Expression of the homeobox protein CDX1 is lost or reduced in a significant proportion of colorectal carcinomas (CRCs) but the underlying mechanism for this is unclear. We have demonstrated absence of CDX1 mRNA expression in 7 of 37 CRC cell lines and shown that all 7 cell lines have a methylated CDX1 promoter. Twenty-five cell lines showed both CDX1 mRNA expression and an unmethylated CDX1 promoter. The five remaining cell lines had a partially methylated CDX1 promoter and all expressed CDX1 mRNA; when treated with the demethylating agent, 5-aza-2-deoxycytidine, these five cell lines all showed increased CDX1 expression. No mutations were found in the promoter and coding regions of CDX1. One polymorphism was demonstrated in each of the promoter, 5 UTR, and coding region of exon 1 of CDX1, but there were no associations between CDX1 mRNA expression and different polymorphic genotypes. Similarly, there was no association between CDX1 mRNA expression and loss of heterozygosity at the CDX1 locus. In conclusion, absence or reduction of CDX1 expression in CRC seems to be primarily regulated by promoter methylation and is probably selected for because of its impact on the differentiation of colonocytes. C DX1 is a homeobox protein involved in regulating normal development of intestinal epithelium in utero (1). In adulthood, CDX1 expression is maintained in small and large intestinal epithelium (1, 2) although its exact physiological role at this time remains uncertain. That the protein has an oncosuppressor function is supported by in vitro and in vivo evidence of loss of CDX1 expression in colorectal carcinogenesis (3, 4). The mechanism underlying this loss is, however, unclear. Possible roles for genetic and͞or epigenetic controls must therefore be considered. There is an ever growing list of oncosuppressor genes whose expressions are silenced through specific methylation of gene promoter CpG islands (CGIs). These genes all show 5Ј regions that are rich in CGIs (5). In sharing this feature, CDX1 represents a prime candidate for promoter methylation control. Indeed, half way through our study, Suh et al. published preliminary data supporting this hypothesis (6).The most comprehensive way of analyzing a gene for evidence of promoter methylation control is by direct sequencing after bisulfite modification of target DNA (so called ''bisulfite sequencing'') (7). Ideally, this analysis should be accompanied by looking for reexpression of the silenced gene after treatment with the demethylating agent, 5-azacytidine or the less toxic 5-aza-2Ј-deoxycytidine (5aza2). Suh et al. (6) showed CDX1 promoter methylation status to explain CDX1 mRNA expression among eight colorectal carcinoma (CRC) cell lines and also successfully used 5aza2 treatment to induce or increase CDX1 mRNA expression in six of the cell lines. Nonetheless, two important questions regarding control of CDX1 expression in CRC remain unanswered. First, possible contributions of gene mutation and͞or loss of heterozygosity (LOH) have not yet been excluded. Secon...

The CD45 antigen is a haemopoietic cell specific tyrosine phosphatase essential for antigen receptor mediated signalling in lymphocytes. Expression of different patterns of alternatively spliced CD45 isoforms is associated with distinct functions. We recently identified a polymorphism in exon 6 (A138G) of the gene encoding CD45 (PTPRC) that results in altered CD45 splicing. The 138G allele is present at a high frequency among Japanese (23.7%), with 5.1% individuals homozygous for the G allele. In this study we show that the A138G polymorphism is the cause of altered CD45 isoform expression, promoting splicing towards low molecular weight CD45 isoforms. We further report that the frequency of A138G heterozygotes is significantly reduced in number in cohorts of patients with autoimmune Graves' disease or hepatitis B infection, whereas G138G homozygotes are absent from a cohort of Hashimoto's thyroiditis patients. We also show that 138G individuals exhibit altered cytokine production in vitro and an increased proportion of memory T cells. These data suggest that the 138G variant allele strongly influences these diseases by modulation of immune mechanisms and may have achieved its high frequency as a result of a natural selection probably related to pathogen resistance.

CD45 (leukocyte common)T he leukocyte common antigen CD45 is an abundant tyrosine phosphatase expressed on all leukocytes (1). The phosphatase activity of CD45 is essential for lymphocyte antigen receptor signal transduction. CD45 can also function as a Janus kinase phosphatase, negatively regulating cytokine receptor signaling (2). Both CD45 knockout mice (3, 4) and humans lacking CD45 expression (5, 6) are severely immunodeficient, with very few peripheral T lymphocytes and impaired T and B cell responses. These studies provide evidence for the crucial role of CD45 in the proper functioning of the immune system. Multiple CD45 isoforms can be generated by alternative splicing of exons 4 (A), 5 (B), and 6 (C) of the extracellular domain (7). CD45 alternative splicing is highly conserved between species and is tightly regulated. In humans, naive T cells express high-molecular-weight CD45 isoforms recognized by CD45RA mAbs. Activation of the cells results in a change of expression to low-molecular-weight isoforms detected by a CD45R0 mAb (8).These two major subsets of T lymphocytes expressing CD45RA and CD45R0 have been termed naive and memory cells. A polymorphism (C77G) in exon 4 of CD45 causing abnormal CD45 splicing has been described in humans (9). Activated or memory lymphocytes in these individuals continue to express both high-CD45RA and low-molecular-weight CD45R0 isoforms, in contrast to the normal pattern of lowmolecular-weight CD45R0 isoform expression. Recently, another point mutation in exon 4 of CD45 (C59A) causing aberrant splicing has been identified, but it appears to be relatively rare (10).The C77G polymorphism and abnormal CD45 splicing have been further linked to the development of multiple sclerosis in German (11) and Italian (12) patient cohorts, although other studies do not support such an association (13,14). We have shown an increased frequency of the C77G variant allele in HIV-1-infected individuals in the United Kingdom (15). All of these observations suggest that abnormal CD45 splicing is associated with altered immunological function, autoimmunity, and viral infections.Here we report a polymorphism in exon 6 A138G in the gene encoding CD45 with a very high prevalence in Japanese and Korean populations. We analyzed the expression of CD45 isoforms in peripheral blood mononuclear cells (PBMC) of individuals homozygous and heterozygous for the A138G variant. Our results show that T cells in individuals carrying the A138G allele display altered cell-surface CD45 isoform expression because of changes in alternative splicing. Analysis of exon 6 A138G and exon 4 C77G variants in different populations showed striking differences in the frequency and distribution of these mutations, suggesting effects of natural selection. Materials and MethodsMaterials. One hundred seventy-five Japanese genomic DNAs were collected from Osaka City University Medical School (Osaka), of which 49 were from patients with malignant gynecological cancer. PBMC were isolated by centrifugation on a Ficoll-Paque (Amers...