Title: Development and validation of a multiplex-pcr assay for X-linked intellectual disability

Transcription

1 Author's response to reviews Title: Development and validation of a multiplex-pcr assay for X-linked intellectual disability Authors: PAULA JORGE (paula.jorge@insa.min-saude.pt) Bárbara Oliveira (barbara.oliveira@insa.min-saude.pt) Isabel Marques (isabel.medeiros.marques@gmail.com) Rosário Santos (mrn.santos@gmail.com) Version: 3 Date: 7 December 2012 Author's response to reviews: see over

2 To the Executive Editor BMC Medical Genetics RE: Revised manuscript Dear Dr. Tim Sands, We have prepared a revised version of the manuscript with reference number , entitled Development and validation of a multiplex-pcr assay for X- linked intellectual disability. The reviewers comments were greatly appreciated and we have given a point-by-point cautious consideration, as indicated in the Response to the Reviewers. We have addressed the major and minor revisions raised by the reviewers and rewrote the paper accordingly. Based on their queries, the authors consider that the data are now more clearly presented in the manuscript, two Tables were added and the Figure was split in two and arranged to include sequencing data. We have also carried out some basic statistical analyses in order to answer the reviewers concerns. All authors have been advised and agree with this revised version of the manuscript. We trust the current version meets with your approval, with that of the reviewers as well as adhering to the journals formatting requirements. Waiting for you answer. Sincerely, Paula Jorge (Corresponding author) Address: UI&D-P Centro de Genética Médica Dr. Jacinto Magalhães, INSA I.P. Praça Pedro Nunes 88, Porto, Portugal phone: (+351) , Fax: (+351) jorgpaula@gmail.com;

3 Response to the Reviewers Referee #1 Reviewer: Ching-Cherng Tzeng General suggestions: 1. This multiples assay reported in this study could be consider as screening test for the subjects with XLMR or nonspecific intellectual disability (ID). Particularly useful is the inclusion of the hot mutation spots in the ARX gene. However, the whole manuscript needs to be reorganized and also has to provide more specific data, including Tables and Figures, that are valuable for the readers working in this field. The followings are my suggestions. 2. In BACKGROUNS, it could divided into 5 paragraphs to briefly summarize the current information we known in the fields of XLMR, FMR1, AFF2, and ARX and, lastly, the aims of this study. 3. In METHODS, it is necessary to provide a Table that summarize the repeat/size distribution of the 100 (confirmed by sequencing analysis) control samples. 4. In RESULTS, it has to focus on the screening results of the 5000 test samples of each gene and described each in separate paragraphs, along with appropriate a Table and Figures. Besides the favorable sides of this assay, it is also necessary to let the readers know clearly the potential limitation(s) and/or pitfall(s) of this assay. 5. In DISCUSSION, each/continuous paragraph(s) has to focus on a single gene, unless needed, such as using AFF2 as internal control for the amplification of the FMR1 gene. 6. In CONCLUSION, it should be concise and clearly describe the potential usefulness as well as the limitation and potential pitfalls of the assay. We have considered all of these points in the revised version, most of which are listed below in response to the referee s suggestions for major and minor revisions. We have also carried out some basic statistical analysis, added two Tables and former Figure 1 was split in two (Figures 1 and 2). Sequencing data was also included in the newest Figure 1. Overall, based on the very welcome suggestions, the authors consider that the data are now more clearly presented in the manuscript. Major Compulsory Revisions: 1. The ABSTRACT has to provide briefly the specific data that could let the readers easily judge the potential usefulness and the limitation of this new assay. These data include the repeat/size range of the three genes detected in the control group (n=100) in the 2nd paragraph (Methods) and in the testing group (n=5000) in the 3rd paragraph (Results). Lastly, in the 4th paragraph (Conclusions) should focus on the usefulness and the limitation of this new assay. The abstract was reformulated accordingly. 2. In Methods (2nd paragraph), the authors have to describe the repeat/size distribution of the three genes in the control group (n=100), or summarize into a Table. These samples had been confirmed by sequencing analysis. Based on these data, the readers could be able to judge the reliability of this multiplex PCR assay they reported. The comment of the reviewer is most pertinent and of particular importance. We have detailed the multiplex-pcr validation data in a new Table (Table 1). 1

