Author's response to reviews

Transcription

1 Author's response to reviews Title: Dystonia, facial dysmorphism, intellectual disability and breast cancer associated with a chromosome 13q34 duplication and overexpression of TFDP1: Case report Authors: Mariana Moscovich (marimoscovich@hotmail.com) Mark S LeDoux (mledoux@uthsc.edu) Jianfeng Xiao (jianfengxiao@gmail.com) Garrett L Rampon (grampon@uthsc.edu) Satya R Vemula (svemula@uthsc.edu) Ramon L Rodriguez (ramon.rodriguez@neurology.ufl.edu) Kelly D Foote (foote@neurosurgery.ufl.edu) Michael S Okun (okun@neurology.ufl.edu) Version: 2 Date: 1 April 2013 Author's response to reviews: see over

2 Title: Dystonia, facial dysmorphism, intellectual disability and breast cancer associated with a chromosome 13q34 duplication and overexpression of TFDP1:Case report We thank the reviewers for their very helpful comments, which have improved the presentation of our data. Based on your reviews, we have altered the manuscript and point-by-point responses are provided below: Reviewer: Daniela Turchetti Reviewer's report: The authors describe the case of a 36 year-old woman with developmental delay and dysmorphic features, carrying a chromosome 13q34 duplication inherited from the healthy father. At age 30, she developed a psychiatric disturbance and was treated with antipsychotic drugs. After that, she manifested dystonia and at age 35 underwent right mastectomy for breast cancer. Major Compulsory Revisions. Although the case is very interesting, the causal link between the CNV and the clinical phenotype remains largely undemonstrated. In particular, late manifestations such as dystonia and breast cancer look more likely to be secondary to the antipsychotic treatment. To try to address this issue, the authors should provide the following information. 1) Segregation of the CNV in the family. The assessment of the CNV in other family members is needed to provide support in favor or against its causative role in the patient's phenotype. Since the proband is the fourth child and the siblings are reported as healthy, it would be crucial to test them, as well as other potential carriers in the paternal side of the family, for the presence of the CNV and the expression of the genes involved. This could also allow to test the hypothesis, raised by the authors, of a gender dependence of TFDP1 expression. Response: The siblings live in another country and could not travel to the United States. However, they were interviewed via Skype and showed no evidence of dysmorphism, cognitive impairment or dystonia. Moreover, videotapes of these siblings were evaluated by two movement disorders specialists (Rodriquez and Okun) and dystonia was excluded. The pedigree is provided as a figure. The CNV did not co-segregate with dystonia. 2) Breast cancer features. A more detailed description of the tumour is required to support a relationship between the genomic defect and the development of cancer. Indeed, amplification of genes in chromosome 13q34 (particularly TFDP1) in breast cancer has been associated with negative estrogen receptor immunoreaction, high histologic grade and unfavourable prognosis. Conversely, breast carcinomas arising in patients treated with neuroleptics, prolactin-releasing drugs, are reported to be more frequently estrogen receptor positive, rarer histologic types such as mucinous type have been described in these patients; moreover positive immunoreaction for prolactin receptor is very common (Fujita et al, 1994). The authors should report at least the biological features of the carcinoma that are routinely assessed for diagnostic purpose, such as grading, estrogen and progesterone receptors, proliferation index; if possible, prolactin receptor staining should be performed. In addition, they should report if serum prolactin levels had been assessed in the patient. Finally, the possibility that breast cancer could have been favoured by the therapy should be discussed. Response: In 2010, the proband was diagnosed with breast cancer during a routine exam and was submitted to a radical right mastectomy with preservation of the areola and nipple. Serum prolactin levels were not obtained. She was treated with tamoxifen, post-operatively, and has shown no evidence of local recurrence or metastasis of her breast cancer. The pathology revealed an encapsulated mucinous carcinoma grade I. The tumor cells inside mucous lakes expressed estrogen and progesterone receptors and did not over-express Her2Neu. Discretionary Revisions 3) The clinical presentation is skewed toward neurological manifestations and treatment, with little description of the other features. The paper would benefit of a more balanced description; for instance,

