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The authors have addressed the issues previously raised by the reviewer and the editor.
# PeerJ Staff Note - this decision was reviewed and approved by Pedro Silva, a PeerJ Section Editor covering this Section #
Please review and address the comments I made on the previous decision email from 10/15/2018.
The authors have greatly improved their manuscript as noted by Reviewer 2. However, Review 1 does raise some valid concerns which necessitates revising the manuscript further. There is definitely a difference between the detection rate of CMA (13%) and MLPA (4.35%). This would appear to be significant and would indicate some potential pathogenic variants could be missed solely using MLPA. Also, as pointed out, as more individuals are tested, particularly using CMA, more dups/dels will be identified. It will be difficult for MLPA probemixes to keep up. Lastly, as the authors indicated in their rebutted letter, MLPA may be better suited for laboratories or regions of the world where CMA usage may be prohibitive because of cost. This is not emphasized in the paper; rather the authors focus on the “comparability” of data for the two technologies when in fact they are not (13% is much greater than 4.35%).
Therefore, the authors should modify their part to better address these issues.
The authors have compared findings by CMA with those from selected probe mixes to detect clinically relevant variants by MLPA in 92 ASD patients and demonstrated that both methods accurately detect pathogenic variants, but CMA is about 3 times more likely to detect likely pathogenic variants (13% vs 4.35%). This may be significant, since use of MLPA probe mixes is necessarily limited to existing knowledge. As new knowledge grows the interpretation of pathogenic variants is likely to grow, as it did in this study regarding DOCK8. It is difficult for me to understand why a less-revealing study would be preferable. The speed with which results are delivered is not a significant advantage when there is a loss of precision. The use of English language is much improved and the literature review is adequate. Please use the term Asperger syndrome for for Asperger's disorder.
This is an observational study which is adequately explained and interpreted.
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Dear Dr Srovnal & co-authors, you are commended for the improved clarity of the manuscript throughout. These data are presented well and the summary of your experiences are valuable. The discussion is now quite long (6 pages) and though it is generally interesting it could be trimmed a little to make the manuscript sharper. I would suggest the discussion of the incidental variants occurring in the individual with FRAXA would be a good candidate for shortening. This is just a suggestion and it can be to the discretion of the editor and you as to the length of the final manuscript.
Minor corrections:
Page 8: Line 127 : The percentages of rare CNV in the males (60.32%) and females (58.62 %) of our cohort were similar. >> Females 48.62% ?
The use of dystrophinopathia is uncommon, I suggest using muscular dystrophy or dystrophia
I am in agreement with the comments made by the reviewers: the manuscript does suffer from multiple deficiencies and thus would need to undergo a major revision before resubmitting it for consideration. The authors certainly should address each of the points addressed by the reviewers, particularly those noted by Reviewer 2. Additionally, I would suggest 1) the title be altered as the evidence for at least CTNNA2 being associated with an increased risk for ASD is weak (in fact the authors spend more time on PARK2), 2) the authors have to clearly state why they felt there was a need to compare CMA to MLPA in detecting alterations in genes in patients with ASD, 3) the authors should concentrate on preparing a tight, coherent presentation of their findings and a coherent conclusion based on their results and, 4) the authors’ manuscript should reflect that their study is really an “observational” study as noted by Reviewer 1.
The text is somewhat scattered and lacks a clear coherent underlying message. The literature is adequate. The cohort is relatively small and heterogeneous which contributes to my difficulty in identifying a clear message.
This is primarily an observational study. The research questions are not well-defined.
The data are relatively sparse and given the lack of clear research questions, it was hard for me to understand the authors' conclusions.
I have nothing further to add.
Clear and unambiguous, professional English used throughout:
No. Corrections to the English are needed
Literature references, sufficient field background/context provided:
No. Key references are missing
Professional article structure, figs, tables. Raw data shared.
Yes
Self-contained with relevant results to hypotheses.
Aims and hypotheses are not clearly stated
Original primary research within Aims and Scope of the journal.
Yes
Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
No. A minimal increase in knowledge is presented in this work.
Rigorous investigation performed to a high technical & ethical standard.
Findings appear to be rigorous in that multiple confirmatory methods are used
Methods described with sufficient detail & information to replicate.
Yes.
Data is robust, statistically sound, & controlled.
No. Evidence for candidacy of DOCK8, CTNNA2 and genes involved in parkinsonism is not shown. Gender balance of controls may not match a typical ASD cohort where more males than females are expected.
Conclusion are well stated, linked to original research question & limited to supporting results.
Conclusions are not clear. Emphasis for using MLPA over microarray as a frontline test is not strong. Discussion states conflicting opinions on this topic
A summary of findings from 92 individuals affected by autism spectrum disorders tested for structural variants by karyotyping, FRAX PVCR test, MLPA of specific targets and chromosomal microarray. Overall 18 individuals with pathogenic or likely pathogenic variants were detected. All pathogenic or likely pathogenic variants were detected by CMA except for one FRAX mutation.
