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Keywords:

  • ITP;
  • FcγIIA;
  • FcγIIIA;
  • polymorphism;
  • therapeutic response

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Clearance of autoantibody-sensitized platelets through Fcγ receptors on phagocytic cells is one of the main mechanisms of thrombocytopenia in idiopathic thrombocytopenic purpura (ITP). We examined the FcγRIIA-131R/H and FcγRIIIA-158V/F polymorphisms in 104 adult chronic ITP patients, and in 59 healthy control subjects using polymerase chain reaction-based allele-specific restriction analysis. The frequency of FcγRIIA genotypes (131H/H, H/R, R/R) was not significantly different between patients and controls, and did not correlate with the responsiveness to treatment. In contrast, among FcγRIIIA genotypes, frequency of 158F/F homotype was smaller in ITP (P < 0·05). Furthermore, in FcγRIIIA-158V/V homotype, the complete remission (CR) rate with medication (treatment with corticosteroid or other immunosuppressive agents) was significantly higher (60%) than that in 158V/F (10%) or 158V/F plus 158F/F, (P < 0·01, P < 0·05). Conversely, the CR rate after splenectomy in 158F/F and 158V/F types (64·3% and 54·6%) was higher than in 158V/V (25%). Our results indicate that the polymorphism of FcγRIIIA, but not FcγRIIA, influences the response to treatment in ITP.

In idiopathic thrombocytopenic purpura (ITP), an immunological mechanism has been under consideration (Fujimura et al, 1996; Shimomura et al, 1996; Karpatkin, 1997; George & Raskob, 1998) since the autoantibodies against platelet membrane glycoprotein (GP)Ib, GPIIb/IIIa or GPVI were detected using radio or enzyme immunoassay. Although several immunological platelet destruction mechanisms have been proposed, the clearance of sensitized platelets by phagocytic cells is thought to be one of the main mechanisms of thrombocytopenia. That is, the Fab portions of the autoantibodies bind to platelet-specific membrane antigens, and these sensitized platelets are phagocytosed through the Fc portion of these antibodies by monocytes and/or macrophages in spleen or liver which express the Fc receptors. The effectiveness of splenectomy to remove the platelet destruction organ and intravenous immunoglobulin treatment (IVIG) to block the Fc receptor for IgG in the phagocytic cells support the clinical importance of the clearance mechanisms carried out by Fc receptors.

The Fcγ receptors are expressed on the effector cells that have a phagocytic function of autoantibody-sensitized platelets and antibody-dependent cell-mediated cytotoxity function (Newland & Macey, 1994). Three different families of Fc γR exist: FcγRI, FcγRII and FcγRIII, which are quite diverse in both their structure and function (Schreiber et al, 1992; van de Winkel & Capel, 1993; Indik et al, 1995; van der Pol & van de Winkel, 1998). Fc receptors are members of the immunoglobulin superfamily found on many different cells (neutrophils, macrophages, lymphocytes and platelets), and form a critical link between the humoral and cellular immune responses (Voura et al, 1997). FcγRI has a very strong affinity for monomeric IgG, whereas FcγRII and FcγRIII only bind effectively to IgG in the form of immune complexes. FcγRII class is encoded by three genes (IIA, IIB and IIC) and FcγRIII class is encoded by two genes (IIIA and IIIB). Several genetic polymorphisms of FcγR were reported and, in some of them, amino acid differences may alter the affinity of the receptors to bind immunoglobulins. The polymorphism of FcγRIIA is caused by a single base substitution at nucleotide position 494. The variant allele of 131 histidine (494 A) is characterized by a high affinity for human IgG2 and a low affinity for murine IgG1, whereas the other allele of 131 arginine (494G) has the opposite binding properties (Salmon et al, 1992). In FcγRIIIA, which is mainly expressed on mononuclear phagocytes, polymorphism of T to G substitution at nucleotide 559 changes phenylalanine (F) into valine (V) at position 176 in the membrane proximal EC2 domain (De Haas et al, 1996; Tamm & Schmidt, 1996). This domain influences ligand binding because FcγRIIIA-V/V homozygotes bind more IgG1 and IgG3 than F/F type. These FcγR polymorphisms suggest the influence of antibody-mediated phagocytic activity or antigen presentation activity.

