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Human Molecular Genetics Advance Access originally published online on August 27, 2004
Human Molecular Genetics 2004 13(20):2377-2384; doi:10.1093/hmg/ddh276
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Human Molecular Genetics, Vol. 13, No. 20 © Oxford University Press 2004; all rights reserved

Disease associations and altered immune function in CD45 138G variant carriers

Sally Boxall1,{dagger}, Tara Stanton2,{dagger}, Kouzo Hirai3,{dagger}, Victoria Ward2, Tomoyo Yasui3, Hideki Tahara3, Akihiro Tamori3, Shuhei Nishiguchi3, Susumu Shiomi3, Osamu Ishiko3, Masaaki Inaba3, Yoshiki Nishizawa3, Ritu Dawes1, Walter Bodmer2, Peter C.L. Beverley1 and Elma Z. Tchilian1,*

1The Edward Jenner Institute for Vaccine Research, Compton, Berkshire RG20 7NN, UK, 2Cancer and Immunogenetics Laboratory, Weatherall Institute of Molecular Medicine, Cancer Research UK, John Radcliffe Hospital, Oxford OX3 9DU, UK and 3Osaka City University Graduate School of Medicine, Abeno-ku, Osaka 545-8585, Japan

Received June 28, 2004; Revised August 2, 2004; Accepted August 19, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The CD45 (leucocyte common) antigen is a haemopoietic cell specific tyrosine phosphatase essential for antigen receptor mediated signalling in lymphocytes (1,2). Mice and humans lacking CD45 expression are severely immunodeficient. Expression of different patterns of alternatively spliced CD45 isoforms in lymphocytes is associated with distinct functions, but the role of each isoform is unknown (3). In humans, naive T cells express high molecular weight isoforms containing the fourth or A exon (‘CD45RA’ cells), but following activation the low molecular weight CD45R0 isoform is expressed (‘CD45R0’ cells). Point mutations in the A exon of CD45 have been suggested to be associated with multiple sclerosis, autoimmune hepatitis, systemic sclerosis and HIV infection in humans (4–8). These polymorphisms (C77G and C59A) prevent the splicing of exon 4, resulting in the presence of an increased proportion of lymphocytes expressing CD45RA-containing isoforms (9,10). Although the C77G variant appears to be associated with certain diseases, the results from these studies are difficult to interpret because of the low frequency of the variant allele (11–14).

We recently identified another polymorphism (A138G) in exon 6 of CD45. The polymorphism results in an amino acid substitution of Thr-47 to Ala in exon 6 and interferes with CD45 alternative splicing (15). In contrast to the C77G variant, peripheral blood T cells from individuals carrying the 138G allele show an increase in the proportion of CD45R0+ cells and a decrease in naive phenotype T cells expressing CD45RA. The 138G allele is present at a high frequency among Koreans (7.3%) and Japanese (23.7%). In Caucasoids, the allele appears to be relatively rare [1.7% in Italians (14) and 0.4% in the UK (15)]. The high allele frequency of this variant in the Japanese population suggests a selective advantage for survival, and we have proposed that the changes in CD45 splicing in the 138G carriers might lead to altered immune responses and associations particularly with autoimmune and viral diseases (15). Here we investigate whether the 138G variant is associated with thyroid autoimmune disorders (Hashimoto's thyroiditis and Graves' disease) and viral infections (hepatitis B and hepatitis C). We studied the phenotype and cytokine production of lymphocytes from individuals with the 138G variant and, using a minigene containing the A138G mutation, we examined the effect of the variant on CD45 splicing.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A minigene containing the A138G mutation shows increased splicing of exon 6
We have previously shown by both RT–PCR and flow cytometric analysis that 138G carriers have increased CD45R0 expression in peripheral blood mononuclear cells (PBMCs) (15). We suggested that the mechanism for the altered isoform expression is the A138G substitution in exon 6, as it has been reported previously that changes in this region of exon 6 affect isoform expression, promoting skipping of exon 6 (16). Indeed, Tsai et al. (16) have shown that mutations of nucleotides 134–144 at the most 3' end of exon 6 (LS37 minigene construct) resulted in CD45 mRNA that did not include the exon 6 sequence, regardless of whether it was synthesized in B cells or in thymocytes. In contrast, the control pSV-mini-LCA30 construct could be alternatively spliced, and in EL-4, produced both kinds of transcripts, one including the exon 6 sequence and one without (16) (Fig. 1A and B). In order to examine the role of 138G variant on CD45 splicing, we introduced the 138G mutation into pSV-mini-LCA30 generating the LS138G construct. We then compared the pattern of splicing of the three constructs pSV-mini-LCA30, LS37 and LS138G in Cos-7 cells. Six individual transfections were performed with each of the constructs and the splicing analysed by RT–PCR (Fig. 1A and B).