The aims as stated in the text "The aim of the study was to assess the efficiency of CMA relative to MLPA in terms of clinical significance and to identify new candidate genes for ASD in the studied cohort of ASD-diagnosed patients" are not clear. There are several interpretations for this: Aim to assess the efficiency of CMA relative to MLPA to detect clinically significant CNV; and/or there are different patient outcomes as a result of using different methods. CMA is established as the frontline test for neurodevelopmental disorders over other methods
Why were CNV less than 10kb excluded? MLPA at least should be able to accurately detect much smaller CNV than this.
You suggest "CNV encompassing the region 9p24.3 were identified in several earlier studies, but none of these works identified DOCK8 as being potentially associated with ASD." Glessner et al 2017 demonstrate well the potential involvement of DOCK8 with ASD as well as other neurodevelopmental disorders.
How do you conclude that DOCK8, Parkinsonism and CTNNA2 are associated with ASD without providing a calculation of enrichment of any of the loci to be involved significantly with ASD? Exact tests with appropriate multiple testing corrections to assert novel loci are associated could be performed.
It is asserted that MLPA using 6 probe sets is more cost effective than microarray for excluding loci involved in syndromic autism but no cost benefit analysis is shown to support these claims. I am not convinced that by the time labour is factored in that there is a significant difference.
Why is exclusion of potentially pathogenic loci by MLPA desired over probable detection by CMA (discussion page 9)?
Controls used for DOCK8 are equal numbers of males and females, does this reflect the gender balance in the 92 samples?
What were the selection criteria for inclusion in this study? What fraction of individuals tested by this department over the 2012-2016 time period does this represent?
Suggest the four CNV that were detected by MLPA but not by CMA should be verified to be real or not using either alternate MLPA probes or quantitative PCR of genomic DNA in those regions.
The individuals with pathogenic CNV have a mixed bag of syndromic neurodevelopmental disorders. It would be good to have a section in the discussion explaining how they were regarded as primarily ASD or PDD-NOS disorders as opposed to the syndromes described in OMIM. My interest is, do they in hindsight of molecular diagnosis have the classical syndromic features of those disorders (particularly the male with Becker muscular dystrophy and the female with 22q deletion syndrome) or are the behavioural phenotypes more pronounced than expected in these individuals that made these diagnoses based on phenotype alone difficult?
Was segregation attempted for any of the CNV identified in individuals with affected siblings or family members in the cohort? This may add evidence towards the likely pathogenicity of those variants.
Suggest remove CTTNA2 from the abstract because no convincing evidence is shown in the manuscript to support it as a genuine ASD candidate gene. There is another report of an individual with an intragenic CTNNA2 deletion and speech delay that may be similar to the individual you describe (Paganelli et al. 2017)?
Minor Corrections
page 3 line 67: MLPA analysis is fast and easily interpretable, and can be used ... >> MLPA analysis is fast, easily interpretable and can be used ...
Page 4 line 69: ...CNV in a cohort of 92 children treated for ASD. It is not clear what treated for ASD means as to my understanding the disorder can't be treated. Would 'diagnosed' be an accurate term?
Page 4 line 71: The aim ... >> The aims ...
Page 6-7 lines 133 - 145: This paragraph beginning with "MLPA and CMA revealed 8 pathogenic CNV in..." seems out of place here. I suggest this discussion of the overlap between different detection methods would be better placed later in the manuscript. Only highlighting what was detected by karyotyping and FRAX testing in this first section would make this easier to follow.
Table 1: 'Normal'. Suggest to use 'negative'.
Table 1: "n = number of the patients with the CNV" is not referring to anything, i.e. the abbreviation (n) does not appear elsewhere in the table.
page 8 line 181: "Of the genes listed above, CTNNA2 and DOCK8 are potentially associated with ASD." References required.
Supplementary table contains multiple spelling errors.
Page 13 Line 3012 counselling >> counseling
References:
1: Glessner JT, Li J, Wang D, March M, Lima L, Desai A, Hadley D, Kao C, Gur RE, Cohen N, Sleiman PMA, Li Q, Hakonarson H; Janssen-CHOP Neuropsychiatric Genomics Working Group. Copy number variation meta-analysis reveals a novel duplication at 9p24 associated with multiple neurodevelopmental disorders. Genome Med. 2017 Nov 30;9(1):106.
2: Paganelli V, Giordano M, Meazza C, Schena L, Bozzola M. An intragenic deletion
within CTNNA2 intron 7 in a boy with short stature and speech delay: A case
report. SAGE Open Med Case Rep. 2017 Feb 15;5:2050313X17693967.
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