Dear Editor,
Sub: Re: 1522406254834089 - Investigating highly replicated asthma genes as candidate
genes for allergic rhinitis.
We have looked at the comments from the reviewers and appreciate their acknowledgment of
us addressing their comments/concerns in the 1st Review. We also would like to thank them
for this round of review and have given below a point-to-point response of their
comments/concerns from the 2nd Review.
Reviewer 1
Reviewer's report
Title: Investigating highly replicated asthma genes as candidate genes for allergic rhinitis
Version: 3 Date: 12 February 2013
Reviewer: IGNACIO DAVILA
Reviewer's report:
Comments and changes from authors answer questions and recommendations.
Nevertheless, there is still a concern: as 30% of patients had asthma, there is a risk of biasing
the final results. I recommend performing a statistical analysis of AR patients without asthma
in order to check the appropriateness of the results
Authors’ response: We appreciate the constructive comment and have performed the
suggested analysis. The results of this analysis are included in Table E3 comparing the results
before and after the elimination of AR patients with asthma. The results clearly indicate that
asthma do not contribute to the significant results detected in the present study. We have
however added a paragraph in the results section and short comments also in the discussion
and the abstract. This would enable the reader to assess if there is a bias due to the asthma.
Reviewer 2
Reviewer's report
Title: Investigating highly replicated asthma genes as candidate genes for allergic rhinitis
Version: 3 Date: 4 March 2013
Reviewer: Giovanni Malerba
Reviewer's report:
The authors responded to the best of their ability all the questions indicated. However, a
response underlines the serious shortcomings of the paper and the knowledge of association
studies. In fact, the authors explain the fact that you have used different markers because the
study is based on the gene. This may be true when conducting linkage studies but not
association studies (unless you have conducted a detailed study on the structure of linkage
disequilibrium of the gene). If it were true what they say, then, in the case of association, all
SNPs should show association, and we know that is not true. We could invoke the allelic
heterogeneity, but we have no information about what the SNP worthy of being studied.
However this has no impact on the final results (but in the final considerations) in the sense
that finally the authors studied two AR samples with different SNP reporting the results of
SNP (this is a limitation of the study).
Authors’ response: We do not understand the remarks made above. We have discussed and
commented all previous comments (Major, Discretionary and Minor) made by this reviewer
(see below in italics). We have also decided to include a test for eventual confounding due to
the presence of asthma among the patients. This includes the addition of Table E3 and
paragraphs in the results, discussion and abstract sections of the manuscript.
In addition, we would like to point out that both SNP collections used in this study are sets of
tagSNPs covering the complement of genes studied in a way appropriate for this kind of
study. In our opinion, it is not at all unreasonable to use the gene level for investigating the
eventual associations between genetic variation in the investigated genes and AR since: 1) We
analyze populations with different ethnicities and allele frequencies, and 2) we search for AR
associations using genes previously associated with asthma. Thus, this is no attempt at a bona
fide replication of associated SNPs, but is rather a straightforward strategy for leveraging the
wealth of information about asthma-associated genes in an AR association study. We use the
resulting information to highlight genes, not SNPs, for further evaluation concerning their
involvement also in the development and severity of the AR phenotype.
Authors’ response for Review 1 from this reviewer.
Major Compulsory Revisions
1) The study sounds a little confusing. Authors tested two populations with different SNPs
hoping to get the same results because the SNPs fall on the same genes. In addition different
allergens were considered when testing the sensitization to various allergens in the 2
populations. This seems a matter of convenience rather than a choice imposed by the study
design (eg choice of the most common allergens, seasonal or perennial, for different
populations).
Authors should discuss these points and rearrange the paper according to their observation.
