Title:The effect of CD14 and TLR4 gene polimorphisms on asthma phenotypes in adult Turkish asthma patients: a genetic study

Transcription

1 Author's response to reviews Title:The effect of CD14 and TLR4 gene polimorphisms on asthma phenotypes in adult Turkish asthma patients: a genetic study Authors: Fusun Sahin (fusunsahin19700@hotmail.com) Pinar Yildiz (pinary70@yahoo.com) Aysegul Kuskucu (aysegul.kuskucu@yeditepe.edu.tr) Mert Ahmet Kuskucu (kuskucum@gmail.com) Nilgun Karaca (karaca.nilgun@gmail.com) Kenan Midilli (kmidilli@gmail.com) Version:3Date:18 November 2013 Author's response to reviews: see over

2 REVISION LETTER: Reviewer: Kamel Hamzaoui 1-TLR4 and CD14 polymorphism were studied. Because: Numerous studies have implicated TLR2, TLR4 and TLR9 in the pathogenesis of asthma or atopy [7,8,9]. The TLR4 single polymorphisms nucleotide A896G and C1196T (result in the amino acid substitutions. Asp299Gly and Thr399Ile, respectively) and affect the extracellular domain of the TLR4 receptor [7,11]. These two polymorphisms modify the receptor s response to endotoxin, which is an important trigger of asthma [7]. Similarly, the CD14 polymorphism C-159T has been shown to influence serum IgE levels and skin prick test responses to allergens [7,12]. I added these sentences to INTRODUCTION in the revised manuscript(red boldface). 2-I corrected title of article: The effect of CD14 and TLR4 gene polimorphisms on asthma phenotypes in adult Turkish asthma patients: a genetic study. 3-I indicated the methodology of our the Abstract with red boldface. Real time PCR (RT- PCR) were used for analysis. 4-I changed the presentation of the INTRODUCTION in the revised manuscript. 5- A power analysis was performed for each gene polymorphism (CD14-159, TLR4 299, TLR4 399) at the beginning of the study (to determine the necessary sample size). We determined the allele frequencies of all polymorphisms (CD14-159, TLR4 299, TLR4 399) according to the results of the studies from our region [13,14] and a meta-analysis (that included European, East Asian and Indian populations) [15]. The allele frequency of CD14 C- 159T was approximately 75% in the asthma group, 53% in the control group. The power analysis was performed according to these values. For the C-159T polymorphism, the required sample size was 74 for each group (Approximate Test). The allele frequencies for TLR4 299 and TLR4 399 were approximately 20% in the asthma group, 5% in the control group, an again the reguired sample size was determined to be 70 for each group (Approximate Test). The number of patients in our study met these criteria. I added these sentences to METHODS- Study Subjects in the revised manuscript with red boldface.

3 Power analyses are in following: Test for Equality of Proportions with Alternative 'not equal' CD P1 : 0,530 P2 : 0,750 Effect Size : 0,464 Approximate Test Effect Size : 0,220 Approximate Test , , , , , , , , , ,801 Total Sample Size = 148 Total Sample Size = 150 Test for Equality of Proportions with Alternative 'not equal' TLR4 P1 : 0,050 P2 : 0,200 Effect Size : 0,476 Approximate Test Effect Size : 0,150

4 Approximate Test , , , , , , , , , ,804 Total Sample Size = 140 Total Sample Size = All of the study subjects are Turkish Caucasian. IN ADDITION; The number of tables were changed as in folowing: * Table 5 was changed as Table 1. * Table 1 was changed as Table 2. * Table 2 was changed as Table 3. * Table 3 was changed as Table 4. * Table 4 was changed as Table 5. New table was added as Table 6-Logistic regression analysis for total IgE level >98 ku/l. I corrected the references in Table 5 ( Table 5 was changed as Table 1 ). *The numbers of the reference were changed after the first six reference in the revised manuscript. New refrences were added. These changes in the references were shown with the red color. *I corrected the grammer mistakes in our article (The corrected grammer mistakes were shown as strikethrough with red boldface).