In this study, we examined the involvement of polymorphism of FcγRIIA and FcγRIIIA in the effectiveness of treatments, retrospectively, and the presence of platelet antibodies in ITP.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Subjects Blood samples were taken from 59 healthy control subjects (30 men, 29 women, median age 26 years) and 104 chronic ITP patients (28 men, 76 women, median age 54·2 years) who were followed-up for 1 year, and had volunteered to be part of the study. No children (< 18 years old) were included in this study. The patients comprised various clinical states from complete remission (CR) to refractory cases, and treatments had been continued in most patients except CR patients. Splenectomy had been carried out in 40 cases (Splenectomy group) and 47 patients had been treated with prednisolone or immunosuppressive agents only (Medication group). The remaining 17 cases were followed-up, without medication, from the beginning. The effectiveness of the treatment procedures was evaluated at least 1 year after starting the treatment.

Initial diagnosis of ITP was determined based on the diagnostic criteria of the Japanese study group for intractable haematopoietic diseases as follows (Shimomura et al, 1996): decreased platelet count (< 100 × 109/l); normal cellularity on bone marrow aspiration or biopsy with normal or increased number of megakaryocytes; absence of splenomegaly; absence of any aetiological factor; and absence of underling disease. Response criteria were as follows (Fujimura et al, 1996): complete remission (CR); platelet count ≥ 100 × 109/l without concurrent treatment; partial response (PR), platelet count ≥ 100 × 109/l with concurrent treatment; minor response (MR), platelet count < 100 × 109/l with increase in platelet count ≥ 20 × 109/l; non-response (NR), platelet count < 100 × 109/l with increase in platelet count < 20 × 109/l.

Platelet antibody assay Citrated blood samples were collected with informed consent, and platelets and buffy coats were separated using centrifugation at 850 r.p.m. for 20 min. Direct MAIPA (monoclonal antibody-specific immobilization of platelet antigens) assay was performed according to Kiefel et al (1987).

FcγRIIA genotyping Genomic DNA was isolated from peripheral blood using the phenol–chloroform–isoamyl alcohol method. Exon 4 encodes the second extracellular domain of FcγRIIA, which contains the functionally important polymorphism that corresponds with a single base substitution (A or G) at nucleotide 494. This encodes amino acid 131 which determines affinity for specific IgG subclasses. The polymorphism was determined as described previously (Jiang et al, 1996). A 191 bp fragment of the FcγRIIA gene containing nucleotide 494 in exon 4 was amplified using polymerase chain reaction (PCR) directly from the genomic DNA. A mutagenesis sense primer (5′-GGA AAA TCC CAG AAA TTC TCG C-3′) corresponding with the nucleotides from 472 to 493, which terminated just before the polymorphic A/G nucleotide at 494, was designed to introduce allele-specific restriction enzyme site for BstU1 (CGCG) on the 5′ end only in the case of G at 494 (131R). Antisense primer was identical to the nucleotides from 644 to 662 (5′-TGT CCA TCC CCT CTT CTC C-3′). The PCR products were digested with BstU1 at 60°C for 2 h. The samples were applied to 8% polyacrylamide gel and stained with ethidium bromide after electrophoresis. In the case of FcγRIIA-131H/H homotype, one band at 191 bp was shown, whereas the digested 168 bp band and a small fragment were observed in 131R/R homotype, and both non-digested 191 bp and digested 168 bp bands were observed in 131H/R heterotype respectively. As a control experiment, PCR products from all three types, which had been found in normal volunteers after nucleotide sequencing, were simultaneously digested in each experiment. Data were then collected only when the control PCR fragments were completely digested.

