Variants of inflammatory mediators: alpha-1-protease inhibitors, cortisol binding globulin, interleukin-1-receptor antagonist and beta-2-adrenergic receptor genes in atopic asthmatic and non-asthmatic South Africans.

Pillay, Visva
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According to current concepts asthma is primarily an inflammatory condition of the bronchi which results from the complex interactions between heterogenous genetic and environmental factors. Although the environmental allergens are fairly well known, little information concerning the genetic differences between atopic and non-atopic individuals is available, A number of candidate genes have been proposed, including genes for protease inhibitors, interleukins and the beta-2-adrenergic receptor ((32AR) The present study was undertaken in order to determine whether molecular variation of certain atopy candidate genes (alpha-1-protease inhibitor, cortisol binding globulin, interleukin-1- receptor antagonist and p2AR) may be associated with atopic asthma in black and white asthmatic South Africans. Alpha-1-protease inhibitor (ctjPI) has been implicated in the pathogenesis of emphysema, if it is present in one of its deficient phenotypic forms. Given that a ^ I is also an acute phase reactant in humans, it is possible that an association exists between the manifestations of asthma and a,PI or its deficiency. This investigation looked at the various phenotypes of cqPI in black and white asthmatic and control individuals by making use of isoelectric focusing as well as to make use of the polymerase chain reaction (PCR) which allowed for the identification of two haplotypes of the Ml phenotype ipf a,PI,viz. Ml (Ala213) and MI (Val213), There was a significant increase in the M l (Ala213) haplotype in the black groups as compared to a white groups. A novel finding was the identification of a new variant, the M1E(j0HANNESBURG)_ A significant difrerence was also found when comparing patients with severe asthma who had the rarer variants of a,PI as compared to mild or moderate asthmatic patients with the M1M1 phenotype indicating that UjPI plays a role in the pathogenesis of asthma.No mutation was found in exon 2 (an amino acid substitution in this exon was shown to be responsible for abnormal CBG steroid binding activity) of the cortisol binding globulin (CBG), (as determined by sequencing analysis) in black and white asthmatic and control individuals in the present study. However, taking the fact that CBG and oqPI share more than 40% homology of amino acid sequence, it would be advisable to continue the search for possible mutations in other exons of the gene which might act as markers for mutations in other genes that are closely linked to the CBG and which play a role in asthma.An important role player in the control of the inflammatory process could be the IL-1 Ra since it is a powerful endogenous anti-inflammatory molecule that competitively inhibits IL-1 a and IL-ip. The allelic frequency of the polymorphism in intron 2 of the IL-1 Ra gene was studied in black and white patients with asthma and control individuals, The plasma IL1 Ra concentration was also determined using a standardised Elisa kit. No significant differences in IL-1 Ra VNTR allelic frequencies were noted in the clinical groups and controls in each of the two population groups, However the 410 bp allele was increased in all black subjects as compared to all white subjects while the 240 bp allele was markedly reduced in all black subjects as compared to all white subjects. Significant differences were observed when we compared the levels of severe patients with patients classified as having mild astlima. Significant differences were also observed when comparing moderate asthmatic children with the mild asthmatic children. Our results indicate, a distinct racial difference in the EL-1 Ra gene polymorphism and although this polymorphism is unlikely to be an important determinant of overall disease susceptibility in asthma the IL-1 Ra plasma concentrations could act as a marker of disease severity in asthmatic patients.The p2 adrenergic receptor (p2AI<) is an important factor in the control of the inflammatory process in asthma. The gly 16 polymorphism of the (32AR which appears to impart enhanced down regulation of receptor numbers has been found to have a higher prevalence in nocturnal white asthmatics. The allelic frequencies of the gly 16 polymorphism was studied in black and white asthmatic children control individuals. Genotyping was performed by making use of PGR and the presence of the mutation was analysed on agarose gels using ethidium bromide staining and confirmed by DNA sequencing. There was no difference in the prevalence of the gly 16 polymorphism of the (32AR between the black and white control individuals. There was a significant increase in the frequency ofthe gly 16 polymorphism of the P2AR between severe and moderate asthmatics. There was also a significant increase in the prevalence of the gly 16 polymorphism in those patients who required a long acting beta stimulant to gain symptomatic control, We did not find a difference- in the prevalence of the gly 16 polymorphism between nocturnal vs. non-nocturnal asthmatics. The gly 16 polymorphism of p2AR was predictive of more severe asthma in this study. It was also predictive for those patients who needed a long acting beta stimulant to attain symptomatic control. This polymorphism may act as a disease modifier in asthma and represents one of the many genetic variables involved in the pathogenesis of asthma. In conclusion, the present study demonstrates the extent and complexity of genetic susceptibility to atopic asthma and it highlights the need for more refined association and functional studies that will identify additional atopy'loci and their association with asthma. Part of the work in this dissertation was the subject of conference p resentations. Pillay V, Gaillard M.C., Dewar J.B, Hirsch G.P and Song E, “Differences in the Interleukin-1 Receptor Antagonist (IL-IRa) Gene Polymorphism In Black And White Patients With Asthma Or Rheumatoid Arthritis And Control Individuals.” 37"' Annual Congress of the Federation of South African Societies of Pathology. Sea Point, Cape Town. 29 June - 2 July 1997. Pillay V, Gaillard M.C, Halkas A and Song E. “The GLY 16 Polymorphism of the P2 Adrenergic Receptor in South African Asthmatic Children.” Biochemistry In Africa: 2nd Of) FASBMB and 15,h SASBMB. Potchefstroom, South Africa. 29 September - 3 October 1998.