Molecular Basis And Polymorphism Of Kell Glycoprotein

Kell blood system is the most important blood group besides ABO and Rh blood systems.Kell blood system is polymorphic and 27 antigens have been defined to date. The different Kell phenotypes usually are resulted from point mutations. An unusual phenotype, which is defined as the complete lack of all of the Kell antigens, has been designated as the Kell-null or K₀ phenotype. K antigen expresses antithetically to k antigen with low frequency. K has a frequency of about 7% in the white race.Because of its great immunogenicity,Kell blood system is thought to be an essential test item before blood transfusion in western countries.In Chinese,Kell blood system is always considered to have low polymorphism, the antigen frequency of K is about 0.07% .As a result,screening of Kell antigens is not carried out before transfusion in China and few research reports on Kell blood system can be refered.K,k and K₀ samples in Chinese were investigated to reveal the KEL genotype. 19 exons in KEL gene were amplified and sequenced or clone-sequenced to analyse their molecular basis. All the K samples had C698T mutation in exon 6,which resulted in Met193Thr. k had the same gene sequence with that of the wild type. 2 K₀ samples were screened from 87,665 O Shanghainese. Both K₀ samples had two heterozygous novel null alleles. At one chromosome, exon 3 nt185 T insertion resulted in TCT→TTC and Ser62Phe. In addition,the T insertion caused frame shift and resulted in a premature stop codons in exon 4. Another chromosom carried G715T in exon 7 and changed a GAA codon encoding glutamic acid at position 239 to TAA which is a stop codon.Further investigations were carried out to find out the KEL gene transcript structure in different blood cells.Transcripts of K and k in reticulocyte cells had only one normal KEL transcript faithful to the mRNA structure and the transcript with K gene mutation could be found. But K₀ reticulocyte cells did not have complete or normal KEL gene transcripts. Alternative splicing of mRNA were detected in total RNA which mostly came from the leucocytes,including intron retention and exon skipping, all these samples had multiple kinds of transcripts.Most of them were abnormal and composed of different exons or introns splicing.The Flow Cytometry was used to detect the expression of Kell antigens on red blood cells and leucocytes.T lymphocytes,B lymphocytes and NK cells were further marked to find out whether the Kell antigens expressed on the cell membrane. The Flow Cytometry was used to detect the strong expression of Kell antigens on red cells except for K₀ sample. Kell antigen expression couldn't be found on K₀ leucocytes. Surprisingly,there were low but definitely positive expression of Kell antigen on leucocytes in all samples except K₀. Other 3 antibodies confirmed the results. Further study implied that leucocytes which had Kell antigen expression were mainly CD4+T lymphocytes and CD19+B lymphocytes while there were little Kell antigen expression on CD8+T lymphocytes and CD19+B lymphocytes.The sibship screening of K₀ proved that the proband's parents both showed normal Kell phenotype and they each carried a mutated KEL gene.The proband's K₀ phenotype was caused by the two mutations separately inherited from mather and father.The proband's sister carried the mutation from her mother while the other chromosome that with normal KEL gene was from her father. The serology confimed that she was of normal Kell phenotype.The proband's brother was of normal Kell phenotype,too.He inherited his parents' chromosomes without KEL gene mutation. Since the proband inherited the two mutated KEL genes,she had the rare K₀ phenotype. KEL gene mutation in only one chromosome can not cause rare Kell phenotype.Sequence specific primers were designed and PCR-SSP (sequence-specific primer polymerase chain reaction , PCR-SSP) were carried out to distinguish the difference between K and k gene.Sequencing were used to evaluate the effectiveness and reliablity of this kit. Then the K/k PCR-SSP kit was successfully applied in examination of the Kell genotype of a group of foreign pregnant women and two samples of ISBT(International Society Blood Transfusion)2008 blood group genotyping workshop.