4 3. RESULTS AND DISCUSSION (1st paragraph): Theoretically, due to the presence of stutter bands, the green signals of the PCR products for the expanded AFF2 allele and ARX allele may have chance(s) of overlapping. Have you ever seen this situation and how to handle these situations? No, there were no coincident overlap between the ARX 2p fragment and an AFF2 allele in any of the samples analyzed. We recognize that this overlap is likely and following what was pinpointed by the reviewer we have included a proposal of improvement for the multiplex-pcr assay; use of NED labelling instead of HEX in either genes (Results and Discussion, final paragraph). As previously mentioned in the manuscript, any anomalous result should be verified by another methodology. A simplex amplification of both the ARX and AFF2 fragments would allow a straightforward answer to an overlapping situation. 4. RESULTS AND DISCUSSION (1st paragraph): Preferential amplification is a common and tough problem in the amplification of the genes rich in CG repeats. It is critical important in screening the subjects who have a normal allele and also an allele with much expanded CG-repeats. The readers working in this field should be very interested to know if this assay could reliably resolve this problem, particularly the unexpected identifying a mosaic male who carries 51 and ~350 repeats. It would be better to show the results detected by this assay and also the Southern blot hybridization. The authors are pleased that the reviewer showed an interest in this case. What we meant was that the multiplex-pcr assay identified an unexpected intermediate-size allele in a sample that was archived as a full mutation carrier. We decided to carefully relook to the Southern blot films despite being obtained more than 16 years ago: no intermediate band was present. The Southern blot (performed with an FMR1 specific 32 P-labelled probe) may not have had sufficient sensitivity to pick up low levels of mosaicism or the expected intermediate band could have become imperceptible to the naked eye. We have reformulated this part of the manuscript and we believe that data concerning this mosaic case is now properly transmitted. The multiplex-pcr result obtained in this mosaic was added in Figure 1 (a). 5. RESULTS AND DISCUSSION (1st paragraph): Based on this assay, the authors could retrospectively detected 2 (or 3, mistyping?) males with small premutation FMR1 alleles (58 and 61 CGG-repeats). These premutation could not be easily detected in their Southern blot assay using autoradiographic film. However, in our hand, it is not a problem to detect all allele larger than 54 CGG-repeats by using a high-resolution, non-isotopic Southern blot assay. Notably, Southern blot assay still has it unique role in this testing that could not be reliably replaced by other assay thus far. As a screening tool to detect alleles with FMR1 mutated, Southern blot assay has rarely been used for males. It is just used for confirmation of those showing ambiguous or fail results in PCR assay. Importantly, it is still often used in many laboratories to screen female samples. It could reliably distinguish those who carry two normal alleles but identical in repeat size from those having a large premutation or full mutation that may not be reliably detected by PCR approaches. (in response to CONCLUSION, 3rd paragraph) The authors admit that the Discussion and Conclusions may be confusing and incomplete, so these sections were completely rearranged and rewritten. As shown in the newly added Figure 2 (b), one of the small premutations picked-up by this multiplex- PCR assay (female heterozygous for 47 and 61 CGG alleles) falls within the generally accepted empirical Southern blot limitation (~30bp discrimination). 6 CONCLUSION: The section has to describe concisely and specifically the findings (and limitations) found in this study and do not redundantly repeat those that have been mentioned in the above sections. 7. CONCLUSION (1st paragraph): It could be the component in the first paragraph of DISCSSION. 2

5 8. CONCLUSION (2nd paragraph): It could be separated into three parts and moved to appropriate paragraphs of DISCSSION. 9. CONCLUSION (3rd paragraph): It has to delete many statements that have been mentioned already before, and to reorganize into a single paragraph CONCLUSON, specially focusing on the potential usefulness and limitations/pitfalls of this new assay. We appreciate these remarks and have tried to reconcile the suggestions of all reviewers. The statements have been redistributed accordingly. An effort has been made to avoid redundancy and the conclusion is now more concise. Minor Essential Revisions: 1. (...) For the reference No. 1, up to the first 30 authors should be listed before adding 'et al.'. This reference was changed accordingly. Discretionary Revisions: 1. (...) A wider space between each paragraph has to be uniformly applied in the whole manuscript, particularly for the Journal with a format that requests no indentation at the beginning of each paragraph. 1. (...)According the the format of this Journal, there is no indentation at the beginning of each paragraph. Please remove these indentations in the following paragraphs, including BACKGROUND (2nd), METHODS (3rd), RESULTS AND DISCUSSION (1st and 2nd), and CONCLUSION (1st, 2nd, and 3rd). The format was changed accordingly. 3

6 Referee #2 Reviewer: Laia Rodriguez-Revenga General comment: (...) Although the methodology proposed is new and might be worth it to publish there are few issues that should be considered before considering its publication. We appreciate the reviewer s suggestions and have rewritten some of the manuscript sections accordingly. Major Compulsory Revisions: 1. The authors state that FMR1, AFF2, and ARX are three of the most common genetic causes involved in X-linked intellectual disability. The AFF2 gene is the cause of nonsyndromic X-linked intellectual disability (XLID) in 1 of 50,000 newborn males, and therefore it can not be considered as one of the most common genetic cause of ID. There authors should modify this statement. The statements were rewritten in the Background sections in order to reconcile all reviewers suggestions. 2. The Material and Methods section should be rewritten since it is not fully clear. Based on the results section a co-amplification of two polymorphic regions from the X-chromosome is also performed in the same reaction. This is not mentioned in the material and methods section. Moreover, the authors mention that a subgroup of 100 samples, with known FMR1 CGG allele sizes, was used to validate the technology. What are the allele sizes? Which is the highest CGG repeat number the technique is capable of amplify? Furthermore, among the DNA samples tested, how many were male samples and how many females? Were they previously tested for the AFF2 expansion and for the ARX duplication? The authors believe that this new version of the manuscript embraces the suggestions and issues raised by all reviewers. We recognize that a basic statistical analysis was missing and results data was not efficiently transmitted in the text. We have modified the Results section considerably and to a lesser extent also the Methods section. The (...) a co-amplification of two polymorphic regions (...) was not clear, and was therefore misinterpreted. It was meant to highlight the advantage of the multiplex-pcr assay with respect to fortuitous detection of sex-chromosome anomalies. A new Figure was prepared exemplifying what is stated (Figure 2, panels e and f). Moreover, the authors mention that a subgroup of 100 samples, with known FMR1 CGG allele sizes, was used to validate the technology. What are the allele sizes? Which is the highest CGG repeat number the technique is capable of amplify? Two Tables were added: one with results obtained in the 100 samples (validation group) and another with the allele sizes of FMR1 and AFF2 repetitive regions, and ARX ex2p variants obtained in the test group (5000 samples). Concerning the question: (...)Were they previously tested for the AFF2 expansion and for the ARX duplication? (...) No, we selected 5000 gdna samples formerly tested by Southern blot for an FMR1 gene expansion (Fragile-X testing). Those samples were previously neither tested for AFF2 nor ARX genes. 4