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  		Figure 1. Effect of the exon 6 138G mutation on splicing. (A) Schematic of the minigenes used. The wild-type pSV-mini-LCA30 construct includes variable exon 6 flanked by the constitutive exons 2 and 8. The exons are represented by open boxes and introns are represented by lines. The introns and exons are not drawn to scale. For the mutant version of the minigene LS37, the hatched box within exon 6 represents the 10 bp substitutions made at nucleotides 134–144 at the most 3' end of the exon. For the mutant LS138G, the line within exon 6 represents the A to G substitution at position 138. (B) The possible alternative splice products are indicated with the expected sizes. (C) RT–PCR analysis of three representative clones expressing the minigenes indicated. Bands in each lane were quantitated and densitograms are shown above the gel, corresponding to the respective minigene splice product. The ratio between the intensity of the ex2–6–8 and ex2–8 products is shown beside the gel and represents the mean result from six transfections for each minigene. A slower migrating band was detected, which represents unspliced RNA, as determined by sequencing.

 
Consistent with previous studies, the pSV-mini-LCA30 can produce both ex2–6–8 and ex2–8 transcripts in a ratio of 12.1, whereas with LS37 the skipping of exon 6 is significantly increased; almost all the product is ex 2–8, and the ratio is 0.6 (Fig. 1C). Analysis of transcripts derived from the LS138G construct shows an increase in exclusion of exon 6, resulting in more ex 2–8 product and a decrease in the ratio to 6.4.

Taken together, these data provide direct evidence that the 138G mutation is the cause of the altered CD45 phenotype in 138G carriers.

Frequency of the 138G variant in autoimmune and infectious diseases
We studied the frequency of the 138G variant in cohorts of Japanese patients with thyroid autoimmune conditions. In Hashimoto's thyroiditis, cellular and humoral responses to thyroid antigens lead to destruction of the organ and hypothyroidism, whereas Graves' disease (GD) is characterized by hyperthyroidism, caused by stimulatory thyrotropin receptor antibodies. We analysed 126 Hashimoto patients and found 50 A138G heterozygotes (allele frequency, 19.8%), comparable to the frequency in the control population (23.7%) (Table 1). Interestingly no G138G homozygotes were detected amongst the Hashimoto samples, although five were expected according to the Hardy–Weinberg law (P=0.02). We found 31 heterozygotes (frequency 8.9%) and no homozygotes out of the 175 Graves' samples. The difference between the controls and Graves' disease is very significant (P<0.001) and corresponds to a relative risk of 0.44 for the A138 versus the 138G allele. This suggests a dominant effect for the 138G allele in Graves' disease.


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  		Table 1. Frequency of CD45 exon 6 A138G alleles in control and disease groups
 
We further analysed the frequency of the variant in two important viral infections, hepatitis B and hepatitis C. We found 23 A138G heterozygotes and two homozygotes among 113 hepatitis B samples (allele frequency, 11.95%). The difference between the controls and hepatitis B is significant (P<0.005), corresponding to a relative risk of 0.55 for the A138 versus the 138G allele. In hepatitis C, we found 48 A138G heterozygotes and eight G138G homozygotes in 173 samples, figures that are as expected according to the Hardy–Weinberg law. No significant association of the 138G variant and severity of disease was detected in hepatitis B or hepatitis C. However, studies in larger cohorts of patients are clearly needed in order to establish whether there is any correlation with disease progression. Taken together, these results show a significant protective effect of the 138G allele in Graves' disease and hepatitis B, and a possible recessive effect in Hashimoto's disease.

