 1.198-3.906, P<0.01).</p><p><b>CONCLUSION</b>Above analysis has identified certain protective and risk factors for CML from the KIR-HLA system, which may provide a clue for the pathogenesis of leukemia and development of individualized immune therapy.</p>

Study of polymorphisms of HLA class Ⅰ (-A, -B, -C) and class Ⅱ (DRB1, DQA1, DQB1, DPA1, DPB1) genes among ethnic Hans from Southern China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the genetic polymorphisms of human leukocyte antigen (HLA)- A, B, C, DRB1, DQA1, DQB1, DPA1and DPB1among ethnic Hans from southern China.</p><p><b>METHODS</b>481 randomly selected individuals were genotyped using a polymerase chain reaction (PCR) sequence-based typing (SBT) method for the above genes. Their allele frequencies were determined by direct counting.</p><p><b>RESULTS</b>In total, 28 HLA-A, 57 HLA-B, 28 HLA-C, 40 HLA-DRB1, 18 HLA-DQA1, 17 HLA-DQB1, 6 HLA-DPA1and 21 HLA-DPB1alleles were identified. Among these, common alleles (with allelic frequencies > 0.05) included A*1101, A*2402, A*0207, A*3303, A*0201, B*40:01, B*46:01, B*58:01, B*13:01, B*15:02, C*01:02, C*07:02, C*03:04, C*03:02, C*08:01, C*03:03, C*04:01, DRB1*09:01, DRB1*15:01, DRB1*12:02, DRB1*08:03, DRB1*03:01, DRB1*04:05, DRB1*11:01, DQA1*01:02, DQA1*03:02, DQA1*03:03, DQA1*06:01, DQA1*01:03, DQA1*05:05, DQA1*01:04, DQA1*03:01, DQA1*05:01, DQB1*03:01, DQB1*03:03, DQB1*06:01, DQB1*05:02, DQB1*03:02, DQB1*02:01, DQB1*03:02, DQB1*06:02, DPA1*02:02, DPA1*01:03, DPA1*02:01, DPB1*05:01, DPB1*02:01, DPB1*13:01, DPB1*04:01and DPB1*02:02.For each of the locus, the overall frequencies of common alleles were 75.57%, 52.81%, 78.28%, 62.16%, 86.70%, 77.23%, 95.32% and 81.59%, respectively.</p><p><b>CONCLUSION</b>The allelic frequencies of the 8 selected HLA loci among ethnic Hans from southern China may served as a reference for anthropology, legal medicine, transplantation and disease association studies.</p>

Cloning and sequencing of KIR2DL1 framework gene cDNA and identification of a novel allele / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop an assay for cDNA cloning and haplotype sequencing of KIR2DL1 framework gene and determine the genotype of an ethnic Han from southern China.</p><p><b>METHODS</b>Total RNA was isolated from peripheral blood sample, and complementary DNA (cDNA) transcript was synthesized by RT-PCR. The entire coding sequence of the KIR2DL1 framework gene was amplified with a pair of KIR2DL1-specific PCR primers. The PCR products with a length of approximately 1.2 kb were then subjected to cloning and haplotype sequencing.</p><p><b>RESULTS</b>A specific target fragment of the KIR2DL1 framework gene was obtained. Following allele separation, a wild-type KIR2DL1*00302 allele and a novel variant allele, KIR2DL1*031, were identified. Sequence alignment with KIR2DL1 alleles from the IPD-KIR Database showed that the novel allele KIR2DL1*031 has differed from the closest allele KIR2DL1*00302 by a non-synonymous mutation at CDS nt 188A>G (codon 42 GAG>GGG) in exon 4, which has caused an amino acid change Glu42Gly. The sequence of the novel allele KIR2DL1*031 was submitted to GenBank under the accession number KP025960 and to the IPD-KIR Database under the submission number IWS40001982. A name KIR2DL1*031 has been officially assigned by the World Health Organization (WHO) Nomenclature Committee.</p><p><b>CONCLUSION</b>An assay for cDNA cloning and haplotype sequencing of KIR2DL1 has been established, which has a broad applications in KIR studies at allelic level.</p>

