Molecular Study On The Relative Antigens Of KEL Gene Exon7～9 In Chinese

Kell blood group is a complicated system and important in clinical medicine,behind of ABO and Rh blood group. It has displayed 27 antigens,with 5 point mutations in exon 8,coding 9 antigens, Kpa/Kpb/Kpc,KEL11/KEL17,RAZ, VLAN, KTIM and one new unnamed antigen, and 2 point mutations in exon9,coding 2 antigens, KEL13, K22.Currently no point mutation in exon 7 has been reported. Some antigens could cause sever transfusion reaction and hemolytic disease in newborn infants (HDN). KEL gene is highly polymorphic in molecular basis and mainly single base mutation causes amino acid change,leading to antigen expression change.In this study,blood samples were collected from 1015 unrelated healthy Han nationality random donors in Shanghai Blood Center between March 2006 to January 2007 and other 55 samples from Xinjiang in May 2006.PCR-RF-SSCP was set up and used to screen in large-scale with serology reagents and positive sample unavailable. Firstly, KEL, gene exons 7～9 of these samples were amplified,among them 150 samples were with known normal KEL sequence;Secondly, PCR products were digested with PvuII enzyme;Thirdly,heating to denature and suddenly cooling to form single strand conformation,polyacrylamide electrophoresis followed with silver dying utilized to screen different single strand conformation patterns. Any sample with abnormal or undiscernable patterns would be sequenced further.3 samples were found with unique pattern,their sequencing results showed that one had a nt1080 G→A synonymous mutation in exon9;the second had a C→A mutation in intron7;the last one had a nt835 G→T mutation in exon7, which changed Glu codon to the termination codon,generating a premature termination codon.These 3 new mutations had got the GenBank accession number, DQ905957,DQ923321 and EF208901.For further study of this individual with nt835 G→T mutation in exon7 and her family ,seology, RT-PCR, PCR,cloning and sequencing were used to explore molecular basis of this mutaion. Seology showed that the individual was K₀ phenotype,but Kell antigens were normally expressed in her father and mother's red blood cells.Beside nt835 G→T mutation, K₀ individual also had a T insertion mutation at nt304 in exon3,bringing premature termination codon in exon4, with the GenBank accession number,EF208900.These two heterozygous novel mutaions of the KEL gene may explain the K₀ phenotype. The family study showed the mother had the same T insertion at nt304 in exon3 and the father had a same nt835 G→T mutation in exon7. The results of RT-PCR and cDNA sequencing suggested that K₀ individual had one transcript, which was with the deletion of total exon3. The candidate transcript with mutation in exon7 was not found even with repeated experiments.Generally,mRNAs containing premature termination codons are often thought to be rapidly degraded by nonsense-mediated mRNA decay. Transcript with mutation in exon7 maybe degraded so rapidly that we could not discovered it. But the possibility that this candidate transcript with premature termination codon in exon7 was shielded by the prevailing transcript of exon3 deleton couldn't be ruled out.