The Chi-square test was used to compare the genotype and the allelic frequency distribution of the ACE gene for patients with Kawasaki disease and also for control subjects.
A Chi-square test was also used to compare groups revealing the presence of a coronary-artery aneurysm as compared to those who did not. A value of p < 0.05 was considered to represent significant difference between tested populations.
RESULTS
The distribution of the I/D, A-240T and G2350A polymorphism of the ACE gene is revealed in Tables 2, 3 and 4. The analysis of ACE D/D genotype distribution in the control group and KD group revealed16.8% and 5.6% respectively. The analysis of ACE 2350 G/G genotype distribution in the control group and KD group revealed 16% and 5.6% respectively.
The analysis of ACE D/D genotype and ACE 2350GG genotype distribution revealed a statistically-significant difference between KD patients and controls. As shown in Tables 5, 6, 7 and 8, no significant difference as regards I/D, A-240T and G2350A polymorphism of the ACE gene between patients with and without coronary-artery aneurysm was observed.
The distribution of the IL -1 ß promoter, IL -1 ß exon 5 and IL -1 Ra polymorphism gene is revealed in Tables 9, 10, 11, 12, 13, 14. The analysis of genotype and allelic frequency distribution of IL -1 ß promoter and IL -1 ß exon 5 revealed no significant difference between Kawasaki-disease patients and controls. The I/II genotype and II allele of the IL -1 Ra gene were found with a significantly higher frequency in KD patients compared with controls. The distribution of genotype and alleic frequency of IL -1 gene polymorphisms between patients with and without coronary-artery aneurysm was no significant difference.
The distribution of the polymorphisms of IL -4 promoter and IL -4 intron 3 gene is
revealed in Tables15, 17. The analysis of genotype and alleic frequency distribution revealed no significant difference between Kawasaki-disease patients and controls. As shown in Tables 16, 18, no significant difference as regards IL -4 gene polymorphisms between patients with and without coronary-artery aneurysm was observed.
The distribution of the IL -6 promoter gene polymorphisn is revealed in Tables 19. The analysis of genotype and alleic frequency distribution revealed no significant difference between Kawasaki-disease patients and controls. As shown in Tables 20, no significant
difference as regards IL -6 gene polymorphism between patients with and without coronary-artery aneurysm was observed.
The distribution of the TNF-a gene polymorphism is revealed in Tables 21. The distribution in control group revealed 6.8% A-308 allele homozygote, 29.1 heterozygote and 64.1 % G -308 allele homozygote. The genotype distribution in KD group reveled 15.9 % A-308 allele homozygote, 22.4 % heterzygote and 61.7 % G-A-308 allele. The allelic frequencies in the control group were 21.4% A and 78.6% G. The analysis of genotype and alleic
frequency distribution revealed no significant difference between Kawasaki-disease patients and controls. As shown in Tables 22, no significant difference as regards TNF-a gene
polymorphism between patients with and without coronary-artery aneurysm was observed.
The distribution of the TGF-ß1 gene polymorphism is shown in Tables 23. The analysis of genotype and alleic frequency distribution revealed no significant difference between Kawasaki-disease patients and controls. As shown in Tables 24, no significant difference as regards TGF-ß1 gene polymorphism between patients with and without coronary-artery aneurysm was observed.
DISCUSSION
Tomisaki first described Kawasaki disease in 1967 (42). The exact cause of Kawasaki disease would appear to still be largely unknown. Serious complications to the coronary
artery may occur as a result of this disease (43). Kawasaki disease is also one of the leading causes of acquired heart disease(s) in the pediatric field. In a number of previous reports, the genotype DD of the ACE gene may have constituted a risk factor for heart disease including coronary-artery disease, thus, here, we have attempted to establish the relationship between Kawasaki disease-suffering patients and ACE gene polymorphisms.