3 dysmorphic features should be described in more detail, and target stature given (to give a measure of how much the stature is abnormally tall in this patient). Response: Dysmorphic features are now described in greater detail:... facial dysmorphism with hypertelorism, down slanting palpebral fissures, mild ptosis and large low-set ears with mild posterior rotation, hirsutism, low anterior hairline, brevicollis, scoliosis, arachnodactyly, tall stature (182.9 cm), and a weight of 62.3 kg (body mass index = 18.6). Family stature: mother ( cm) and father ( cm). 4) Some comments on CNVs inherited from healthy parents, based on the recent literature, would be useful in the discussion. Response: The topic of CNV penetrance has been added to the discussion section of the manuscript. Reviewer's report Title: Dystonia, facial dysmorphism, intellectual disability and breast cancer associated with a chromosome 13q34 duplication and overexpression of TFDP1: Case report Reviewer: Lorenzo Melchor Reviewer's report: Overall, the study carried out by Moscovich and colleagues is a well-conducted analysis relatively important in its field, as it is the first report to describe a potential connection between chromosomal region 13q34, TFDP1, and dystonia. The report firstly describes the patient clinical symptoms, to further show the finding of the genomic duplication and the definition of its boundaries using very appropriate techniques for such purposes. The discussion is also well structured with an emphasis on the mechanism of TFDP1 upregulation as well as its gender specificity. Having said this, some minor and discretionary changes need to be addressed to improve the quality of this interesting manuscript, and thus to ensure acceptance. The authors need not only to improve the genomic definition of the 13q34 duplication by adding chromosome 13 SNP array profiles to the Figure 2, but also to carry out statistical analysis in the comparative analysis of copy number values and gene expression levels. Response: Array CGH was performed by a commercial laboratory (Quest) and they did not provide an array CGH profile picture and were not willing to do so when contacted regarding the results. In any case, the duplication is small and a picture would add little substance to the presentation. These statistical comparisons are not possible given the biological sample sizes (N) of 1. It is almost standard in the field to use mean or median values derived from technical triplicates to perform parametric and non-parametric comparison of biological groups. Cross comparison of technical replicates to biological replicates is not scientifically valid. A list of discretionary changes is also provided, with suggestions for additional information or a new figure/table to show the clinical evolution of the patient at a quick glance. Response: We have added a table to show the clinical evolution of the patient. MINOR ESSENTIAL REVISIONS 1) Genomic characterization of 13q34 duplication. The genomic characterization of 13q34 duplication seems well explained in the text, but remains scarce in Figure 2. This referee would suggest additional sections in Figure 2 to show the genomic aberration definition as follows: Figure 2A: First, in the manuscript section Case Presentation page 5, the authors mention ( ) Karyotype analysis exposed a chromosome 13q34 duplication ( ). If picture of the conventional karyotype is available for the authors and the segment duplication can be noted, which may be difficult as is only 218Kb, it would be beneficial to show the abnormal and the normal chromosomes 13 with arrows pointing out the duplicated segment. However, if the sentence refers to results given by the SNP Array analysis, it should be changed to ( ) Molecular cytogenetic analysis exposed a chromosome 13q34 duplication to prevent misunderstandings. Response: Array CGH was performed by a commercial laboratory (Quest) and they did not provide an array CGH profile picture and were not willing to do so when contacted regarding the results. These statistical comparisons are not possible given the biological sample sizes (N) of 1. It is almost standard in the field to use median values derived from technical replicates (e.g., triplicates) to perform parametric and

4 non-parametric comparison of biological groups. Cross comparison of technical replicates to biological replicates is not scientifically valid. The sentence in question has been changed to Molecular cytogenetic analysis... Figure 2B: The authors identified a copy number aberration in 13q34 using Affymetrix Genome-Wide Human SNP Array 6.0. It is common in clinical reports identifying small genomic imbalances to show such aberrations with an array CGH profile picture (See for instance Capra et al BMC Med Gen 2012 or Carrascosa Romero et al. Am J Med Genet 2012). An image of the SNP Array profile of the chromosome 13 will add value and accuracy to the genomic definition. Response: Array CGH was performed by a commercial laboratory (Quest) and they did not provide an array CGH profile picture and were not willing to do so when contacted regarding the results. We agree that a profile picture of the genomic region in question is visually appealing and often used in publications describing structural variants. Figure 2C: Current Figure 2 could comprise the third section of a modified Figure 2. References to each of these sections should thus be included across the text when convenient. Response: Although our method of presentation is not standard, it does convey the necessary and critical information related to the structural variant identified in our pedigree. 2) Statistical analyses Table 2 and Table 3 show the results of the quantitative PCR of genomic DNA and the relative gene expression level analyses in leukocytes from peripheral blood of normal controls, patient, her father (carrier) and her mother. Although there are differences in copy number values and gene expression levels between subjects, statistical tests should be carried out to ensure significance. The U-Mann Whitney Test could be used for pair wise comparisons (patient versus control, patient versus father, patient versus mother, father versus control, father versus mother, mother versus control) or the Kruskal-Wallis Analysis for multiple group comparisons. These statistical tests should be performed for each assay and their p- values shown in the tables. Response: These statistical comparisons are not possible given the biological sample sizes (N) of 1. It is almost standard in the field to use mean or median values derived from technical replicates (e.g., triplicates) to perform parametric and non-parametric comparison of biological groups. Cross comparison of technical triplicates to biological replicates (neurologically-normal controls) is not scientifically valid. DISCRETIONARY REVISIONS 1) Minor typos can be found throughout the text: Page 6. Identification of the duplication 1st paragraph: Four genes were involved in the genomic alternation Substitute alternation for alteration. Response: Changed to alteration. Page 8. ( ) allowed us to identify the 5 and 3 breakpoints (Chr13:114,020,670 and Chr:114,239,014) ( ) Substitute last coordinate for Chr13:114,239,014. Response: Corrected. 2) Follow the Guidelines for Gene nomenclature when citing gene/protein names. Page 10. ( ) Neddylation is the process by which Nedd8 is conjugated to target proteins. Nedd8 is an ubiquitin-like modifier of protein function ( ) Unless it is referred to a non-human study, use capital letters for NEDD8 and in italics, if referred to the gene. Response: This change has been added to the manuscript. 3) Further description of the family The authors may choose to expand the information of the family, if consent is provided, to find out whether more cases of breast cancer and/or dystonia were seen in the paternal side of the family. The patient was very young at the time of diagnosis (35 years old) of a mucinous carcinoma, usually reported in older patients, which thus may suggest an inherited germ line predisposition. More breast cancer cases in

