Title:MS in South Asians in England: early disease onset and novel pattern of myelin autoimmunity

Transcription

1 Author's response to reviews Title:MS in South Asians in England: early disease onset and novel pattern of myelin autoimmunity Authors: Richard S Nicholas (r.nicholas@imperial.ac.uk) Vassiliki Koastadima (vkostadima@yahoo.com) Maya Hanspal (mah83@cam.ac.uk) Bejamin R Wakerley (benwakerley@fastmail.fm) Ruhena Sergeant (Ruhena.Sergeant@imperial.nhs.uk) Saskia decuypere (sdecuypere@ichr.uwa.edu.au) Omar Malik (malikomar@hotmail.com) Rosemary J Boyton (r.boyton@ic.ac.uk) Daniel M Altmann (d.altmann@ic.ac.uk) Version:4Date:14 April 2015 Author's response to reviews: see over

2 13 April, 2015 Dear Prof. Smiljana Risti Re. MS: MS in South Asians in England Thank you for sending referee comments on the above manuscript. We here summarise the author responses to these reviews. We agree that with these changes the manuscript is considerably strengthened, and hope that you will now find it acceptable for publication. Referee 1. The paper is clear; the findings are novel and important. No revisions are needed Response: We thank this referee for the enthusiastic comments. Referee 2. We thank referee 2 for detailed appraisal of the manuscript and hope that our responses and amendments have yielded a much stronger, clearer manuscript. Major Compulsory Revisions 1) I have clearly problems to understand the recruiting strategy of this study. There is no clear statement in the methods. Were all patients included that were treated with a first episode that were treated within a certain period at the same clinic? If not, why is there no matching for age (the Caucasian MS patients being significantly older?) Or were there other selection criteria to chose these patients in this study? Response 1. There were no additional selection criteria beyond the description as we had documented in the Methods; that is, during the period of study, patients were recruited sequentially who were attending clinic who agreed to participate and who were either of South Asian or Caucasian origin. We have now expanded this description as follows: Study Population Ethical approval was obtained from the Thames Valley Multicentre Ethics Committee (05/MRE12/8). Clinical data included: age, sex, disease onset, family history, clinical course, clinical status, first and present symptoms and signs, treatment and Extended Disability Severity Scores (EDSS); using this data the MS severity score (MSSS) was calculated (Table 1). Diagnosis was based on McDonald criteria [22]. Patients of S. Asian origin were asked ethnicity, place of birth, age of immigration to UK, previous country of residence and grandparents language; patients confirmed that both parental

3 origins were from S. Asia. Subjects were recruited for this study sequentially from those patients attending clinic who agreed to participate and who were either of South Asian or Caucasian origin. (The added text appears in the manuscript on page 5, lines ). 2) The authors do not discuss any possible confounding effects for the observed earlier onset of the disease in S.Asian vs. Caucasian patients. It might well be that the late-onset S.Asians are not observed because of different numbers of immigration (more immigrants in the 1970ies than in the 1950ies excluded?). It seems to me that this observation needs more consideration and that the conclusion is taken too fast that S.Asians have earlier MS in the UK. Response 2. We have modified the Discussion to more carefully annotate our conclusions about earlier onset in South Asians. The number of individuals in Greater London defining their ethnicity as Asian has indeed risen in sequential censuses, reflecting both immigration and the growth of these communities (for example, 13.2% of Greater London respondents in the 2001 Census compared to 18.4% in the 2011 Census). This referee suggests that a lack of late-onset MS seen in South Asian patients may reflect a skewing in the agedistribution in the South Asian community due to lower immigration figures in the mid-20 th century. If we have understood the critique correctly, a caveat is thus that onset may be skewed to an earlier age in the South Asian individuals due to a relatively smaller pool of elderly South Asians in the UK community in whom we could have picked up later onset disease? We have analysed the census demographics (see below) and agree that there is indeed a dissimilar age-spread in the UK population defining themselves as White British or Asian British, the latter encompassing Indian, Pakistani and Bangladeshi. However, please note that our figure relates simply to reported age at first diagnosis White British' Asian British' Figure 1. Percent of White British or Asian British UK 2009 population falling within the given age ranges. The Discussion has now been modified as follows at page 12, lines :

