Concordance of identifying the presence or absence of KIR genes by Flow-rSSO and PCR-SBT methods: a comparative study / 中国输血杂志

【Objective】 To study the concordance of identifying the presence or absence of KIR genes using flow reverse sequence-specific oligonucleotide probe (Flow-rSSO) hybridization and sequencing based typing-PCR (PCR-SBT) methods. 【Methods】 A total number of 131 cases of DNA samples from Han population were subjected to identify the presence or absence of all 16 KIR genes by Flow-rSSO method, and then sequenced at coding sequence for all 14 functional KIR genes using our in-house KIR PCR-SBT assay. The concordance of identifying the presence or absence of all functional KIR genes by Flow-rSSO and PCR-SBT was analyzed. Samples with inconsistent initial results were re-tested using the Flow-rSSO commercial kits with different Lot number, and further tested using the PCR-SSP commercial kit. 【Results】 The presence or absence of 14 functional KIR genes for 129 of 131 samples were completely in accordance via the PCR-SBT and Flow-rSSO methods. Two samples, one with 3DL1 negative, the other with both 2DS3 and 2DS5 negative initially-identified by Flow-rSSO, were actually all positive tested by PCR-SBT. Further retest by Flow-rSSO commercial kits with different Lot number and PCR-SSP commercial kit indicated that the two samples were all positive, which agreed well with PCR-SBT results. 【Conclusion】 In this paper, the initial test results of the presence or absence of KIR genes identified by Flow-rSSO for 2 samples were wrong, which indicated the importance of carrying out the quality control for reagents in KIR gene testing.

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.

Polymorphism of KIR2DL4 gene among northern Chinese Han population / 中国输血杂志

【Objective】 To investigate the polymorphism of KIR2DL4 gene in northern Chinese Han population. 【Methods】 A total of 327 DNA samples were isolated by magnetic beads from unrelated individuals of northern Chinese Han population. The coding sequence (CDS) of KIR2DL4 were amplified using four pairs of KIR2DL4-specific PCR primers developed by our own KIR sequencing-based typing patent, and each exon of KIR2DL4 carried by the four PCR amplicons was then subjected to DNA Sanger sequencing in both directions. The genotype of each sample was assigned using the Assign 4.7 software. 【Results】 Among 327 individuals, 8 different kinds of KIR2DL4 alleles were detected, with observed frequencies ranked as KIR2DL4*00102 [77.1%(252/327)], *00501 [35.8%(117/327)], *011 [20.2%(66/327)], *00602 [12.5%(41/327)], *00801 [8.6%(28/327)], *00103 [4.9%(16/327)], *00503 [1.5%(5/327)] and *00504 [0.9%(3/327)]. In this study, the 10A type alleles which can encode a full membrane-bound receptor include 2DL4*00102, *00103, *00501, *00503, *00504 and *00602, whereas the 9A type alleles which produce truncated forms of receptor include 2DL4*00801 and *011. The observed frequencies for 10A and 9A type KIR2DL4 alleles were 97.6% (319/327) and 27.8% (91/327), respectively. The ratio of 10A to 9A type was 3.5: 1. The observed frequencies of KIR2DL4 alleles in northern Chinese Han population showed no significant difference compared with southern Chinese Han population (P>0.05). 【Conclusion】 The allelic diversity of KIR2DL4 elucidated in the present study can provide valuable data for KIR-associated disease studies and human evolution.

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.

Solutions to cross-matching incompatibility in clinical: retrospective study of 1 770 cases in Shenzhen / 中国输血杂志