Phenotypic analysis of PBMC
Because altered immune phenotypes and function are significant features of both animal model and human autoimmune diseases, we next sought evidence for this in individuals carrying the 138G allele. CD45RA and CD45R0 expression defines subsets of CD4 and CD8 cells that have been termed naive and memory cells (17). We have already reported that the proportions of these subsets are altered in individuals carrying the 138G variant allele (15). Here we examined other markers that either distinguished naive and memory cells or the proportions of memory subsets. Our aim was to determine whether only CD45 isoform expression was affected or whether the CD45RA and CD45R0 cells in 138G individuals also exhibited characteristic ‘naive’ and ‘memory’ phenotypes. PBMCs from healthy G138G homozygotes, A138G heterozygotes and A138A control homozygotes were analysed by flow cytometry. All the G138G variant samples showed the previously described increased proportion of CD45R0+ T cells (15); among CD8 cells the mean was 49.4±8.9%, when compared with 18.9±9.3 in controls (P=0.03) (Fig. 2A), and in the CD4 subset the mean was 48.4±9.3 versus 32.8±9.3% in A138A controls. A138G heterozygotes show an intermediate CD45R0+ phenotype for CD8 and CD4 cells (data not shown). Furthermore, G138G individuals exhibit decreased expression of CD27, CD28, CD62L and CCR7 and increased expression of CD11a and CD95 in CD8 cells (Fig. 2B). Less exaggerated changes in expression of these markers were detected in the CD4 cells (Fig. 2C). These changes suggest that the most prominent effect in 138G carrying individuals is an increase in the proportion of memory T cells.



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  		Figure 2. Expression of CD45 isoforms and activation markers on CD4 and CD8 cells from four healthy G138G homozygous and six A138A homozygous control individuals. (A) The proportions of CD8 and CD4 T cells from G138G and A138A control individuals that are CD45R0+. (B) Proportions of CD8 and (C) CD4 T cells that express CD11ahi, CD27, CD28, CD62L, CD95 and CCR7. Means and standard deviations of data expressed as the percentage of CD8 and CD4 T cells from four G138G and six A138A control individuals are shown. Four healthy A138G heterozygotes were also assayed in this experiment and showed intermediate phenotypes (data not shown). Differences between G138G and A138A indidviduals for CD8+CD45R0+, CD8+CD95+, CD8+CCR7+ and CD4+CD62L+, CD4+CD95+ cells are statistically significant by Mann–Witney, P=0.03.

 
Cytokine production by PBMC
We next analysed cytokine production in PBMC from 138G carrying individuals. Intracytoplasmic flow cytometric analysis after stimulation by phorbol myristate (PMA) and ionomycin showed that G138G cells have a significantly higher frequency of intracytoplasmic IFN-gamma positive CD4 and CD8 cells (30.7 versus 18.9% in CD4 and 35.3 versus 9.7% in CD8 cells, Table 2). Heterozygotes showed an intermediate frequency of cytokine-positive cells in both T cell subsets (Table 2 and Fig. 3). Under the conditions of these assays using cryopreserved PBMC, intracytoplasmic staining for cytokines other than IFN-gamma proved unreliable. These preliminary results show that expression of the variant 138G allele is associated with an increased frequency of CD4 and CD8 cells showing intracytoplasmic staining for the Th1 cytokine IFN-gamma. Clearly, further analysis on fresh PBMC for other cytokines will be needed to understand fully the immune function in these individuals.


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  		Table 2. Frequency of IFN-gamma producing CD4 and CD8 T cells of individuals with different 138G alleles
 


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  		Figure 3. Frequency of intracytoplasmic IFN-gamma positive CD4 or CD8 T cells. PBMC from G138G and A138A homozygous or A138G heterozygous individuals were stained for CD4 or CD8 and for intracytoplasmic IFN-gamma after activation with PMA and ionomycin. Data from 12 individuals analysed in one experiment are shown. Horizontal lines show the mean for each group. Similar results were obtained in a repeat experiment.