The human ACE gene has been cloned and localized to chromosome 17q23 (44). A 287 bp insertion/deletion polymorphism in intron 16 has been identified for use as a genetic marker (45). The A-240T polymorphism is on the promoter region of the ACE gene. The G2350A polymorphism is located in intron 17. Circulating levels of ACE are under substantial genetic control, there being ample evidence to suggest that the I/D polymorphism is in strong linkage disequilibrium with a major gene effect at the ACE gene locus, this locus being suggested to control up to 44% of the variability in ACE levels (46). Philippe. et al. (1998) analyzed the distribution of mean serum ACE activities according to ACE I/D genotypes for different age groups of Emirati subjects, these authors finding that the correlation between the ACE I/D dimorphism and circulating serum ACE activities was able to be quite easily documented. Mean ACE levels were lowest amongst II homozygotes, intermediate for ID heterozygotes, and highest amongst DD homozygotes (47). Further studies have shown that the T allele of the A-240T polymorphism and the G allele of the G2350A polymorphism were associated with an increase in circulating ACE concentration (48, 49).
ACE is a zinc metalloprotease that cleaves angiotensin I into the angiotensin II molecule which is a promoter molecule for vasoconstriction. Further, ACE is produced mainly by vascular endothelial cells (50), it being frequently reported that the vascular lesions that
typically arise amongst sufferers of Kawasaki disease are associated with endothelial-cell damage of, particularly, small and medium blood vessels the ACE level depression arising subsequent to blood-vessel endothelial-cell injury (51). Falcini et al. (1996) reported ACE values were significantly lower amongst active Kawasaki-disease sufferers than was the case for healthy children, it being suggested that the decreased ACE levels under such
circumstances may be linked to a diffuse vascular inflammatory process (52). Since we didn’t attempt to measure the ACE activity level amongst our test-group individuals, the relationship
between the ACE gene polymorphism and ACE levels was not able to be clearly elucidated.
For this case-controlled study, the ACE gene polymorphisms noted were
hypothesized to be associated with the coronary artery aneurysms associated with Kawasaki disease. Our results suggest no evidence of an association between ACE gene
polymorphisms and the formation of coronary-artery aneurysms. This result differs from that contained in the report of Takeuchi in which Kawasaki-disease patients also suffering
coronary-artery aneurysm reflected the presence of the genotype II ACE polymorphism more frequently than was the case for non-aneurysm sufferers amongst a Japanese
sub-population (53).
In our study, the distribution of the DD genotype, G2350G of the ACE gene polymorphism amongst Kawasaki-disease patients appeared to differ to that for the control group, there existing significant difference between both groups (Tables 2 & 4). In this study, the increased risk of Kawasaki disease associated with the A allele of the G2350A
polymorphism is also observed. According to the interpretation of Frossard (1998) and Zhu (2001) data, it would appear that expression of the DD genotype of the ACE gene and the G allele of the G2350A polymorphism were associated with an increase in ACE activity
(47,49).These fragments is located on an intron and thus cannot affect the expression of mRNA directly. It is hypothesized that the insertion/deletion and G2350A fragments are in
linkage disequilibrium with a still unknown DNA fragment that acts as a silencer fragment.
Our study group of Kawasaki-disease patients did reveal a lower level of the DD genotype and the G allele of the G2350A polymorphism than was the case for the control group, for which case, the ACE concentration should be lower than is the case for the control case. The ACE inhibition reduced free radical expression, however, its role in modulating the
inflammatory response has not been clearly defined. Alternatively, renin-angiotensin systems may influence KD pathogenesis via more global effects in other tissues. For example,
vascular wall might modulate the response to systemic inflammation. All such hypotheses are worthy of further study. Contrasting our patients suffering coronary aneurysm with those who did not, we do not appear to demonstrate any difference in distribution of the ACE-gene polymorphism between these two groups; such a result suggesting that ACE activity does not relate to severity of KD.
For all 107 Kawasaki-disease patients, the distribution of the I/D ACE genotype was II:0.28, I/D:0.66 and DD:0.05, such results being comparable with the results of Takeuchi (1997), who reported for 36 Japanese Kawasaki-disease patients, that the distribution of the I/D ACE genotype was II:0.42, I/D:0.50 and DD:0.08 (53). The allele frequencies were 0.61 for the I allele and 0.39 for the D allele amongst our Kawasaki-disease patients (Table 2), similar to corresponding levels of 0.66 for the I allele and 0.34 for the D allele amongst the Japanese sub-population as reported by Takeuchi in 1997 (53). If the Kawasaki-disease patient in Asia reflects a similar ACE gene polymorphism, then further, more-expansive studies would appear to be needed.