7 3. The results section, as for the FMR1 gene, three premutation alleles were identified, however the authors only provide alleles sizes for two. Are they considering the intermediate allele as permutated? In such a case it should be corrected since it can not be considered as a premutation allele. The authors mention an 8% increase of sensitivity with this PCR multiplex methodology compared with the Southern Blotting. How was this 8% calculated? Are they also considering the identification of the full mutated allele? Was this full mutated allele detected by this multiplex methodology? If so, can they include it in the image as an example? There are in fact only two premutated alleles; we thank the reviewer for pointing this out. No full mutation was detected with this assay, neither was this an objective for this multiplex-pcr. 4. In the results section, it is mentioned that a Klinefelter case was detected with a full-methylated FMR1 allele. How was this allele detected by the multiplex-pcr methodology? Is this technology sensitive for methylation? If so, this is not mentioned in the material and methods section. This methodology has no aim to detect the methylation status of FMR1 gene. Reference to the Klinefelter case in the Results and Discussion section is now more explanative. Following the reviewers comment a new Figure was prepared in order to include examples of those results (Figure 2, panels e and f). 5. Figure 1 shows profiles for control samples. It should be stated in the Figure legend. Moreover, it would be of interest to provide images showing the profiles of the samples they are reporting or a table gathering all the remarkable findings. We admit that the term control samples could be misleading, as it was meant to designate samples used to validate the multiplex-pcr assay, and not necessarily normal controls. Accordingly, these samples designation was changed to validation group. A Table describing significant results obtained in the 5000 samples (Test Group) was included (Table 2). A new Figure (Figure 2) was prepared focusing relevant multiplex-pcr assay results of the mutational hotspot of FMR1-CGG alleles, AFF2-CCG alleles and of the ARX ex2p in the test group. 6. How do they discriminate between a female carrying an homozygous FMR1 normal CGG allele and a heterozygous female with one FMR1 expanded allele and a normal CGG allele? This is one of the limitations of this multiplex-pcr methodology, further discussed in the Results and Discussion section. Which is the percentage of cases that do not amplify and need to be further characterized by Southern Blotting? We recognize that these figures were not efficiently transmitted in the results. We have reformulated this part of the manuscript in order to include some statistical data. We carefully reviewed all the records and present new data organized by gene and separated by males and females, in order to avoid further confusion. Minor Essential Revisions: 1. Mental retardation should be replaced by intellectual disability The term mental retardation was replaced. 2. In the conclusions the authors describe that this methodology is useful to diagnose fraxopathies. What do they understand for fraxopathies? It should be described in the paper. The word fraxopathies intend to encompass the fragile X syndrome as well as the other genetic disorders concerning the FMR1 gene (according to pubmed the first publication using this term is from Filomena Pirozi, AJMG, 2011). This term has been removed in the current revised version of the manuscript. 5

8 Referee #3 Reviewer: denise M christofolini General comment: The manuscript brings us a new methodology to view an old problem, the identification of the genetic cause of intellectual disability. Techniques as conventional PCR and Southern blot now can be replaced by more modern and precise ones based on modern molecular biology. However, this methodology has already been used for the identification of FMR1 mutation in isolate. And some details must be stated. The authors are grateful to the reviewer for the positive feedback. We appreciate the reviewer s questions and have rewritten some of the manuscript sections accordingly. -Minor Essential revisions Background (...) X-linked intellectual disability (XLID) represents a common cause of monogenic mental retardation. The affirmation is not correct. XLID are a group of different diseases, caused by mutations in various genes located at X chromosome, as described in the conclusion. The way it is written it seems that is only one gene that is responsible for all the diseases. Rewrite de sentence. The sentence was rewritten in both of the Background sections in order to accommodate suggestions raised by all the reviewers. Methods Determine how many samples from boys and from girls were tested. Data of samples and of the results are now presented in more detail, including gender. Discussion Authors should discuss about the limitations of the methodology described. The authors conciliated the suggestions of all reviewers and rearranged the conclusion significantly; limitations of this methodology were explained with respect to each gene, separately. 6