 
To exclude the possibility of altered cytokine staining due to the freezing and shipping of the samples, we carried out experiments to compare fresh and frozen cells from six local Chinese and Japanese donors. No differences between fresh and frozen samples were observed in the phenotype of lymphocytes or the frequency of cytokine positive cells. The range for IFN-gamma staining in fresh CD3+ cells was 11.1–21.2% and 10.1–21.4% for frozen cells. Paired results from fresh and frozen samples from each donor were almost identical.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study we show a protective effect of the 138G variant in cohorts of patients with autoimmune Graves' disease and hepatitis B infection. We provide direct evidence that the A138G mutation is a cause of altered CD45 isoform expression and preliminary evidence that 138G individuals exhibit increased numbers of memory/activated lymphocytes and increased IFN-gamma production.

There are several possible explanations for the effect of the 138G variant. An important factor in the pathogenesis of autoimmune diseases is a change in the balance between Th1 cytokines, which promote cell mediated immunity, and Th2 cytokines, which promote humoral immunity. In Graves' disease, there is a shift towards Th2 cytokine responses (18,19), whereas Hashimoto patients show Th1 activation (20). It is possible that increased IFN-gamma production in 138G carriers would counteract the Th2 cytokine deviation in Graves' disease. Furthermore, it has been suggested that activated (IFN-gamma producing) CD8 cells may reduce the pathogenic Th2 dominance in Graves' disease (19,21). In contrast, increased IFN-gamma production in the 138G variant might not affect the disease course and already polarized Th1 cytokine balance in Hashimoto's thyroiditis. However, the lack of G138G homozygotes in Hashimoto's thyroiditis suggests the possibility of a specific recessive effect in homozygotes, which needs further investigation. Further analysis of immune function in diseased individuals may provide insights into the role of CD45 variant alleles in protection against disease or disease pathogenesis.

The contribution of CD8 cells to the control of hepatitis B virus infection is well documented (22). In addition to clearance of infected cells by cytolytic CD8 cells, the anti-viral effect of IFN-gamma produced by these cells has been shown to be an important protective mechanism (23). It is very likely that the increased proportions of activated T cells and IFN-gamma production in A138G neonates would limit amplification of the virus. Furthermore, it has been suggested that neonates have Th2 biased immune responses (24,25). It is therefore possible that the prevalence of Th1 cytokines in A138G infants would be beneficial at this early stage of life in controlling hepatitis B infection, whereas it would not have such a significant impact later in life. This might be the case for the hepatitis C cohort we have studied. Whatever the mechanism, comparisons of immune responses of individuals carrying or lacking the 138G allele may provide insights into the molecular mechanisms underlying the interactions between hepatitis B and hepatitis C viruses and IFN-gamma. Clearly, further independent cohort studies, family based approaches and immune functional studies in normal individuals and patients, need to be performed to confirm our preliminary functional data and elucidate the role of this polymorphism in disease incidence and progression.

The expression of CD45 is essential for normal development and function of lymphocytes. Both mice and humans lacking CD45 expression are severely immunodeficient (26–29). However, notwithstanding the clear evidence that CD45 expression is essential for normal lymphocyte function and that N-terminal alternative splicing is conserved in fish, birds and mammals (30,31), there is no clear understanding of the role of CD45 isoforms in lymphocyte function. In mice, a point mutation in the cytoplasmic domain has been associated with a lupus-like autoimmune disease (32) and the authors postulated that this was due to an effect on dimerization of CD45 molecules. Others have shown that a large extracellular domain is required for TcR signalling in transfected cell lines (33). On the other hand, evidence from transgenic mice expressing single CD45 isoforms suggests that different isoforms reconstitute immune function equally well if they are equally well expressed (34). Here we show that a relatively subtle alteration in alternative splicing is associated with altered disease susceptibility and increased IFN-gamma production. The mechanism of these effects is not known, but CD45 has been reported to affect cytokine signalling pathways (35). We speculate therefore that specific combinations, rather than individual CD45 isoforms, may have the most profound effect on signalling. In 138G individuals, excess expression of low molecular weight CD45 isoforms may lead to altered signalling and increased IFN-gamma production. This in turn may affect disease susceptibility.