Studies of cytokines have been reported that proinflammatory cytokines such as IL -1, IL-4, IL-6 and TNF-a are elevated in the sera of KD patients (12, 13). These results suggest that cytokines play an important role in the onset of this disease. The inflammatory reaction produced by a number of factors, including the type of stimulus, the dose of stimulus, and
genetic characteristics of the host. The stimulus for the inflammatory reaction in children with KD remains unknown. Some cytokine polymorphisms might be related to KD.
Interleukin 1 belongs to a cytokine family modulating cellular proliferation and has the capacity to induce other cytokines. It is a primary mediator of the inflammatory response and has been shown to induce prostaglandin synthesis (54). The IL -1 genes are associated with several immunoinflammatory diseases (55). Interleukin I exists in 2 forms, IL-1α and IL-1β, which are encoded by distinct genes but share the same receptors and biological
properties( 56). The loci for IL -1α and IL-1β are located on the proximal region of the long arm of chromosome 2 ( 57). The IL -1β polymorphism has been correlated with IL -1β
expression( 58). Different polymorphisms have been described in the IL -1ß gene, and at least 2 of them could influence protein production : one located in the promoter region at position-511(IL-1β-511) and the other in exon 5(59, 60). The allele E2 of IL -1 ß exon 5 has been described to be associated with an IL -1ß high secretor gene.
The IL-1Ra is structurally related to IL -1 a and IL-1βand competes with these molecules for occupation of IL -1 cell surface receptors. The presence of the IL-1Ra allele II was associated with enhanced IL -1βproduction in vitro(61). The type II IL -1Ra allele has been previously found in association with a variety of autoimmune diseases: alopecia areata, lichen sclerosus, systemic lupus erythematosus, ulceractive colitis, and late-onset psoriasis.
These genes code several proteins that may be key components in the pathogenesis of KD.
In this study, we observed that the IL -1 Ra genotype I/II and II allele are associated with higher susceptibility to KD. These observations suggest that genes that contribute to
regulating the level of IL -1 production may be useful in predicting the occurrence of KD. The IL-1 Ra genotype and II allele may be involved in the formation of KD through a complex pathway. Such as change the pathway of signal transduction between cells and then change
inflammatory mechanism in human disease. IL -1 Ra binds to IL -1 receptor with an avidity equal to that of IL-1, so fails to stimulate the cells. In our KD patients, an imbalance exists in this system, because the relative levels of production of IL -1 Ra are not adequate to
effectively block the proinflammatory effects of IL -1.The IL-1ß promoter and IL -1ß exon 5 gene polymorphisms are not useful in predicting the susceptibility to KD. Previous report (55) showed that IL -1 genes may have a role in the severity of the disease rather than in
susceptibility to the disease itself. But in this study, we observe no association of IL -1 gene polymorphisms between patients suffering coronary aneurysm with those who did not.
IL-4 is a helper T cell type 2 cytokine involved in the promotion of humoral immunity.
The IL-4 -590T allele has been shown to be associated with an enhanced IL -4 activity as measured by IgE production in Jurkat cells. This suggests that a C- to- T exchange at position -590 enhances IL -4 production or activity by T cells. In the study by Hunt et al., the frequency of the T allele of the C/T polymorphism of the IL -4 gene was 14 % in the control subjects. In our study, we found that the frequency of the T allele was 77.7% in controls to be different to Hunt et al. The difference in the result may reflect several factors. There may be genuine geographical differences in the data sets, however, this is unlikely as all controls.