Authors’ response: Since this study investigates asthma genes for their eventual contribution
also to the AR phenotype and since there are inherent differences in the genetic architecture
between the two populations, we use the gene as the replication unit and not the individual
SNPs. The reason for investigating the response to these specific allergens is because they are
the most common allergens in the respective population. Our study aim to investigate a set of
highly replicated asthma genes for their possible involvement in AR. In doing so, we have
analyzed one typical Swedish population with AR as it appears in Sweden and one typical
Chinese population with AR as it appears in Singapore. The study investigates two different
populations, which differ both with respect to ethnicity and with respect to the allergens that
are operative. We have tried to clarify this in several parts of the manuscript.
2) Population sample and asthma
Authors performed a candidate gene study for AR by selecting a list of gene been previously
associated with asthma. What is the prevalence of asthma of the 2 samples? Authors should
discuss this issue.
Authors’ response: In the two materials used in this study, none of the patients suffer from
severe asthma and less than 10% have moderate asthma symptoms with continuous
medication. About 30% of the patients experience seasonal asthma symptoms (allergic
asthma), which in some cases requires periodic asthma medication. In our opinion, this will
not affect the results in this study since the absolute majority of patients with asthma
symptoms have asthma that is conditioned by allergic rhinitis. In our AR populations with
100% allergic rhinitis cases, it would be very unlikely that an observed association would be
due to asthma given the rather low prevalence of 10% in the populations. We have added a
comment on this issue in the materials and methods section.
3) SNPs of the studies
Of the 192 (Swedish pop ) and 429 SNPs (Chinese pop) , how many SNPs are shared by the 2
studies? Authors should discuss about their confidence in finding common associations
(between the 2 studies) even if testing different SNPs (eg: due to different allele freq in the 2
pops (?))
Authors’ response: We use the gene as the replication unit and not the individual SNPs, since
this study investigates asthma genes for their eventual contribution also to the AR phenotype
and since there are large allele frequency differences between the two populations. Both sets
of SNPs represent the genetic variation of these genes in the respective population.
4) Study of rs324981 in a third sample group (?)
Authors state that “As a proof of concept, we investigated the functional NPSR1 coding
variant …..” I don’t understand why they studied the marker rs324981 and which sample was
tested. Authors should describe in more detail this part of the study and clarify it.
Authors’ response: We have tried to clarify this in the result section of the manuscript.
Minor
Statistical genetic analysis
In the 2 samples, for each SNP two tests of association were computed (by allele and by
genotype). Why did the authors use 2 different tests (doubling the number of statistical tests
performed) Authors should comment this issue.
Authors’ response: It is customary to perform both allele and genotype tests in this type of
study.
Discretionary Revisions
I am not sure that Kruskal-Wallis rank sum test is the most appropriate choice to test the
association between genotype and degree of sensitization since it treats the “degree of
sensitization” as categorical variable whereas it should be treated as ordinal variable.
Authors’ response: The Kruskal-Wallis test is a standard non-parametric test that is intended
to be an alternative to the regular ANOVA, i.e. the F-test of the means of multiple groups for
a quantitative variable. As is well known, the F-distribution is derived under the assumption
that the quantitative variable is normally distributed within each group (category). The KW
test uses ranks of the observed variable and uses these to test the hypothesis. By doing this,
the dependence of normality is removed. The cost is a loss of power, if the original variable
was normally distributed. It should be pointed out that the loss of power is relatively limited.
The KW test is thus often used as a conservative alternative in the situation encountered here.
We have addressed the comments and suggestions raised by the reviewer to the best of our
abilities. In addition to the amendments as mentioned above, we have also edited the
manuscript for clarity. We are thus resubmitting the manuscript for your consideration and
hope for acceptance and publishing soon.
Yours sincerely,
Dr. Chew Fook Tim, Associate Professor,
Department of Biological Sciences, Science Drive 4, Singapore 117543, Faculty of Science,
National University of Singapore; Phone: +65-65161685; Email: dbscft@nus.edu.sg;
Dr. Lars Olaf Cardell, Professor and Head of Division,
Division of ENT Diseases, CLINTEC, Karolinska Institutet, Huddinge, SE-141 57
Stockholm, Sweden, Phone: +46 8 58581453, Fax: +46 8 4474907,
Email: lars-olaf.cardell@ki.se