Progress in research on genetic polymorphisms and sequence-based typing of KIR genes / 中华医学遗传学杂志

Killer cell immunoglobulin-like receptors (KIRs) are members of the immunoglobulin superfamily expressed on natural killer (NK) cells and a subset of T cells. Given the receptor-ligand relationship between certain KIR and human leukocyte antigen (HLA) classⅠmolecules, the KIRs are involved in the regulation of NK cell activation through conveying activating or inhibitory signals, which plays an important role in immunities involved in transplantation, tumor, infection as well as autoimmune diseases. This paper has provided a review for the research on KIR gene polymorphisms and summarized the characteristics of the sequence-based typing method for KIR genes.

Sequence analysis of a novel allele HLA-C*01:78 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the genetic basis for a novel allele HLA-C*01:78.</p><p><b>METHODS</b>Genomic DNA was extracted from peripheral blood using a QIAGEN quick DNA extraction kit. The regions encompassing HLA-C from exon 1 to intron 3 and intron 3 to 3'UTR were amplified and cloned using a cloning sequencing kit in order to split the two alleles apart. Selected clones were sequenced to include exons 2 to 4.</p><p><b>RESULTS</b>Sequencing results have indicated the HLA-C alleles of the proband to be a novel C*03:04 allele. The sequence has been submitted to GenBank (KF049216). BLAST analysis has confirmed the novel allele to have one nucleotide difference as C*01:03 at genomic nt316C>A (codon 82CGC>AGC) in exon 2, which has resulted in replacement of one amino acid (82R>S).</p><p><b>CONCLUSION</b>The novel allele has been officially named as C*01:78 by the WHO Nomenclature Committee. The HLA allele type of the proband was therefore A*02:07, 24:02; B*40:01, 46:01; C*01:78, 03:04; DQB1*05:02, 05:02; DRB1*16:02, 16:02.</p>

Methylation of CpG island in ABO gene promoter coding glycosyltransferase with dual donor specificity / 中国组织工程研究

BACKGROUND:During the research of ABO blood type antigen, the overwhelming majority samples of same ABO gene express a normal and same ABH antigen. But a certain amount samples with the same ABO genetic background show different antigen intensity expression as for different family or individuals. The ABO blood type has complex expression regulation mechanism. Analysis of ABO blood group serology and genetic background of these rare bi-specific AB phenotype specimens, and further studying on epigenetics may partly revealed ABO gene expression mechanism. OBJECTIVE:To study methylation of CpG island and explore the relationship between ABO gene promoter coding glycosyltransferase with dual donor specificity and ABH antigen expression. METHODS:Six samples detected as CisAB or B(A) phenotype were studied in this paper. The whole code sequences and promoter sequence of ABO gene were amplified respectively. The level of CpG methylation in promoter of ABO gene was further detected with bisulfite treatment method. RESULTS AND CONCLUSION:Among the six bi-specific AB phenotype samples, two previously-identified CisAB05/B(A)06 al eles with nt803C>G on the basis of B101 al ele sequence could be seen, and three additional methylated sites nt-33(30%), nt+27(50%) and nt+49(50%) were found between the two regions of CpG island in promoter of ABO gene. Two CisAB01 al eles with nt803C>G mutation on the basis of A101 sequence were found at nt-26C(10%). Other two B(A)04 al eles contained nt640A>G mutation on the basis of B101 sequence were found in the whole code sequences regions, and six additional methylated sites nt-33(10%), nt+16(50%), nt+57(60%), nt+59(60%), nt+68(60%) and nt+74(60%) were found between the two samples. No abnormity was identified in the promoter region of ABO gene. Our results indicated that the differential methylation levels in the CpG island of ABO gene promoter region may affect ABH antigens expression on the red cel membrane even if the samples had the same ABO genetic background.