IL-4 intron 3 RP1 and RP2 alleles are located in a polymorphic region in the third intron and are characterized by 2 and 3 tandem repeats, respectively, of a 70-bp sequence (37). More recently, a third allele has been reported, including 4 tandem repeats, but we do not observe this rare allele in our population. Many of the polymorphisms have been related to different levels of cytokine production, but the functional incidence of the intron 3
polymorphism of the IL -4 gene is not known. It can be hypothesized that distinct numbers of variable number of tandem repeats (VNTR) copies may affect the transcriptional activity of the IL-4 gene. The human gene for IL-4 has been mapped to the q arm of chromosome 5, in a cluster of cytokine genes which code proteins that are important in the control of different
aspects of immune response (62). Our data shows that the C-T promoter and intron 3
RP1/RP2 polymorphism of the IL-4 gene do not confer protection against the development of KD and coronary aneurysm formation.
IL-6 is a multifunctional cytokine expressed in many tissues, including adipose tissue, skeletal muscle and hypothalamus. We examined the IL -6 gene promoter, G/C polymorphism at position -174 has been reported to be associated with an altered IL -6 promoter activity and with different plasma levels of IL -6 in healthy men (39). The recently described C-174G
promoter polymorphism of the IL -6 gene has been found to influence plasma IL -6 levels in patients with systemic-onset juvenile chronic arthritis (63) and in patients with primary
sjören’s syndrome. Although KD patients demonstrate a drastic increase in serum IL -6 during acute phase, there were no significant differences in the nucleotide sequence between the KD patients and normal control group.
The stimulus in KD for the increased serum levels of TNF-a remains unknown. The gene coding for TNF-a lies within the MHC on chromosome 6 (64). A number of genetic polymorphisms upstream from the coding sequence for TNF-a have been described. The presence of the A allele at the –308 site is associated with elevated TNF-a production in response to endotoxin in whole blood cell cultures (65). An increased frequency of the TNF-a-308 A allele has been associated with poor outcome after certain infections. We tested the hypothesis that children with KD may have a higher frequency of the A allele at the TNF-a-308 sites, which is associated with elevated serum levels of TNF-a after an inflammatory stimulus. The results of our study demonstrate that the TNF-a-308 gene A allele is not associated with KD and aneurysm formation.
Transforming growth factor-beta (TGF-β) is a multifunctional cytokine involved in many cellular processes, such as cell proliferation, embryonic development, tumorigenesis, wound healing, fibrosis, and immune and inflammatory cell responses (66,67). Its
immunoregulatory effects include the inhibition of immunocompetent cells and the regulation of cytokine production (66).
KD is an acute febrile illness and one of the most important forms of systemic vasculitis occurring in early children. Vasculitis associated with KD is characterized by initiation of a pro-inflammatory cytokine cascade that results in dramatic immune activation.
Matsubara et al. reported the decrease in the concentrations of TGF-β1 in the sera of patients with KD (68). The decrease in TGF-β1 may result in an increase in TNF-α, because TGF-β1 can suppress TNF-α production from macrophage/monocytes. The mechanisms leading to the decreased TGF-β1 levels remain unknown. We postulated the possible role of TGF-β1 to be involved in the pathogenesis of KD. In our case-controlled study, the TGF-β1 gene C-509T polymorphism, the frequencies of the T allele and TT genotype in the control population were 54 and 31 %, respectively- somewhat different from those in the Caucasian population (24 and 5 %, respectively) (69). The distribution of the C-509T polymorphism was studied in the KD and control groups, and studied in the patients suffering coronary aneurysm and those who did not. The results showed no evidence of any association of TGF-β1 gene C-509T with KD.
In conclusion, we have found that the DD genotype; G2350G of the ACE gene polymorphism and the G allele at G2350A are present at a significantly-lower frequency amongst patients suffering Kawasaki disease than is the case for controls .The I/II genotype and II allele of IL-1 Ra are associated with KD. These data illustrate that a different gene regulation of KD pathogenesis. It is highly unlikely that polymorphism alone represents the susceptibility gene for the development of disease. In addition, there appears to be no
significant association between ACE gene, IL -1 gene, IL-4 gene, IL-6 gene, TNF-a gene and TGF-ß1 polymorphisms and the potential for the formation of a coronary-artery aneurysm
formation for Kawasaki-disease sufferers.
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