It is an overstatement to say "This study aims to investigate

1: This is a report of antibody levels in children with autism and ADHD which is an interesting topic that would be of interest to the readership of N******oscience Letters. The manuscript has problems with inappropriately discussing the state of the field. The authors also make overstatements about their own study. Lastly, the methods include limited details that would be necessary to evaluate the results and conclusions. Abstract: It is an overstatement to say “This study aims to investigate the association between degeneration of the cerebellum and the etiologies of ASD and ADHD.” Introduction: It is unclear how the authors are utilizing the literature about Tourette syndrome and OCD (references 5,6) to support their introduction about autism. This is the first of several citations for which is was unclear how the authors were using the citation to support their discussion (i.e. reference 12 which is a mouse study when authors have been discussing human symptoms; references 15 and 16 which are not about comorbidity measures to support high comorbidity of ASD and ADHD). Another significant problem with the introduction is that no previous findings on antibody levels in ADHD and ASD are described, despite many studies being published. Particularly of interest that are neither described in the introduction nor cited in the discussion are: Rout et al 2012, Haukanes et al 2015, Bayram et al 2016. However, studies which did not focus on the antibodies of interest here are also important to describe to address the idea of auto-immunity in general in childhood disorders In the abstract, introduction and throughout the paper, it is not appropriate to call these antibodies as a group “cerebellar antibodies” The authors do not introduce what the Yo or GAD antigens refer to, which is particularly a problem because they use the term “PCA” or “cerebellar antibodies” for the anti-Yo antibodies multiple other places in the manuscript Page 4: It is too strong a statement to say “The most widely known abnormalities associated with ASD are atrophy of the cerebellum and selective loss of Purkinje cells.” Page 5: The authors mis-cite the conclusions of the ADHD meta-analysis “A meta-analysis study showed a frequency of 5-29% among children” The average frequency was 5.29% and ranged from 2% to 12%. Page 5: It is a mis-statement to say “Presence of Yo antibodies is the most common and well defined pathway of cerebellar degeneration.” Clearly—this is a rare etiology for cerebellar degeneration and is not well-defined. Page 5: it is unclear what the authors mean by “Additionally, distribution of glutamic acid decarboxylase (GAD) in the n******oendocrine tissues, and antibodies against GAD act upon the cerebellar pathways” Do the authors mean to say that GAD is distributed in n******oendocrine tissues? If so, why is this relevant? The authors should clarify that anti-GAD antibodies act upon multiple brain regions, one of which is cerebellar pathways. Methods: Were samples run as single measures or in duplicate or triplicate? From what source were ELISA products obtained? What is range of technical error of the assay… the sensitivity and inter-assay variation? The authors describe the reference level using one kind of measure (IE/ml) and report results in another measure (pmol/L). What conversion was used? Because of the lack of clarity, it is difficult to independently evaluate what the average and range of measured levels of each antibody suggest and so difficult for this reviewer to evaluate the results and conclusions. The authors should use a Bonferroni correction for the correlations Results: I cannot fully evaluate the results because of the methods issues I pointed out What categorical variable was used with chi square in table 2? Page 8: What do the authors mean by “Poor positive correlation”? Do the authors mean “anti-GAD levels” at the bottom of page 8 where they say “GAD levels” Page 9: what do the authors mean when they say “However, no correlation was found for groups” at the top of page 9 Discussion I cannot fully evaluate the discussion because of the methods issues I pointed out There seems to be a contradiction in the following two sentences “According to the findings, no significant differences in antibody levels were observed among the 2 groups. However, children with ADHD reported significantly higher levels of Purkinje cell antibodies compared to the healthy subjects.” It is an overstatement to say “Most recent studies on ADHD have established associations of the disorder with abnormalities in Purkinje cells, as reduced cerebellar volume, and developmental differences [20-23].” Using “Some” instead of “Most” would be appropriate. Also relevant to this sentence is how the cited references demonstrate abnormalities in Purkinje cells. This appears to be an overstatement When the authors reference Palmen et al 2004, they describe this as a “structural imaging study” which is not accurate It is incorrect to say “The potential role of immune system in ASD etiology have been consistently supported by evidences…” given the fact that there are inconsistencies in the field, which are apparent in the references that the authors selected (#41, #42) It is inaccurate to say “higher levels of antibodies were found in the ASD group when compared to the controls in the present study.” Given that there was no significant difference, any difference in the means should not be described as higher or lower Reviewer #2: This study reports on the quantitation of anti-Yo and anti-GAD antibodies in samples of serum from persons with ASD, ADHD, and controls (about 40 subjects per group). The primary experimental finding is an elevation of anti-Yo antibodies in subjects with ADHD vs, controls. This is an interesting observation. However, the paper suffers from several deficiencies and an overall poor presentation with much missing and confusing information. 1. The authors repeatedly use the term “anti-cerebellar antibodies” and make a major point of linking their results to the cerebellum. I do not understand why they do this as (a) they measure only samples of blood and not cerebellum expression, and (b) GAD is certainly expressed in most brain regions and other organs besides the brain. Also, what is evidence that “Yo” in specific for the cerebellum?. 2. In fact, there is no explanation as to what Yo is and its function. The Intro section describes irrelevant info but does not address these key points. 3. A description of methodology in terms of the key blood measurements using the ELIZAs is virtually absent. A paragraph of the antibodies used and their specificities is needed. 4. In the key tables, tables 2 and 3, the authors parachute in the acronym “PCA”. There is no explanation as to what this is, although it might refer to Yo??? 5. In table 2, the results of the quantitations are given. The only significant difference is in PCA controls vs ADHD with p < 0.017. Yet looking at the numbers shown – means 2.9 vs 1.6 and extremely large SDs 8.3 and 19.3 and wide ranges given it seems difficult to understand how the difference is so significant. The authors should double-check their calculations. Also, a dot plot histogram might be helpful to visually grasp the variance. 6. The English needs to be much more polished. 26-JAN-2018 02:10 PM

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