5 REVISION LETTER Reviewer: Susanne Schulz Major Comments: 1-I gived clinical and demographic characteristic of the healthy controls as stated for asthma patients in Table 1 with red boldface. ( Table 1 was changed as Table 2 ). 2-A power analysis was performed for each gene polymorphism (CD14-159, TLR4 299, TLR4 399) at the beginning of the study (to determine the necessary sample size). We determined the allele frequencies of all polymorphisms (CD14-159, TLR4 299, TLR4 399) according to the results of the studies from our region [13,14] and a meta-analysis (that included European, East Asian and Indian populations) [15]. The allele frequency of CD14 C- 159T was approximately 75% in the asthma group, 53% in the control group. The power analysis was performed according to these values. For the C-159T polymorphism, the required sample size was 74 for each group (Approximate Test). The allele frequencies for TLR4 299 and TLR4 399 were approximately 20% in the asthma group, 5% in the control group, an again the reguired sample size was determined to be 70 for each group (Approximate Test). The number of patients in our study met these criteria. I added these sentences to METHODS- Study Subjects in the revised manuscript with red boldface. Power analyses are in following: Test for Equality of Proportions with Alternative 'not equal' CD P1 : 0,530 P2 : 0,750 Effect Size : 0,464 Approximate Test Effect Size : 0,220

6 Approximate Test , , , , , , , , , ,801 Total Sample Size = 148 Total Sample Size = 150 Test for Equality of Proportions with Alternative 'not equal' TLR4 P1 : 0,050 P2 : 0,200 Effect Size : 0,476 Approximate Test Effect Size : 0,150 Approximate Test , , , , , , , , , ,804 Total Sample Size = 140 Total Sample Size = 154

7 3-I included logistic regretion analysis (multivariate analysis) for IgE to RESULTS in the manuscript: To determine factors affecting total IgE levels, we performed a logistic regression analysis using the backward stepwise method for variable selection based on the likelihood ratio. Because the median IgE level in the asthmatic patients was 98 ku/l, this value was used as the threshold for calculations in the logistic regression analysis (Table 6). A significant correlation was found between IgE levels and allergic dermatitis, eosinophil counts, and skin prick test results. New table was added as Table 6-Logistic regression analysis for total IgE level >98 ku/l. 4-*We didn t use CT+TT. We used CC+CT (C allele) in Table 4 for statistical analysis. We analysed C allele for mild, moderate and severe asthmatics statistically. I showed these points in Table 4 with red boldface. p=0.048 should be p=0.049 in abstract as with Table 4. p=0.049 is shown in Table 4 as black boldface. ( Table 4 was changed as Table 5 ) *I included the allele characteristics (C allele CC+CT, T allele TT) in Table 3 and 4 ( Table 3 was changed as Table 4 and ( Table 4 was changed as Table 5 ); I added the CC+CT genotype in Table 2 ( Table 2 was changed as Table 3 ) with red boldface. * There was no significant difference regarding CD and TLR4 299, TLR4 399 genotype distributions comparing asthma and control groups by chi-square test (p>0.05). This was shown in Table 2 (Table 2 was changed as Table 3). C allele (CC+CT) had a tendency to low IgE levels (according to median log IgE:1.99), but there was no statistically significant difference in all asthma group (p=0.09, Figure 1A). There was a statistically significant difference between median log IgE levels (according to median log IgE:1.99) and C allele in atopics (p=0.04, Figure 1B). In all asthma patients, there was not a significant association between the severity of asthma and CC+TT and TT (p=0.10, Figure 2A). In atopy group, there was a significant association between the severity of asthma and CC+TT and TT (p=0.049, Figure 2B).