4 Is it possible, however, that a lack of late-onset MS seen in South Asian patients may reflect a skewing in the age-distribution in the South Asian community? Reference to U.K. demographic data relative to self-declared ethnicity (see for example yynfjb0k1cljvofe&hl=en#gid=0) indicates that there is indeed a relatively smaller pool of elderly South Asians in the UK community in whom we could have picked up later onset disease, the UK Asian population being somewhat more skewed to individuals under the age of 50 than the UK Caucasian population. However, note that our data relate simply to reported age at first diagnosis. 3) Is the effect in Fig. 2 really specific for myelin peptides? Have they done a control with a non-myelin peptide, e.g. tetanus toxoid or birch pollen. Can the authors exclude that some patients are more prone to IFN-g production than others? Did they check if e.g. proliferation showed a similar pattern? Response 3. While we have not done detailed epitope mapping for nonmyelin antigens, we now state in the Methods that responses to control antigen, purified protein derivative (PPD) of Mtb were not significantly different between groups and there is no evidence to support an intrinsic difference in frequency of IFNγ secreting CD4 T cells. We have added the following text to page 9, lines : We did not observe global differences in T cell immunity between the groups in assays against other antigens such as PPD. 4) I have serious concerns about the presentation and the statistical analysis of the T cell reactivity data as presented in Fig. 3 and 4. First, the graphical representation is not sufficient. It is hardly possible to recognize anything in these figures due to the size and the lack of a legend. Furthermore, the strength of a mean antigen response would be more informative than the percentage of claimed positive responses. It would to my understanding make more sense to show a stimulation index (x times baseline value). In addition, statistical analysis should be done over all epitopes. Response 4. We have redrawn the graphs to make them clearer changing the orientation and increasing the size. Figure legends were indeed present in the original manuscript, but we do hope that the referee will agree that we have altered the legend to Figures 3 considerably to make it more self-explanatory.

5 To give us coverage of what are relatively large myelin peptide panels with which to screen CD4 T cell responses using a relatively limited peripheral blood sample, we used the well-established approach of combining peptides into cocktails of 6 peptides and tested in triplicate, as has previously been described (Yonkers et al., 2006, J. Inf Dis. 194, 391). This approach yielded 30 cocktails, each individual peptide present in two separate groups with 5 other peptides. The 5 other peptides were either from the other molecule tested (MBP or MOG) and from separate areas of the same molecule. To have a positive response the peptide had to fulfil the response criteria in both groups that the peptide was present in thus increasing the certainty of a positive response. As each peptide was with different peptides it is not possible to assess the baseline level of activity, as it is determined in addition by the presence of the other peptides; thus we cannot derive a stimulation index, only the extent to which it is above its control. As a result of the rigor in determining a positive response we utilised similar statistical methods as used in Yonkers et al., 2006 [ref 23]. Each comparison utilised as a comparison the group as a whole. We did not correct for multiple testing as peptide sequences both overlap and are related to each other on the molecule and positive responses were derived when the peptides was analysed with peptides from different areas of the MBP and/or MOG molecule. Furthermore when using Chi-squared/Fisher s test in this context the appropriate correction is unclear from the literature as Bonferroni s correction is too conservative. As this was an exploratory work and we have described the raw results but have modified the discussion to take account of our methods of analysis. We have expanded the methods to address this issue and modified the discussion to describe the technique that was used in this work [ref 23]: Yonkers NL, Rodriguez B, Post AB, Asaad R, Jones L, Lederman MM, Lehmann PV, Anthony DD. HIV coinfection impairs CD28-mediated costimulation of hepatitis C virus-specific CD8 cells. J Infect Dis Aug 1;194(3): Epub 2006 Jun 30 The relevant Methods paragraph, at page 5, lines , now reads as follows: Pools of 6 peptides in triplicate were utilised, each peptide in two pools: To give us coverage of what are relatively large myelin peptide panels with which to screen CD4

6 T cell responses using a relatively limited peripheral blood sample, we used the wellestablished approach of combining peptides into cocktails of 6 peptides and tested in triplicate, as has previously been described [23]. This approach yielded 30 cocktails, each individual peptide present in two separate groups with 5 other peptides. The 5 other peptides were either from the other molecule tested (MBP or MOG) and from separate areas of the same molecule. To have a positive response the peptide had to fulfil the response criteria in both groups that the peptide was present in thus increasing the certainty of a positive response. PBMC responses to peptide were assessed by IFNγ ELISpot. Minor Essential Revisions 5) The discussion lacks the appraisal of epidemiologic data from the 1960ies/70ies concerning MS risk and migration. Here, several potential confounding factors have been described for e.g. younger age at onset. Response 5. We have now addressed this in more detail in the Discussion (page 12, lines ) Referee 3. The manuscript is complete. The data on the differences in adaptive immune T cell epitopes in the South Asian population are fascinating Level of interest: An article of outstanding merit and interest in its field. Response: We thank this referee for the enthusiastic comments. In summary, we have responded in detail to the referees comments and hope the manuscript may now be considered acceptable for publication. Yours sincerely Prof DM Altmann