passages: PMC-2709655-sec-17__text
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passages: Discussion
  Because Netherton syndrome exhibits allergic phenotype, it is reasonable to speculate that SPINK5, which is mutated in Netherton syndrome, may act as a candidate gene for asthma and other allergic diseases [9]. An extensive search for single nucleotide polymorphisms in the SPINK5 led to the identification of a number of SNPs, including six nonsynonymous SNPs in coding region that might perturb its immune function. Subsequent genotyping of three nonsynonymous SNPs, A1103G, G1156A and G1258A in two independent panels of British families showed a significant association between SNP G1258A (Glu420Lys) and atopy, atopic dermatitis, elevated serum IgE levels and asthma [10]. This association was confirmed in a large German population and two Japanese populations [16-18]. Kato et al analyzed eight SNPs in exon 13 and 14 of the SPINK5 including G1258A (Glu420Lys), and found a positive association of seven SNPs with atopic dermatitis in a Japanese study sample using a case-control study design [16]. Nishio et al surveyed five of six previously reported nonsynonymous SPINK5 SNPs in Japanese atopic families identified through asthmatic children or subjects with atopic dermatitis and found that SPINK5 was associated with development of atopic dermatitis but not asthma [17]. Kabesch M et al. analyzed G1258A (Glu420Lys) in a German population of school children, and found its association with asthma as well as a concomitant occurrence of asthma and atopic dermatitis [18]. However, two subsequent studies failed to replicate the original SPINK5 findings for allergic diseases [19,20]. Folster-Holst et al genotyped four nonsynonymous SNPs (Asp106Asn, Asn368Ser, Asp386Asn, and Glu420Lys), and detected no association between SPINK5 and atopic dermatitis in populations of Northern German origin [20]. Jongepier H et al. failed to detect any association between SPINK5 and asthma, atopic phenotypes and atopic dermatitis in a Dutch population [19]. These discordant findings probably reflect different genetic and environmental backgrounds in various populations. To determine whether nonsynonymous SNPs of the SPINK5 are involved in the pathogenesis of asthma in the Chinese Han population, we performed a case-control study by genotyping four nonsynonymous SNPs in the SPINK5. We did not detect any significant association between these nonsynonymous SNPs and asthma in our Chinese samples. With our sample size, we expected a power of at least 80% in detecting an effect of OR >=1.3 for each of these SNPs. Therefore, our failure to detect an association for these 4 SNPs was not due to the sample size. These results suggest that the polymorphisms in the coding region of the SPINK5 are unlikely to contribute to asthma risk in the Chinese Han population. However, because our patients were ascertained for asthma, we could not exclude a role of the coding SNPs of the SPINK5 in atopic dermatitis in our population.
  The variations in the regulatory sequences of genes may determine risks to common diseases by causing different levels of expression. Therefore, the identification and functional evaluation of polymorphisms in promoter region are of great value in understanding the genetic susceptibility to asthma. In order to determine the role of the SPINK5 promoter polymorphism in the pathogenesis of asthma, we genotyped a promoter polymorphism, -206G>A, in 422 asthma patients and 410 controls, and found a marginal association. The frequency of allele G was significantly higher in asthmatic patients than that in controls (p = 0.022). To confirm the association, additional 267 asthma patients and 301 controls newly recruited from the same hospital were genotyped, and the -206G>A polymorphism remained significantly associated with asthma (P = 0.001), even after Bonferroni correction(adjusted P = 0.01). To our knowledge, this is the first report of an association of -206G>A polymorphism with asthma. We further examined the potential functional role of this promoter polymorphism, and found that the G to A substitution at -206 generated a GATA-3 transcription factor binding site.
  Major transcription factors controlling Th1 and Th2 development, such as T-box transcription factor and GATA3, are possibly involved in asthma and atopic diseases. GATA-3, a transcription factor specifically expressed in T helper 2 (Th2) cells, plays a critical role in the differentiation of Th2 cells from uncommitted CD4+ lymphocytes. In addition, GATA-3 is essential for the expression of the cytokines IL-4, IL-5 and IL-13 that mediate allergic inflammation [23]. Our luciferase reporter assay confirmed that SNP -206G>A is associated with the transcriptional activity of SPINK5. The G allele was associated with decreased transcriptional activity of the SPINK5. The mechanism by which the -206G>A SNP affects SPINK5 expression may be explained by the potential differential transcription factor binding of GATA binding factor, since -206G>A is located at the core sequence of GATA binding factor binding site. Electrophoretic mobility shift assay confirmed that the A to G substitution at -206 significantly reduced the binding efficiency of nuclear proteins to this element. Our data suggest that loss of GATA transcription factor regulation with the -206G may decrease the SPINK5 expression and thereby potentially perturb the immunosuppressive function of LEKTI.
  