title: A point mutation within CD45 exon A is the cause of variant CD45RA splicing in humans.
abstract: The leukocyte common antigen (CD45) is alternatively spliced, generating various isoforms expressed on hemopoietic cells. The splicing pattern of CD45 in T cells is altered in some individuals who show abnormal expression of high molecular weight isoforms containing exon A. The variant splicing pattern was shown to be associated with heterozygosity for a silent point mutation within CD45 exon A. This C to G transition is located 77 nucleotides downstream of the splice acceptor junction of exon A (198 bp total length). Here we report that this mutation is the cause of abnormal splicing. To isolate the mutant gene, somatic cell hybrids of lymphocytes with a CD45 splicing defect and a mouse lymphoid line were produced and clones expressing different isoforms of CD45 were isolated. Expression of the high molecular weight isoform containing exon A was associated with the mutation within exon A. All hybrids expressing the low molecular weight isoforms lacking exon A contained the normal allele of CD45 only. In addition, minigenes including this mutation were constructed and transfected into various cell lines (COS-7, HeLa, CHO). Semi-quantitative reverse transcription polymerase chain reaction showed an increase of more than tenfold in splicing to CD45RA (concomitant with a decrease in splicing to CD45RO) when compared with the normal minigene. Taken together, these results demonstrate a causal relationship between the mutation in CD45 exon A and the variant splicing pattern observed. The involvement of trans-acting splicing factors that interact with this region of CD45 pre-mRNA is currently under investigation.
authors.affiliation: Imperial Cancer Research Fund, Tumour Immunology Unit, University College London Medical School, GB. zilch@icrf.icnet.uk
authors.forename: C F
authors.initials: CF
authors.lastname: Zilch
authors.forename: A M
authors.initials: AM
authors.lastname: Walker
authors.forename: M
authors.initials: M
authors.lastname: Timón
authors.forename: L K
authors.initials: LK
authors.lastname: Goff
authors.forename: D L
authors.initials: DL
authors.lastname: Wallace
authors.forename: P C
authors.initials: PC
authors.lastname: Beverley
mesh_terms: D000483:Alleles; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D003412:Cricetulus; D005091:Exons / Q000235:genetics; D005813:Genes, Synthetic; D006367:HeLa Cells; D006801:Humans; D006822:Hybrid Cells; D017493:Leukocyte Common Antigens / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D017493:Leukocyte Common Antigens