FcγRIIIA genotyping Determination of the FcγRIIIA polymorphism was performed according to Koene et al (1997). A portion of exon 4 of FcγRIIIA was amplified, which corresponded to the second extracellular domain and contained the position at nucleotide 559. The 1·2 kb fragment expanding outside of the exon 4 was amplified first and nested PCR was performed using the first PCR products. In the second PCR, primers were designed to amplify 94 bp fragment in which NlaIII recognition sequence (CATG) was created only in the case of G at 559 (158V). Sense primer was from nucleotides 527–558 (5′-ATC AGA TTC GAT CCT ACT TCT GCA GGG GGC AT-3′), and antisense primer was from 589 to 620 (5′-ACG TGC TGA GCT TGA GTG ATG GTG ATG TTC AC-3′). Amplified products from the second PCR were digested with NlaIII at 37°C for 3 h, then applied to 10% polyacrylamide gel and stained with ethidium bromide. One 94 bp band which was not digested by NlaIII in the FcγRIIIA-158F/F homotype, two bands (61 bp and 33 bp) which were produced by NlaIII digestion in the 158V/V homotype, and three bands (94 bp, 61 bp and 33 bp) in the 158F/V heterotype were identified respectively. Control experiments were performed in the same manner as described for FcγRIIA typing.

Statistical analysis Genotype distribution, allelic frequency and correlation between polymorphism of Fcγ receptors and platelet-specific antibodies were analysed using the χ2-test. The influences of polymorphisms of FcγRIIA and FcγRIIIA on the therapeutic response were analysed using the Mann–Whitney U-test.

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Genetic distribution and allelic frequency of FcγRIIA and FcγRIIIA polymorphism in ITP cases and normal healthy controls

FcγRIIA Genotype distribution and allelic frequency of FcγRIIA in ITP cases and normal healthy controls are summarized in Table I. In ITP patients, FcγRIIA-131H/R genotype was present in 54·8% (57/104), whereas H/H and R/R genotype were in 38·5% and 6·7% respectively. The distribution of these three types was not significantly different from normal controls. FcγRIIA-131R/R was the lowest type in both groups. The allelic frequency of FcγRIIA-131R and 131H was also similar in both groups, which is 0·42 and 0·58 in the control group and 0·40 and 0·60 in the ITP group respectively.

Table I.   Distribution of FcγRIIA genotypes and allele frequencies in ITP patients and healthy control subjects
 Control group (n = 59)ITP patient (n = 104)
Genotype distribution
 131R/R7(11·9%)7(6·7%)
 131H/R30(50·8%)57(54·8%)
 131H/H22(37·3%)40(38·5%)
Allelic frequency
 131R(Arg)0·420·40
 131H(His)0·580·60

FcγRIIIA Genotype distribution and allelic frequency of FcγRIIIA in ITP cases and normal healthy controls are shown in Table II. There were significantly fewer (P < 0·05) ITP patients with the 158F/F homotype (26·9%) than control subjects (42·3%). FcγRIIIA-158V/V homotype and 158V/F heterotype were present in 10·6% and 62·5%, respectively, which were higher than that of the control group although the difference was not statistically significant. Allelic frequency of FcγRIIIA-158F and 158V in both groups was not statistically different.

Table II.   Distribution of FcγRIIIA genotypes and allele frequencies in ITP and healthy controls
 Control group (n = 59)ITP patient (n = 104)
  • *

    P < 0·05: Statistically significant difference between patients and control group.

