Associations between Genetic Variants in DAB, PRKAG, and DACH Genes and Gender in Chronic Kidney Disease
, Attila Nagy 8 and Amir Houshang Shemirani 1,9,10,*Round 1
Reviewer 1 Report
The work is interesting, he needs minor modifications.
-lines 24-25: clarifying the study group. Here are described: "200 patients with
chronic renal disease and 250 control individuals. However, in table 2, are described 163 patients with chronic renal disease (68 male, 95 female) and 218 ontrol individuals (89 male, 129 female).
- describe, in materials and methods, the lymphocyte isolation procedure, carried out prior to genomic DNA extraction (ficoll?).
-better describe the paragraph:' 2.4. Sequencing"
Minor editing of English language required in discussion.
Author Response
Response to Reviewer 1 Comments
Comments and Suggestions for Authors
Many thanks to the reviewer for his/her kind comments. We have changed our manuscript as instructed. We highlighted the changes.
The work is interesting, he needs minor modifications.
-lines 24-25: clarifying the study group. Here are described: "200 patients with
chronic renal disease and 250 control individuals. However, in table 2, are described 163 patients with chronic renal disease (68 male, 95 female) and 218 control individuals (89 male, 129 female).
As we started our study, we planned to include 200 patients and 250 controls. That is why we included this information in the abstract. But, after applying exclusion criteria, some patients excluded. As you know, some results should be repeated, specially in our case for triplex PCR. Hence, we had a very limited amount of precious samples for our study, these mandatory re-analyses had been missed. Consequently, we had to exclude some controls and patients. All said, we have two choices for our manuscript, either to describe all in our text or change the numbers in the abstract. We have changed number in the abstract as: (Page 1) Genomic DNA was extracted from buffy coat of 163 patients with chronic renal disease and 218 control individuals.
- describe, in materials and methods, the lymphocyte isolation procedure, carried out prior to genomic DNA extraction (ficoll?).
We have provided more information as instructed. It is added at the first part of 2.3. Genotyping (Page 3).
DNA was extracted with a Genomic DNA Mini Kit (Central European Biosystems). Briefly, genomic DNA was isolated from peripheral blood lymphocytes. This procedure consisted of five steps: red blood cell lysis, cell lysis, DNA binding, washing and elution.
First, we centrifuged the samples for 10 minutes at 800 xg. Then the buff-colored layer was pipetted, being careful not to disturb the other blood components, and up to 200 µL was transferred into a microcetrifuge tube. We discarded supernatant after 10 minutes incubation with RBC Lysis Buffer and centrifugation. We repeated the previous step and resuspended the pellet by adding 200 µL RBC Lysis Buffer. For cell lysis step, we added 250 µL of GB Buffer to the tube and incubated at 70°C for 30 minutes. For DNA binding step, we added 250 µL absolute ethanol to the sample and transferred all of the mixture to the GD Column. GD column then was placed in a new Collection Tube after centrifugation. We performed the washing stepwisely by adding W1 Buffer, centrifugation, adding Wash Buffer and centrifugation. Finally, we added 100 µL preheated Elution Buffer to GD Column and incubated at 37°C for 10 minutes. Samples containing DNA were collected by centrifugation at 16000 xg for one minute.
-better describe the paragraph:' 2.4. Sequencing"!
We tried to describe this part better (page 3).
To confirm the results of HRM analysis, 10% of samples were randomly se-quenced using ABI Prism 310 DNA sequencer (Applied Biosystems). Primers for re-al-time PCR were also used for sequencing analysis. A 5-ml aliquot was analyzed on an agarose gel to verify the specificity of the product, and the remaining material was used for sequencing. All PCR products were purified with Amicon Ultra (Merck Mil-lipore, Schwalbach, Germany) and directly sequenced from both sides using the BigDye terminator V1.1 (Applied Biosystems, Foster City, California, USA). The ob-tained sequences were aligned to the expected target sequences manually.
Best regards
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The study was elegant and compact. I can give some advises for becoming more better then now as below.
1. In introduction DACH1 function should be given clearly.
2. In line 49, a reference style should be changed according to journal rules.
3. In line 28-56, ARIC, ERSD and TGF-β should be given long form. Also, CRP should be written long in line 85.
4. Ethics number should be given. In addition that, it should be mentioned in material method part.
5. Included criteria should be given.
Author Response
Response to Reviewer 2 Comments
Comments and Suggestions for Authors
We thank the Reviewer for the useful comments and suggestions. We have modified the manuscript in light of the comments. The responses to comments are detailed below. These changes have highlighted by yellow color throughout the manuscript.
The study was elegant and compact. I can give some advises for becoming more better then now as below.
- In introduction DACH1 function should be given clearly.
We have added the text to the manuscript (Page 2):
It is well-known as a tumor suppressor protein whose decreased expression closely correlates with a poor prognosis in a number of malignancies [8]. DACH1 protein regulates expression of target gene either by binding to chromatin or by combining with other transcription factors. [9,10]. DACH1 DNA-specific binding results transcriptional repression of the target gene [11].
- In line 49, a reference style should be changed according to journal rules.
We have removed the unnecessary part: “(Bash et al., 2009)”
- In line 28-56, ARIC, ERSD and TGF-β should be given long form. Also, CRP should be written long in line 85.
We have changed our manuscript accordingly.
Page 2, line 48: Atherosclerosis Risk in Communities
Page 2, line 50: end-stage renal disease
Page 2, line 56: Transforming Growth Factor-β
Page 2, line 93: C-reactive protein (CRP)
- Ethics number should be given. In addition that, it should be mentioned in material method part.
It has added as instructed.
Page 2, line 86: The Ethics Committee of the University of Debrecen, Hungary approved the study (DEOEC RKEB/IKEB 3936-2013). The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. All patients and controls provided written informed consent.
- Included criteria should be given.
We changed our manuscript according to the comment. (Page 2, line 82)
CKD is categorized according to stages: eGFR between 60 and80 ml/min/1.73m2, eGFR between 30 and 60 ml/min/1.73m2, eGFR between 15 and 30 ml/min/1.73m2, and eGFR less than 15 ml/min/1.73m2 [15]. We included patient with eGFR less than 60 ml/min/1.73m2.
We tried improve our manuscript grammatically by one of our native English colleague.
Best regards
Author Response File: Author Response.pdf
