Unusual case presentations associated with the CD45 C77G polymorphism

Abstract

CD45, the leucocyte common antigen, is a haematopoietic cell specific tyrosine phosphatase. Human polymorphic CD45 variants are associated with autoimmune and infectious diseases and alter the phenotype and function of lymphocytes, establishing CD45 as an important regulator of immune function. Here we report four patients with diverse diseases with unusual clinical features. All four have the C77G polymorphism of CD45 exon 4, which alters the splicing and CD45RA/CD45R0 phenotype of lymphocytes. We suggest that C77G may be a contributing factor in these unusual cases.

Fig. 1

Flow cytometric analysis of C77G variant individuals. PBMC from two healthy individuals, one C77C (wild-type) homozygote and one C77G heterozygote, were separated by ficoll/hypaque gradient centrifugation and stained with CD45RO-PE, CD45RA-FITC and either CD4 or CD8-APC antibodies pre- (a) and post-stimulation (b) with PHA for 10 days. Analysis was performed on CD4 or CD8 gated cells. Normal expression is characterized by the loss of CD45RA and gain of CD45RO expression upon activation. Variant C77G expression is characterized by the absence of a single CD45RO+ population. Even after stimulation for 10 days C77G cells remain double CD45RA+/CD45RO+. (c) Flow cytometric analysis of CD45 splicing in CVID patients. PBMC were isolated by ficoll/hypaque separation and triple stained with CD3-APC, CD45RA-FITC and CD45-RO antibodies. Analysis of the CD3 gated population is shown. Variant CD45 splicing in the C77G patient with prolonged poliovirus excretion can be identified by the absence of the single CD45RO+ population. The C77C CVID individual shows a depletion of CD45RA (naïve) and increase in CD45R0 (memory/activated) T cells.

Fig. 2

Identification of an exon 4 CD45 G77G homozygote. (a) Sequence analysis of wild-type C77C, heterozygous C77G and homozygous G77G samples. Position 77 of exon 4 is indicated by asterisks. (b) PCR analysis for detection of C77G was performed on wild-type C77C, heterozygous C77G and homozygous G77G genomic DNA, with primers on either side of the site of mutation, amplifying a fragment of 155 bp in wild-type DNA. The C77G transition introduces a new restriction site for MspI, which cleaves the mutant PCR product into two fragments of 72 and 83 bp (34). The absence of an undigested band of 155 bp indicates the presence of homozygous G77G in individual 3.