Genotype distribution
 158F/F25(42·3%)*28(26·9%)*
 158V/F30(50·9%)65(62·5%)
 158V/V4(6·8%)11(10·6%)
Allelic frequency
 158F(Phe)0·620·55
 158V(Val)0·380·45

Combined genotypes of FcγIIA and FcγIIIA were also analysed in ITP. In FcγIIIA-F/F genotype, FcγIIA-R/R type was found in one patient (3·6%), FcγIIA-H/R in 17 (60·7%) and H/H in 10 (35·7%). In FcγIIIA-V/F type, FcγIIA-R/R, H/R and R/R were five (7·7%), 36 (55·4%) and 24 (36·9%) respectively. In FcγIIIA-V/V type, FcγIIA-R/R, H/R and R/R were 1 (9·1%), 4 (36·4%) and six (54·5%) respectively. The distribution of FcγIIA genotypes or allelic frequency among each FcγIIIA genotype was not significantly different.

Correlation between FcγR polymorphism and therapeutic response

We investigated whether or not the FcγR polymorphism could influence the response to ITP treatment retrospectively. The effectiveness was evaluated at least 1 year after splenectomy or starting steroid therapy. The non-effective group included the MR (minor-responder) and NR (non-responder) group. There were no significant correlations between the effectiveness of medication or splenectomy and FcγRIIA polymorphism (Fig 1, Table III).

image

Figure 1.  Relationship between FcγRIIA-131 polymorphisms and response to treatment by medication and splenectomy. The left panel shows the Medication group and the right panel shows the Splenectomy group. White, shadow, and black bars indicate CR (complete remission), PR (partial response) and MR (minor response) plus NR (non response) respectively.

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Table III.   Distribution of FcγRIIA genotypes in ITP patients, subclassified by the clinical response (CR, PR, or MR plus NR) in Medication group (A) and Splenectomy group (B). (A) Medication group.
 CR (n = 9)PR (n = 22)MR + NR (n = 16)Total (n = 47)
131R/R1 (33·3%)1 (33·3%)1 (33·3%)3
131H/R5 (20·0%)12 (48·0%)8 (32·0%)25
131H/H39719
 (15·8%)(47·4%)(36·8%) 
(B) Splenectomy group.
 CR (n = 22)PR (n = 8)MR + NR (n = 10)Total (n = 40)
131R/R2 (50·0%)1 (25·0%)1 (25·0%)4
131H/R13 (59·1%)4 (18·2%)5 (22·7%)22
131H/H7 (50·0%)3 (21·4%)4 (28·6%)14

On the other hand, remarkable differences were observed among the FcγRIIIA genotypes (Fig 2, Table IV). In FcγRIIIA-158V/V homotype, the CR rate in medication was higher than in the 158V/F heterotype or the 158F/F homotype. CR was found to be 60%, 25% and 10% in the V/V, F/F and V/F type respectively. The difference between the V/V and the V/F type (P < 0·01) and between the V/V and V/F plus F/F type (P < 0·05) was statistically significant. In splenectomy cases, however, the 158V/V homotype was less effective than other genotypes. About 80% of 158F/F or V/F type were in CR or PR; most of them were in CR, whereas only 50% of those with 158V/V type were in CR or PR, although the number of 158V/V homotype was relatively small for statistical analysis. Nonetheless, it is worth noting that a reverse phenomenon was observed for the CR rate between the Medication and Splenectomy groups among the FcγRIIIA genotypes.

image

Figure 2.  Relationship between the FcγRIIIA-158 polymorphisms and response to treatment by medication and splenectomy. The left panel shows the Medication group and the right shows the Splenectomy group. CR, PR, MR and NR are as defined in the legend to Fig 1. Statistically significant differences were observed in the CR rates between V/V and V/F type (*P < 0·01) and between V/V and V/F plus F/F type (**P < 0·05) of the Medication group

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Table IV.   Distribution of FcγRIIIA genotypes in ITP patients, subclassified by the clinical response (CR, PR, or MR plus NR) in the Medication group (A) and the Splenectomy group (B). (A) Medication group.
 CR (n = 9)PR (n = 22)MR + NR (n = 16)Total (n = 47)
  1. P-values were calculated using the U-test and the differences among 158F/F, 158F/V and 158V/V genotypes. (*P < 0·01: CR (V/V) versus CR (V/F), **P < 0·05: CR (V/V) versus CR (V/F + F/F) in the Medication group).