【Objective】 To analyze the blood samples sent by hospitals in Shenzhen to solve ABO cross-match incompatibility during 2011 to 2020, so as to find corresponding solutions to improve the efficacy of blood transfusion. 【Methods】 The clinical data of 1 770 cases of cross-match incompatibility in our laboratory from January 2011 to December 2020 were collected and reviewed. The causes of cross-match incompatibility were analyzed, the types of unexpected antibodies were determined. The overall incidence of antibodies was evaluated by statistical method of classified variables. The safety of blood transfusion was safeguarded by ABO homotype plus cross-matching compatibility. 【Results】 1) The 1 770 samples, presenting cross-matching incompatibility, involved 956 patients. The average number of cross-matching per patient from 2011 to 2015 was 1.32(307/232), which increased from 1.27(103/81) in 2016 to 2.23(286/128) in 2018, and remained stable in 2019 and 2020. 2) Among 956 patients, auto-and/or allo-antibody in plasma were yielded in 90.38%(864/956), including auto-antibody plus alloantibody in 42.26%(404/956), solo auto-antibody in 20.71%(198/956) and solo allo-antibody in 27.41%(262/956). Up to 20 kinds of specific allo-antibodies were detected, belonging to 8 blood groups. Among them, 70.82%(551/778) were Rh blood group, such as anti-E(37.15%)>anti-c(20.95%)>anti-C(5.27%)=anti-e(5.27%)>anti-D(2.19%), followed by MNS [11.40%(112/778)], Kidd [5.66%(44/778)], Leiws [3.21%(25/778)], Duffy [1.80%(14/778)], Diego [1.03%(8/778)], P1 [0.39%(3/778)] and H [0.26%(2/778)]. 3) 86%(37/43) of multiple transfusion recipients, aged below 20 years old, were thalassemia, and 1-4 kinds of allo- and/or auto-antibody were yielded. 【Conclusion】 The cross-matching incompatibility were mainly caused by allo- and/or auto-antibodies, which may be induced by blood transfusion, pregnancy or autoimmune diseases such as autoimmune hemolytic anemia.Those suspicious blood samples in clinical should be sent to blood group reference laboratory for further determination, in order to ensure the safety and efficacy of blood transfusion.

Identification of a novel HLA-DQB1*03 allele caused by variant of a single nucleotide / 中华医学遗传学杂志

OBJECTIVE@#To delineate the characteristics of a novel HLA-DQB1 allele identified during routine HLA matching in a leukemia family.@*METHODS@#The mother and brother of the patient were subjected to PCR sequence-specific oligonucleotide probe (SSOP), PCR sequence-based typ1ing (SBT), as well as next-generation sequencing (NGS).@*RESULTS@#PCR-SBT revealed that the patient's mother and brother's HLA-DQB1 sequences did not fully match with any known allele combination. NGS revealed that the novel allele has differed from the closest matched DQB1*03:02 with a T>G substitution at position 233 in exon 2, which resulted in substitution of Valine at codon 46 by Glycine. Pedigree analysis confirmed that the novel HLA-DQB1 allele was inherited from his mother.@*CONCLUSION@#A novel HLA-DQB1 allele has been identified through next generation sequencing and was officially named as HLA-DQB1*03:362 by the World Health Organization HLA Factor Nomenclature Committee.

Long-term clinical evaluation on total parathyroidectomy in patients with secondary hyperparathyroidism / 中华普通外科杂志

Objective:To evaluate the safety and long-term effect of total parathyroidectomy in patients with secondary hyperparathyroidism.Methods:One hundred fifty-four patients with secondary hyperparathyroidism who underwent total parathyroidectomy in Zhongshan Hospital,Dalian University from Mar 2012 to Mar 2018 were followed up for 3-9 years,including the level of iPTH, serum calcium and phosphorus and dosing of calcium supplement.Results:Among the 154 patients, the iPTH level in 149 patients fluctuated within 15-60 pg/ml. After oral calcium carbonate, the blood calcium fluctuated in 1.8-2.4 mg/ml, and serum phosphorus was 0.8-1.6 mg/ml. The level of iPTH in 5 patients was between 80-150 pg/ml, which was higher than the normal value 10-70 pg/ml. The clinical symptoms of all patients were significantly relieved.Conclusion:Total parathyroidectomy is safe and reliable in the treatment of secondary hyperparathyroidism with low recurrence rate and stable long-term effect.