There have been previous reports of altered CD45 isoform expression in disease (36–38), but we describe here what we believe is evidence that genetic variants affecting CD45 isoform expression are associated with autommunity and viral infection. Although one possibility is that the effect of the 138G allele is neutral, the most likely explanation for the high frequency of this variant is natural selection. In contrast to this allele, most neutral polymorphic variants are at a lower frequency and are changes in third base pair positions or in introns. Given the functional effect and high frequency of the 138G variant not only in Japanese but also in China (unpublished data) and Korea (15), natural selection is therefore the most likely explanation for its frequency. The original selection for the 138G CD45 variant may have been with respect to pathogen resistance and what we see now is a residue of this after the pathogen effect has gone. The high frequency of 138G carrying individuals (~40% in Japan) suggests that the presence of allele may also affect susceptibility and pathogenesis to other autoimmune and infectious diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
DNA samples from 175 Graves' and 126 Hashimoto patients were obtained through the Osaka City University Hospital. Hyperthyroidism due to Graves' disease was diagnosed on the basis of history and signs of hyperthyroidism with diffuse goiter and the laboratory findings, including elevated serum-free T4 and T3 concentrations, undetectable serum thyroid stimulating hormone (TSH) and positive TSH receptor antibody. Hashimoto's thyroiditis was diagnosed by positive thyroglobulin and/or thyroid peroxidase antibodies, reduced echogenicity on thyroid ultrasound and normal or elevated TSH level. In total, 113 hepatitis B and 173 hepatitis C samples were collected in the outpatient clinic of Osaka City University Hospital. All of the 113 hepatitis B patients were infected at birth by transmission from their mothers and were positive for hepatitis B surface antigen. Of the hepatitis B patients 22% were carriers (alanine aminotransferase activity within normal range), 58% had chronic hepatitis (elevation of alanine aminotransferase for more than 6 months and liver inflammation histologically proven by tissue biopsy), 16% had liver cirhosis and 4% hepatocellular carcinoma. The hepatitis C patients were infected later in life and were all positive for antibodies to HCV antigen. In all samples, HCV RNA was detected, except for four patients who had cleared the virus (two of these were A138G heterozygous and one G138G homozygous). In the hepatitis C group of patients, 13% were carriers, 58% had chronic hepatitis, 16% liver cirhosis, 10% hepatocellular carcinoma and 3% had cleared the virus. Control genomic DNA samples from 176 Japanese, collected from Osaka City University Medical School, have been described previously (15). All of the control and patient groups studied are of similar ethnic origin and live in the area surrounding the Osaka University Hospital. Approval was obtained from the ethical committee of the City University Graduate School of Medicine Osaka and the patients gave consent for the study.

Amplification refractory mutation system PCR
To detect carriers of the exon 6 A138G mutations, we used the amplification refractory mutation system (ARMS) PCR with two separate reaction mixes, containing one forward primer and one of the two reverse primers as previously described (15). The presence of the 138G variant allele in all of the samples was confirmed by sequencing.

Flow cytometric analysis
Surface phenotypic analysis and intracytoplasmic staining for IFN-gamma were performed on cryopreserved PBMC (shipped from Japan to the UK) from six healthy A138A controls and four healthy G138G homozygous carriers. Cells from four A138G heterozygotes were also analysed (data not shown). The ages of all subjects in this study were between 27 and 58 years. In total, 2x105 PBMC were stained with either allophycocyanine (APC)-conjugated CD4 (S3.3, Caltag, Silverstone, UK) or CD8–APC (clone RPA/T8, Pharmingen, San Diego, CA, USA) along with fluorescein isothyocyanate (FITC)-conjugated CD45RA (clone HI100, Pharmingen) and phycoerythrin (PE)-conjugated CD45R0 (clone UCHL1, Pharmingen) mAbs in a single step at 4°C for 20 min and washed with PBS, containing 0.2% BSA. The following reagents and antibodies were also used to stain cell suspensions: CD11a–FITC (G43-25B), CD28–FITC (CD28.2), CD62L–FITC (Dreg56), CD95–FITC (DX2), CCR7 (2H4), all from Pharmingen; CD27–FITC (LT27), from Serotec (Kidlington, UK).