158F/F3 (25·0%)]5 (41·7%)4 (33·3%)12
158V/F3 (10·0%)]*]**16 (53·3%)11 (36·7%)30
158V/V3115
 (60·0%)(20·0%)(20·0%) 
(B) Splenectomy group.
 CR (n = 22)PR (n = 8)MR + NR (n = 10)Total (n = 40)
158F/F9 (64·3%)2 (14·3%)3 (21·4%)14
158V/F12 (54·6%)5 (22·7%)5 (22·7%)22
158V/V1 (25·0%)1 (25·0%)2 (50·0%)4

Relationship between the presence of platelet-specific antibodies and FcγR polymorphism

Finally, we examined the platelet-specific antibodies (anti-GPIbα, anti-GPIIb and anti-GPIIIa) using a modified MAIPA assay. Anti-GPIbα, anti-GPIIb/GPIIIa, and both antibodies were detected in 19 (18·3%), 13 (12·5%), and 49 (47·1%) cases respectively (Table V). Antibody-negative patients totalled 23 (22·1%). However, there was no correlation between the prevalence of these antibodies and FcγRIIA or IIIA polymorphisms.

Table V.   Distribution of FcγRIIA genotypes (A) and FcγRIIIA genotypes (B) of ITP patients subclassified by the presence of anti-platelet antibodies. (A)
FcγRIIA genotypes131R/R131H/R131H/H 
Both Antibodies (+) (n = 49)3 (42·9%)27 (47·4%)19 (47·5%) 
GPIb ab (+) (n = 19)1 (14·3%)11 (19·3%)7 (17·5%) 
GPIIb/IIIa ab (+) (n = 13)0 (0%)9 (15·8%)4 (10·0%) 
Both Antibodies (–) (n = 23)3 (42·9%)10 (17·5%)10 (25·0%) 
Total (n = 104)75740 
(B)
FcγRIIIA genotypes158F/F158V/F158V/V 
Both Antibodies (+) (n = 49)14 (50·0%)30 (46·2%)5 (45·4%) 
GPIb ab (+) (n = 19)5 (17·9%)13 (20·0%)1 (9·1%) 
GPIIb/IIIa ab (+) (n = 13)3 (10·7%)9 (13·8%)1 (9·1%) 
Both Antibodies (–) (n = 23)6 (21·4%)13 (20·0%)4 (36·4%) 
Total (n = 104)286511 

The relationship between the platelet antibodies and the combined genotypes of FcγIIA and FcγIIIA was also analysed. However, no types were frequently found in antibody-positive or -negative cases.

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The human genome project suggests that a polymorphism occurs approximately once every 800–1200 bp, and the frequency of selected variant alleles of the genes are different among races (Lehrnbecher et al, 1999). When our results were compared with a previous report (Lehrnbecher et al, 1999), the frequency of the variant genotype of FcγRIIA was different from the results analysed in Caucasians and African-American (H/H 22–26%, H/R 50–51% and R/R 24–23%), but was close to the results in the Far East populations (H/H 49%, H/R 47% and R/R 8%). Our data therefore support that the R/R homotype of FcγRIIA is found in a relatively small population, characteristically in people from the Far East, including the Japanese. On the other hand, the frequency of the FcγRIIIA polymorphism analysed in this study is the first to be reported because there are no previous publications about the FcγRIIIA polymorphism in Japanese or Far East populations. However, it was not too different from the reported frequency in Americans except for the V/V genotype (Koene et al, 1997). The V/V type is also a minor allele in American (11%) but was found in a smaller population (6·8%) in this study.