8 Minor Comments: 1-I corrected the grammer mistakes in our revised article (The corrected grammer mistakes were shown as strikethrough with red boldface). 2-I corrected figure 1B, 2A and 2B legends in FIGURE LEGENDS. 3-I corrected p value in the abstract it is stated, that the CC+CT genotype was more frequent in moderate-severe asthma group in atopics. p=0.048 should be p=0.049 in abstract as with Table 4. p=0.049 is shown in Table 4 as black boldface. 4-I included the reference numbers RefSNP (rs) of the SNPs to INTRODUCTION in the revised manuscript. TLR4 Asp299Gly (RefSNP ID: rs ); TLR4 Thr399Ile (RefSNP ID: rs ); CD C/T (RefSNP ID: rs ). IN ADDITION; The number of tables were changed as in folowing: * Table 5 was changed as Table 1. * Table 1 was changed as Table 2. * Table 2 was changed as Table 3. * Table 3 was changed as Table 4. * Table 4 was changed as Table 5. New table was added as Table 6-Logistic regression analysis for total IgE level >98 ku/l. I corrected the references in Table 5 with red boldface ( Table 5 was changed as Table 1 ). *The numbers of the reference were changed after the first six reference in the revised manuscript. New refrences were added. These changes in the references were shown with the red color.

9 REVISION LETTER Reviewer: Lei Wan Major Compulsory Revisions 1-I corrected the grammer mistakes in our revised article (The corrected grammer mistakes were shown as strikethrough with red boldface). 2-A power analysis was performed for each gene polymorphism (CD14-159, TLR4 299, TLR4 399) at the beginning of the study (to determine the necessary sample size). We determined the allele frequencies of all polymorphisms (CD14-159, TLR4 299, TLR4 399) according to the results of the studies from our region [13,14] and a meta-analysis (that included European, East Asian and Indian populations) [15]. The allele frequency of CD14 C- 159T was approximately 75% in the asthma group, 53% in the control group. The power analysis was performed according to these values. For the C-159T polymorphism, the required sample size was 74 for each group (Approximate Test). The allele frequencies for TLR4 299 and TLR4 399 were approximately 20% in the asthma group, 5% in the control group, an again the reguired sample size was determined to be 70 for each group (Approximate Test). The number of patients in our study met these criteria. I added these sentences to METHODS- Study Subjects in the revised manuscript with red boldface. Power analyses are in following: Test for Equality of Proportions with Alternative 'not equal' CD P1 : 0,530 P2 : 0,750 Effect Size : 0,464 Approximate Test Effect Size : 0,220

10 Approximate Test , , , , , , , , , ,801 Total Sample Size = 148 Total Sample Size = 150 Test for Equality of Proportions with Alternative 'not equal' TLR4 P1 : 0,050 P2 : 0,200 Effect Size : 0,476 Approximate Test Effect Size : 0,150 Approximate Test , , , , , , , , , ,804 Total Sample Size = 140 Total Sample Size = 154

11 3-Since the CD14 polymorphism is located in the promoter region, will the polymorphism affect the promoter activity? The CD14 gene is present in the major susceptibility region (5q31-33) for atopy and asthma [17,18]. Studies investigating the relationship between the CD14 gene and atopy and asthma have revealed conflicting results. First, Baldini et al. [12] described several single nucleotide polymorphisms in the CD14 gene, which is a receptor with high affinity to for LPS. The polymorphisms described were found to affect the binding affinity for Sp1, -2, and -3 transcription factors at the GC box in vitro, thus altering the expression of CD14. They also detected a C-T substitution in the promoter region of the gene (the CD14 C-159T polymorphism) and higher CD14 levels and low skin test sensitivity in TT homozygotes [12]. The CD14 C-159T polymorphism involves a C T nucleotide substitution, located -260 bp from the translation start site and -159 bp from the transcription start site, which alters CD14 promoter activity in vitro by decreasing the affinity of Sp protein binding and thus enhancing transcriptional activity [19]. As a result, the CD14 C-159T promoter polymorphism was associated with serum CD14 levels [12,20] as well as the phenotypes of patients with allergy [18,21]. I added these sentences to DISCUSSION in the manuscript with red boldface. IN ADDITION; The number of tables were changed as in folowing: * Table 5 was changed as Table 1. * Table 1 was changed as Table 2. * Table 2 was changed as Table 3. * Table 3 was changed as Table 4. * Table 4 was changed as Table 5. New table was added as Table 6-Logistic regression analysis for total IgE level >98 ku/l. I corrected the references in Table 5 ( Table 5 was changed as Table 1 ). *The numbers of the reference were changed after the first six reference in the revised manuscript. New refrences were added. These changes in the references were shown with the red color.