HLA Polymorphism Analysis, Identification Of HLA Novel Alleles, And Disease Association Study Of HLA In Chronic Myeloid Leukemia

ObjectiveTo study of HLA-A,B and DRB1 genes and haplotypes polymorphism in Jiangsu Han population and identification of HLA-A,B, DRB1 novel alleles in Jiangsu Branch of Chinese Marrow Donor Program(CMDP)as well as the disease association analysis of HLA in chronic myeloid leukaemia(CML).MethodsPCR-SSP and PCR-SSOP were employed in HLA typing for healthy controls and chronic myeloid leukemia patients.Popular genetic statistical software of Arlequin was used to analyze the gene and haplotype polymorphism and disease association study.Molecular cloning and sequence analysis were used to identify the existence of novel alleles for the suspected samples screened by PCR-SSP HLA-typing.Serologic typing and family analysis were also used for the new alleles.ResultsA total of 18 different HLA-A,34 different HLA-B,and 13 different HLA-DRB1 alleles were identified in the popular genetics study.The most frequent alleles were A~*02 in locus A,B~*15 in locus B,and DRB1~*09 in locus DRB1.The most common haplotypes were A~*30-B~*13-DRB1~*07(6.92%)in A-B-DRB1 loci,A~*33-B~*58(5.35%)in A-B loci,B~*13-DRB1~*07(8.17%)in B-DRB1 loci,and the A~*02-DRB1~*09(8.30%)in A-DRB1 loci.Four novel alleles were identified and designated as HLA-B~*0740,DRB1~*1609,DRB1~*1449 and Cw~*0339 by WHO Nomenclature Committee for Factors of the HLA System.B~*0740 had one nucleotide substitution at position 605 from A to T(codon 178 AAG->ATG)resulting in an amino acid change from lysine(K)to methionine(M)comparison to the most homologous allele B~*0705.DRB1~*1609 differed from the most homologous allele DRB1~*160101 by one nucleotide at position 127 where A->T(codon 47ACC->TCC)resulting in a coding change 47 Y to F.DRB1~*1449 differed from the most homologous allele DRB1~*1432 by four nucleotides at nucleotide(nt)position 71 where C->G(codon 28 GAC->GAG)resulting in a coding change 28 D to E;at nt 196 where G->A(codon 70 CGG->CAG)resulting in a coding change,70 R to Q;at nt 244 where T->G and 245 where G->T(codon 86 GTG->GGT)resulting in a coding change 86 V to G Cw~*0339 had identical nucleotide sequence to Cw~*031101 in exon 2 and 3,except for two nucleotide changes from T to G at position 97,and T to C at position 105.The changes leaded to an amino acid alteration from tyrosine to aspartic acid at amino acid residue 9(Y9D),while the alanine at residue 11(11A) remained unchanged.By comparison of the HLA gene distribution characteristics between CML and healthy control populations,differences with statistical significance were found in HLA-A~*30(CML group 5.42%versus control group 9.13%)with odds ratio(OR)0.57,DRB1~*07 (CML group 8.14%versus control group 12.51%;OR=0.62),and B~*81 (CML group 0.51%versus control group 0.09%,OR=5.44).ConclusionHLA polymorphism study showed some distribution characteristics and linkage disequilibrium,which may shade new lights in population genetics and anthropology studies of Han-Chinese.It also serves as foundation for future donor pool selection in Jiangsu stem cell donor registry.Four novel HLA alleles were confirmed by the sequencing based typing and officially designated by WHO HLA Nomenclature Committee, which may contribute to the HLA polymorphism in China.CML and HLA association study suggest that expression of HLA-A~*30,DRB1~*07 might imply a protective effect on CML acquisition,while B~*8 lmight be associated with CML susceptive factors in our population.