Recently, a number of groups have investigated the clinical significance of variant alleles in FcγRIIA and FcγRIIIA in several disease populations (Lehrnbecher et al, 1999). FcγRIIA polymorphism has been associated with various clinical conditions, including heparin-induced thrombocytopenia (HIT), systemic lupus erythematosus (SLE) and bacterial infections (Brandt et al, 1995; Burgess et al, 1995; Salmon et al, 1996; Wu et al, 1997; Carisson et al, 1998). Although these observations showed the correlation of these disease predispositions with FcγR polymorphisms there is still some controversy because of differences in race or number of subjects investigated. In this study, we could not determine the difference between the frequency of FcγRIIA genotypes in healthy control subjects and ITP patients. A report from Ireland (Williams et al, 1998) showed that their group of refractory ITP patients showed an increased frequency of R/R genotype (48%) compared with the healthy control group (18%), and suggested that the FcγRIIA polymorphism may be implicated in the pathophysiology of ITP or may be responsible for modulating the immune response in ITP. In contrast, a report from Canada (Horsewood et al, 1998) presented data that indicated no correlation, and suggested that the FcγRIIA polymorphism did not produce susceptibility to and severity of ITP, as demonstrated here. There have been no reports of FcγRIIIA in ITP patients. We have now shown that the frequency of 158F/F genotype in ITP was significantly lower than in control subjects.

We further analysed the relationship between the Fcγ receptor polymorphism and the response to treatment. Our data did not provide evidence that the FcγRIIA polymorphism has a relationship with the effectiveness of ITP treatment. In contrast, the 158V/V genotype of FcγRIIIA illustrates a trend that medication is more effective than splenectomy compared with other genotypes. Conversely, the 158F/F and V/F types predispose to ineffective response by medication, but not by splenectomy. These results suggest that the polymorphism of FcγRIIIA, rather than FcγRIIA, influences the effectiveness of ITP treatment. It is known that most of the platelet autoantibodies in ITP belong to the subclass of IgG1 or IgG3, but not to IgG2 (Tijhuis et al, 1991). Regarding the affinity of the receptors, it was reported that the FcγRIIA polymorphism influences the binding affinity mainly for IgG2 (Warmerdam et al, 1991), and that the FcγRIIIA-158V/V homotype binds to IgG1 and IgG3 with a higher affinity than the F/F homotype (Koene et al, 1997; Wu et al, 1997). Taken together, FcγRIIIA polymorphism, but not FcγRIIA, may contribute to the different levels of clearance of antibody-sensitized platelets and further supports the role of the FcγRIIIA polymorphism on the response to treatment in ITP. It is not clear why the reverse effect was observed between medication and splenectomy among the different polymorphisms of the FcγRIIIA. One possible explanation is that in the V/V type, which has a higher affinity for antibody-sensitized platelets, medications such as prednisolone are more effective because of suppression of the function of phagocytes carrying this receptor. In contrast, splenectomy is less effective because the other reticuloendothelial organs such as the liver could still destroy platelets with high affinity even after the spleen was removed. In the F/F or the F/V type, however, the effectiveness of the elimination of the main platelet destruction site (the spleen) is more obvious because phagocytes of the other organs have only lower affinity for the platelets, which may induce the compensatory state for the platelet destruction by platelet production.

It is known that the Fcγ receptors regulate not only phagocytic activity but also the antibody production indirectly through the phagocytosis followed by the antigen presentation (Gessner et al, 1998). In this study, no correlation was observed between the presence of platelet antibodies and the polymorphisms of the Fcγ receptors. These results might indicate that the polymorphisms of the Fcγ receptors did not regulate the production of autoantibody against platelets.

In summary, our results suggest the possibility that the analysis of FcγRIIIA polymorphism could indicate the effectiveness of medication and splenectomy, which would be clinically useful information for the selection of treatment in ITP.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We gratefully acknowledge the expert technical assistance of Ms Motoko Yoneya and Miki Ueda. This work was partially supported by the Ministry of Health and Welfare and by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research (No.12670990, No.11671001).

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  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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