For intracytoplasmic staining 1x105 PBMC per well were incubated in U-bottom 96-well plates in 200 µl of RPMI1640+10% FCS in the presence of 50 ng/ml PMA and 0.5 µg/ml ionomycin. GolgiPlug (Pharmingen) was added after 2 h and cells incubated for an additional 12 h. The cells were surface labelled with CD4–APC or CD8–APC antibodies as described earlier and permeabilized with 40 µl Permafix (OrthoDiagnostic, UK) for 40 min in the dark. The cells were washed and stained with FITC conjugated IFN-gamma antibody (clone 25723.11, Pharmingen) for 30 min at room temperature. Isotype matched mAbs were used as controls. In total, 10 000 or 50 000 events per sample were collected on a FACSCalibur (Becton Dickinson, Mountain View, CA, USA) and analysed using WinMDI software.

A comparison of lymphocyte phenotype and the frequency of intracytoplasmic IFN-gamma positive cells in fresh and cryopreserved PBMC was performed on six local Chinese and Japanese donors.

Statistical analysis
Chi-square test, using Yates continuity correction to allow for small numbers, was used to analyse the disease association of the 138G variant allele. For comparison of phenotypic analyses between cell subsets in A138G and control individuals, Student's t-test assuming equal variance was used. In addition, we have also reanalysed the data using the non-parametric Mann–Witney test, because the data may not be normally distributed.

Minigene constructs
The minigene pSV-mini-LCA30 has been described previously (16) (kindly provided by H. Saito, Dana Faber Cancer Institute, Boston, MA, USA) and contains exons 2, 6 and 8 under the control of the SV40 early promoter (Fig. 1A). It has been demonstrated that the pSV-mini-LCA30 can be alternatively spliced to produce two kinds of transcripts, one including the exon 6 sequence and one without (Fig. 1B). The LS37 minigene was also used (Fig. 1A) and contains mutation of nucleotides 134–144 at the most 3' end of exon 6, which have been shown to result in CD45 mRNA that did not include the exon 6 sequence (16).

To study the effect of the 138G mutation on splicing, the 138G substitution was introduced into the pSV-mini-LCA30 construct according to the QuickChange site-directed mutagenesis kit (Stratagene) generating construct LS138G. Clones containing the 138G mutation were identified by sequencing. Exponentially growing Cos-7 cells (5x106 cells) were mixed with 1 µg of the linearized (PvuI digested) respective constructs in PBS, then subjected to electroporation (Bio-Rad Gene Pulser II, Bio-Rad, Hemel Hempsted, UK) at 25 µF and 280 V. After 48 h cells were harvested and resuspended in TRI-reagent (Sigma) for RNA extraction and RT–PCR analysis as described subsequently. Six independent transfections were performed and analysed for each minigene.

RT–PCR analysis
Total RNA was extracted from the transfected cells and cDNA synthesis performed using First Strand cDNA Synthesis Kit (Amersham Biosciences). The cDNA was amplified by primers in exon 2 (ex 2 fw: 5'-TAGGGACACGGCTGGCTTCCAG-3') and exon 8 (ex 8 rev: 5'-CATGTTGGCTTAGATGGAGTAG-3') generating bands for the ex 2–6–8 product of 266 bp and for the ex 2–8 of 122 bp (Fig. 1B). The PCR conditions for amplification included a 4 min incubation at 94°C followed by 25 reaction cycles (1 min at 94°C, 1 min at 55°C, 1 min at 72°C) and final 10 min extension at 72°C. The identity of the RT–PCR products was confirmed by sequencing. RT–PCR products were resolved on Visigel Separation Matrix (Stratagene) and the bands quantitated using Quality One software (Bio-Rad).


   ACKNOWLEDGEMENTS
 
We thank Dr Persephone Borrow for helpful discussions and suggestions, and Dr H. Saito for the CD45 LS37 and pSV-mini-LCA30 minigenes.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +44 1635577929; Fax: +44 1635577901; Email: elma.tchilian{at}jenner.ac.uk

{dagger} The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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