5 the family would support the findings of this manuscript. Nevertheless, this may have already been explored but no additional patients with breast cancer and/or dystonia may have been found. Response: There was no history of breast cancer or dystonia in any first or second degree relatives. 4) A table or figure to sum up the chronology and the patient s clinical symptoms would be useful for a quick glance of the patient evolution. Response: Clinical chronology is summarized in Table 1. Reviewer: Francesco Brancati Reviewer's report: Re: review of the manuscript entitled Dystonia, facial dysmorphism, intellectual disability and breast cancer associated with a chromosome 13q34 duplication and overexpression of TFDP1: Case report The authors report on the identification of a genomic duplication spanning about bp in a girl with syndromic dystonia (psychomotor delay, breast cancer, tall stature, facial dysmorphism) and speculated whether the genes involved in this rearrangement might have a role especially in the pathogenesis of dystonia. The father is also a carrier of the same rearrangement. Indeed, they verified that leucocyte expression of these transcripts was abnormally high (more than an additional 0.5x) for one of these genes (TFDP1) in the proband but not in her father. The authors provided a nice presentation of this gene as a candidate for dystonia, while the possible explanation of its differential expression among the father and his daughter is weak ( could be gender dependent due to differential hormonal effects on gene regulation in either cis or trans ). Response: The statement in question has been modified. This is an interesting and well-written case report. It is true that there are very few patients with chromosomal imbalances manifesting primary dystonia. Also, the authors nicely cloned the duplication breakpoints, where a 69 bp fragment showing 100% identity to a long terminal repeat of the endogenous retrovirus family K was inserted, further highlighting how these rearrangements might be complex. This work may benefit from revision with respect to the point mentioned below: In this reviewer s opinion, the manuscript should be significantly shortened and presented as a case report since there are no definite evidences supporting a pathogenic role of this duplication in the phenotype (and specifically in dystonia). Response: We agree that the pathogenicity of the structural variant is not definite. The manuscript was submitted as a case report and includes case report in the title. Other points to improve: Major: 1. Please clarify which databases have been interrogated (or whether the authors have run a number control samples) to verify if the identified duplication might be a CNV. Response: The structural variant identified in our pedigree appears to be novel - it has not been reported in the Database of Genomic Variants. To avoid semantic confusion, we have avoided use of the term CNV in the manuscript because our variant is complex and some geneticists limit the term CNV to benign changes in copy number. Please clarify if even broader constitutional (not somatic) deletion/duplications comprising this segment at 13q34 have been previously reported in the literature and if patients displayed any of the features observed in your patient. Response: Chromosome 13q34 deletions/duplications have not been associated with dystonia or dysmorphism. Minor: Abstract: it is unclear whether the duplication is de novo or not. Response: We now make it clear in the abstract that the proband s father also harbored the same duplication. It is stated Karyotype analysis exposed a chromosome 13q34 duplication. Please verify this statement considering that the estimated size of the duplication is about 225 kb as assessed by SNP-array.

6 Response: Corrected to Molecular cytogenetic analysis exposed a chromosome 13q34 duplication. I would move Table 1 (Primers) to supplementary file/online methods or make it available on request. Same for Table 2 (qpcr results to confirm the duplication on genomic DNA). Response: The table of primers (Additional file 1) is now provided as an additional file. A table listing breast cancer- and dystonia-associated genes is also provided as an additional file. Given the important of qpcr to this story, we have retained this information in the print portion of the manuscript.