Analysis and verification of a HLA-DQB1*03:90N allele with a single base deletion / 中华医学遗传学杂志

OBJECTIVE@#To verify a HLA-DQB1*03:90N allele and method to improve the accuracy of HLA typing.@*METHODS@#A total of 2265 hematopoietic stem cell donors from Shenzhen Branch of China Marrow Donor Program in 2018 were initially detected by a PCR sequence-specific oligonucleotide probe (SSOP) method. Among these, a rare HLA-DQB1 allele was identified by sequence-based tying (SBT) and Ion Torrent S5 next generation sequencing (NGS).@*RESULTS@#The SSOP typing result suggested the HLA-DQB1 to be a rare allele, while an insertion and a deletion was suspected in its exon 2 by SBT, which were confirmed by NGS as DQB1*03:90N and DQB1*06:01, respectively.@*CONCLUSION@#Rare alleles suspected by the SSOP method should be verified by other methods to ensure the accuracy of HLA genotyping. Rare alleles formed by deletions can be detected by NGS with accuracy.

Identification of a novel KIR3DL3*064 allele by cDNA cloning and sequencing / 中华医学遗传学杂志

OBJECTIVE@#To report on a novel KIR3DL3 allele identified in a southern Han Chinese individual.@*METHODS@#Peripheral blood sample was collected from a voluntary blood donor with inconclusive result by KIR3DL3 sequence-based typing (SBT). Total mRNA was extracted and subjected to reverse transcription to obtain KIR3DL3 cDNA, which was then amplified by PCR with a pair of KIR3DL3-specific primers. The product was subjected to cDNA cloning and sequencing.@*RESULTS@#cDNA cloning and sequencing have identified a wide-type KIR3DL3*00802 allele and a novel KIR3DL3*064 allele. The latter differed from KIR3DL3*00601 by a missense variant at codon 374[c.1184 C>T (p.Thr374Ile)] in exon 9. The novel KIR3DL3 allele has been officially assigned by the KIR subcommittee of World Health Organization Nomenclature Committee for factors of HLA system.@*CONCLUSION@#cDNA cloning and sequencing may be used to distinguish inconclusive results in KIR3DL3 SBT in order to identify novel KIR alleles.

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.

Association of polymorphisms of KIR2DL4 gene with leukemia among ethnic Hans from southern China / 中华医学遗传学杂志

OBJECTIVE To assess the association of polymorphisms of KIR2DL4 gene with leukemia among ethnic Hans from southern China. METHODS A total of 590 leukemia patients were recruited, which included 283 cases of acute lymphoblastic leukemia (ALL), 277 cases of acute myeloid leukemia (AML), and 80 cases of chronic myeloid leukemia (CML). Meanwhile, 306 healthy controls were randomly selected from volunteer blood donors. Both groups were subjected to sequence-based typing of the entire coding sequence of the KIR2DL4 gene using an in-house assay. The genotype for each sample was determined with Assign 4.7 SBT software. The frequencies of each detected KIR2DL4 allele, the 9A type KIR2DL4 allele and 10A type KIR2DL4 allele of the ALL, AML and CML groups were compared with those of the control group. RESULTS Five KIR2DL4 alleles, namely KIR2DL4*001, *005, *006, *008 and *011, were detected in the ALL, AML and CML groups as well as the control group. A significant difference was detected in the frequencies of KIR2DL4*011 and 10A type KIR2DL4 allele between the ALL group and the control group (KIR2DL4*011: OR = 1.66, P = 0.01; 10A type KIR2DL4: OR = 0.42, P = 0.03), but was lost after P correction (Pc > = 0.05). No significant difference was detected in the frequencies of KIR2DL4 alleles, the 9A type KIR2DL4 and 10A type KIR2DL4 allele between the AML and CML patient groups compared with the control group (P > 0.05, Pc > 0.05). CONCLUSION The allelic diversity of the KIR2DL4 locus showed no significant association with leukemia among ethnic Hans from southern China.

An investigation of work-related musculoskeletal disorders among sonographers in a province of China and related influencing factors / 中华劳动卫生职业病杂志

