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REVIEWER - Morton Printz
We thank the reviewer for the comments and suggestions. We have made specific
changes according to the suggestions, as outlined below. We have also made an
extensive revision of the manuscript for grammar and spelling corrections to improve
language.
Major Revisions:
1. We have employed the standard analytical approach for QTL mapping in inbred crosses
as implemented in the QTL Cartographer software. As solicited by this reviewer we have
calculated the exact p-value through 5000 permutations (Figure 1). As shown, the LH value
obtained is highly significant even through this approach.
Figure 1
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2. The data normality was tested using the Kolmogorov-Smirnov test before all parametric
tests and regression/correlation procedures. To clarify we added detailed information in
the “Statistical Analysis” subsection the following sentence (page 6, line 18):
“Kolmogorov-Smirnov test (with Lilliefors' correction) was used to test data
for normality of the estimated underlying population using SigmaStat 3.11
(Systat Software, Inc., San Jose, CA, USA). Normality is assumed for all
parametric tests and regression procedures.”.
3. We agree with the reviewer and to clarify we revised the manuscript to ensure that the
reader understand that we mapped a new QTL localized in chromosome 8 associated with
a negative effect on heart rate in the SHR
Minor Revisions:
1. We revised the Discussion section and some of the speculative paragraphs were
deleted. Only paragraphs that showed directly evidence of the involvement of the
candidate genes and heart rate related phenotypes (i.e., arrhythmia, QT prolongation,
ventricular tachycardia, and syncope) were maintained.
2. Grammar and spelling corrections:
1. The title was corrected;
2. Page 2, line 6 (Abstract): we changed “Using an F2 rats from an intercross of
Spontaneously Hypertensive Rats (SHR) x Brown Norway (BN)...” to “Using an F2
intercross of Spontaneously Hypertensive Rats (SHR) x Brown Norway (BN)...”;
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3. Page 3, line 11 (Background): we changed “Total genome scan to identify
quantitative trait loci (QTL) is a powerful tool...” to “A total genome scan to identify
quantitative trait loci (QTL) provides a powerful tool...”;
4. Page 3, line 16 (Background): corrected as suggested;
5. Page 3, line 17 (Background): we changed “...the identification of the exact
genes related to complex traits specifically for an increase in heart rate associated
with hypertension” to “...the identification of the genes related to complex traits such
as increased heart rate associated with hypertension”;
6. Page 4, line 15 (Methods): we changed “...in male 12-14 week-old” to “...male
12-14 week old animals”;
7. We verified the nomenclature of the marker and APOA02 is correct.
8. Page 9, line 9 (Discussion): we changed “...an increased in heart rate” to “...that
lead to increased heart rate levels”;
9. Page 10, line 1 (Discussion): we changed “...method take into account” to
“...the composite interval mapping method considers the”;
10. Page 10, line 18 (Discussion): we changed “Herewith he mapped a new QTL
localized in chromosome 8 associated with tachichardia in hypertensive animals” to
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“We, herein, mapped a new QTL located in chromosome 8 and associated with a
negative effect on heart rate in the SHR”;
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REVIEWER - Michal Pravenec
We thank the reviewer for the comments and suggestions. We have made specific
changes according to the suggestions, as outlined below. We have also made an
extensive revision of the manuscript for grammar and spelling corrections to improve
language.
1. Reviewer #2 raised an important point to any genome-wide mapping effort: the density
and heterozygosity of genetic markers used in the genome mapping assay. Unfortunately,
our markers panel is not as dense as requested by reviewer #2. In addition, we were not
able to retrieve genomic DNA from these animals to conduct new genotyping experiments,
improving the precision of the mapping effort. We have, however, updated all the mapping
distances of the used markers and this certainly has improved our ability to correctly
identify QTL loci. In Figure 2 we present the updated marker panel.
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Figure 2
More importantly, however, although we agree that a denser set of genetic markers
would certainly improve our power to detect new QTL we are confident that the QTLs
detected in our experiment truly reflect genomic regions with genes responsible for blood
pressure variability in this F2 population. During the last years our group has been able to
isolate each of the previous 4 blood pressure QTLs mapped in this F2 experiment
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(unpublished data) deriving 4 congenic strains. The results are reproducible and, as
expected, in all congenic lines blood pressure phenotype was altered indicating that the
mapped chromosomal region contain gene(s) that influence blood pressure in the SHR.
We do agree that this is a limitation of the present work we have added a paragraph in the
discussion section of the manuscript emphasizing this fact. Methods (Page 5, line 7) and
Discussion (Page 12, line 15) sections:
“Genetic Marker Map
The 188 animals from the F2 progeny were genotyped for 179 genetic
markers distributed along the rat 21 chromosomes according to previous
study (Schork et al., 1995).
Although no new genotypes were
incorporated for this study, we have recently re-accessed the genetic
distances of each genotyped marker (Soler et al., 2006) through
sequential consultation of different internet databases (Rat Genome
Database, http://rgd.mcw.edu/; RatMap, http://ratmap.gen.gu.se/; and
Whitehead
Institute
–
Rat
Genome,
http://www-
genome.wi.mit.edu/rat/public/).”
“One potential study limitation is the number of genetic markers used in
the genome-mapping experiment. Although a large number of F2
analyses have been reported, genes responsible for SHR hypertension
and/or heart rate related phenotypes have not been yet identified. One of
the possible reasons for this fact is the small number of markers
genotyped in these studies. We have used in our mapping experiment
179 markers, polymorphic between the SHR and BN strains. Although
genetic distances and location of these markers have been updated
before conducting our analysis it is possible that QTLs important for the
studied phenotype are still unidentified.”
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2. We could not exclude the possibility that both blood pressure and heart traits are
determined by the same genes. However, we do not believe that the heart rate QTL
identified in the present study is sharing candidate genes with the previously blood
pressure QTL mapped (Shckork et al., 1995). The mapped QTLs are relatively far away
from each other and present a genetic distance of 10.87 cM.