Objective@#To investigate the prevalence of work-related musculoskeletal disorders (WMSDs) among sonographers in a province of China and influencing factors for WMSDs, and to provide a practical basis for the prevention and treatment of WMSDs in sonographers. @*Methods@#From November 2016 to February 2017, stratified cluster sampling was used to select 700 sonographers from 50 hospitals in this province. A self-designed questionnaire for WMSDs in sonographers was used to investigate general data and the prevalence of WMSDs, and the influencing factors for the prevalence of WMSDs were analyzed. @*Results@#The prevalence rate of WMSDs among these sonographers was 80.22%, and the prevalence rates of WMSDs in the shoulder, the neck, the waist, the back, the wrist, the elbow, the hip, the knee, and the ankle were 74.55%, 68.87%, 63.44%, 57.26%, 53.16%, 45.22%, 37.88%, 30.44%, and 29.24%, respectively. There was a significant difference in the prevalence rate of WMSDs between the sonographers with different ages and working years, and the prevalence rate of WMSDs tended to increase with the increase in age and working years (χ2=20.86 and 18.52, P<0.01) . The multivariate logistic regression analysis showed that female sex (odds ratio [OR]=1.798) , working years >16 (OR=1.004) , weekly working hours >40 (OR=1.616) , poor physical conditions (OR=1.690) , and high work fatigue (OR=1.302) were risk factors for WMSDs in sonographers. @*Conclusion@#There are high prevalence rates of WMSDs in the shoulder, the neck, the waist, the back, the wrist, and the elbow. Sonographers should strengthen self-protection awareness, and effective preventive measures should be adopted to reduce the prevalence rate of WMSDs.

Establishment of a quality control system for HLA allele typing and its key points / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To list the key points for quality control during HLA-A, B, C, DRB1 and DQB1 allele typing by taking consideration of hardware, software and experimental procedures.</p><p><b>METHODS</b>A total of 10 167 samples from randomly selected healthy blood donors and donor-recipient pairs from Shenzhen were typed for exons 2-4 of HLA-A, B, C, exon 2 of HLA-DRB1, and exons 2 and 3 of HLA-DQB1 by PCR- sequence-based typing. For 56 cases whose forward and reverse sequences were inconsistent, the samples were re-checked by a PCR-sequence specific oligonucleotide probe method. Novel alleles not included in the IMGT/HLA database were cloned and sequenced using in-house primers.</p><p><b>RESULTS</b>Eight novel HLA alleles were identified. A table for key positions of single nucleotide polymorphisms (SNPs) were generated, which summarized the key points for quality control during HLA-A, B, C, DRB1 and DQB1 allele typing. Among the listed SNPs, 3 were located at the HLA-A locus, 8 were at the HLA-B locus, 6 were at the C locus, 6 were at the DQB1 locus, and 4 were at the DRB1 locus. To ensure the quality control, an unique sample number for DNA transferring tubes in the process of experiment should be considered.</p><p><b>CONCLUSION</b>A protocol for quality control should be enforced by checking all of the key points. The SNPs and critical control points of the alleles should be examined to ensure the accuracy of HLA typing results.</p>

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>

Allelic diversity of KIR2DL4 gene and identification of five novel alleles among southern Han Chinese population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the polymorphisms of the KIR2DL4 gene in a southern Han Chinese population.</p><p><b>METHODS</b>Genomic DNA isolated from 306 unrelated individuals was amplified by using KIR2DL4-specific PCR primers. The PCR products were genotyped for the entire coding sequence by sequencing-based typing (SBT). Assignment of allelic profile was accomplished with an Assign 3.5 software. For samples with inconclusive SBT results, the RT-PCR products covering the entire coding sequence of the KIR2DL4 gene were subjected to cloning and haplotype sequencing.</p><p><b>RESULTS</b>Among the 306 individuals, 11 alleles were detected, of which 5 novel alleles were officially named by the KIR subcommittee of the World Health Organization Nomenclature Committee for factors of HLA system. The observed frequencies for the 11 alleles were KIR2DL4 *00102 (75.5%), *00103 (8.2%), *00501 (34.0%), *00503 (0.7%), *00504 (0.7%), *00602 (14.4%), *00801 (11.4%), *011 (22.2%), *032 (0.3%), *033 (0.3%) and *034 (0.3%). The ratio of 10A type alleles including 2DL4*00102, *00103, *00501, *00503, *00504, *00602, *032, *033, *034 and 9A type alleles including 2DL4*00801, *011 were 97.0% and 33.0%, respectively, with a ratio of 2.9:1.</p><p><b>CONCLUSION</b>The allelic diversity of the KIR2DL4 gene in southern Han Chinese has been elucidated, which may provide valuable data for research on transplantation, reproductive immunity, KIR-associated disease and evolution.</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>