Review Reports - Rates of Divergent Pharmacogenes in a Psychiatric Cohort of Inpatients with Depression—Arguments for Preemptive Testing

Round 1

Reviewer 1 Report

Overall, my opinion is that this manuscript is very interesting for clinicians and readers. There is some data on this important topic, which should be better described in the introduction. Many significant limitations in methods and results should be addressed, especially sampling and statistics should be better explained. The main limitation is the sample size (e.g. not calculated by appropriate statistics, small sample size) and type of the study (no approved study protocol given –STROBE), with which the authors cannot fully answer their very ambitious hypothesis. Results are well presented. Generally, the paper has medium issues with the standard of writing. However, in the current form presented, I suggest a major revision. I hope that I have helped the authors with my comments.

The manuscript could be strengthened by attending to the following matters:

1) GENERAL COMMENTS

Positive:
- Interesting topic

-Important topic

Negative:
- Limitations

- Small sample size and study type

- Unclear patients history

INTRODUCTION

Line 16: like cerebrospinal fluid (Suggest Central circulatory system first)

Lines 15-31: References are needed

Lines 45-50: Please do not label patients »psychiatric«. I suggest using» patients with mental disorders«.

Lines 52-56: The authors should mention some psychotropics, where genetics is extremely important for therapeutic and adverse events (e.g., aripiprazole, lurasidone, cariprazine).

Lines 52-56: What is known about pharmacodynamic drug-drug interactions and genetics?

Line 64: We analyzed our data for divergent genotypes of all metabolizing enzymes included in the stratipharm panel [Table 1]. = move to the Methods

Lines 65-73: I agree with the authors that genetics is essential for desired therapeutic outcomes. However, therapeutic drug monitoring is the first gold standard (TDM). Even in patients with high/low activities of some CYPs, we can use TDM for medication optimization.

Another important issue is a lack of approved standards and protocols and reimbursement questions (e.g., who will pay for this service?). The authors should report how payments take place in Germany for this service.

Especially in psychiatry, we have many important studies where genetics has been studied, although few patients have profited from this research.

Clin Pharmacol Ther. 2021 Sep;110(3):786-793. DOI: 10.1002/cpt.2233. & Association of CYP2C19 and CYP2D6 Poor and Intermediate Metabolizer Status With Antidepressant and Antipsychotic Exposure: A Systematic Review and Meta-analysis. JAMA Psychiatry. 2021 Mar 1;78(3):270-280. & Impact of CYP2C19 Genotype on Escitalopram Exposure and Therapeutic Failure: A Retrospective Study Based on 2,087 Patients. Am J Psychiatry. 2018 May 1;175(5):463-470. doi: 10.1176/appi.ajp.2017.17050550.

AIM

Line 81: Please remove »psychiatric« and change it to »patients with mental disorders«

Lines: 80-84: I think that this hypothesis is too ambitious for this type of research (e.g., type II error, hypothesis testing) = RCT should be conducted

MATERIAL AND METHODS

Line 86: Did the patients pay extra for this service, or was this service complimentary for all study participants?

Line 88: Did the authors check how many treatment-naive and chronic patients were?

Lines 105-112: Please remove statistical tests and establish a separate paragraph.

Did the authors calculate the sample size?

More data on lab tests and standards should be included.

RESULTS

Lines 116-118: Please provide a flowchart.

Line 130 – Figure: Please remove back lines.

Lines 140-146: The authors should provide a separate paragraph in the Methods and explain much better methods. Comparing 108 and 51464 seems to be a source of selection bias.

DISCUSSION

Lines 174: sertraline is metabolized by CYP2D6, CYP2C19, CYP3A4 and CYP2C9. = reference is missing

This collaboration led to fewer medication-related problems, and clinical pharmacists can recognize more problems (even those not recognized by psychiatrists themself).

LIMITATIONS

The authors should include more limitations, including sample size, type II, statistics and TDM.

FURTHER STUDIES

The authors should also focus on collaborative care, including clinical pharmacists' services (available in the U.S.).

CONCLUSION

I suggest the authors remove references from Conclusions and focus more on their results, including limitations.

References: Recheck them because there are some mistakes (N#5 20 bolded etc..…).

Author Response

Reviewer 1

1) GENERAL COMMENTS

Positive:
- Interesting topic

-Important topic

Negative:
- Limitations

- Small sample size and study type

- Unclear patients history

INTRODUCTION

Line 16: like cerebrospinal fluid (Suggest Central circulatory system first) changed accordingly

Lines 15-31: References are needed three references were included

Lines 45-50: Please do not label patients »psychiatric«. I suggest using» patients with mental disorders«.changed accordingly

Lines 52-56: The authors should mention some psychotropics, where genetics is extremely important for therapeutic and adverse events (e.g., aripiprazole, lurasidone, cariprazine).General formulation was included in the introduction section line 21)

Lines 52-56: What is known about pharmacodynamic drug-drug interactions and genetics? Sentence changes accordingly and DDI were added

Line 64: We analyzed our data for divergent genotypes of all metabolizing enzymes included in the stratipharm panel [Table 1]. = move to the Methods was cancelled, because it is already in the methods section.

Especially in psychiatry, we have many important studies where genetics has been studied, although few patients have profited from this research. Perez et al. 2017 and Bousman et al. 2018 were added.as well as the mentioned citations below

AIM

Line 81: Please remove »psychiatric« and change it to »patients with mental disorders« was changed accordingly

Lines: 80-84: I think that this hypothesis is too ambitious for this type of research (e.g., type II error, hypothesis testing) = RCT should be conducted I agree, this is stated in the limitation section, however this was the aim of this retrospective study.

MATERIAL AND METHODS

Line 86: Did the patients pay extra for this service, or was this service complimentary for all study participants? Complimantary, it was added in the text.

Line 88: Did the authors check how many treatment-naive and chronic patients were? Demographic section was added in the results section.

Lines 105-112: Please remove statistical tests and establish a separate paragraph. Done

Did the authors calculate the sample size? This was not done, as it was a retrospectiuve analysis of naturalistic data.

More data on lab tests and standards should be included. ? Table one shows all SNPS that were tested. Infomration on the method (swab test) was included

RESULTS

Lines 116-118: Please provide a flowchart. For what exactly? As this was not a RCT, but naturlaistic data, wthere are no drop outs etc.. Results are shown in Figure 1,2 and 3

Line 130 – Figure: Please remove back lines. You mean the y-axis help lines? Can be changed, once the editor gives feedback on the figres format that the journal wants to use.

Lines 140-146: The authors should provide a separate paragraph in the Methods and explain much better methods. Comparing 108 and 51464 seems to be a source of selection bias. Explanation added in the methods section.

DISCUSSION

Lines 174: sertraline is metabolized by CYP2D6, CYP2C19, CYP3A4 and CYP2C9. = reference is missing AGNP guideline was added as reference.

Lines 188-195: I highly agree that clinical pharmacists should be included in the multidisciplinary team in mental health institutions, including psychiatric hospitals. We have found some relevant papers: Positive evidence for clinical pharmacist interventions during interdisciplinary rounding at a psychiatric hospital. Sci Rep. 2021 Jul 1;11(1):13641. doi: 10.1038/s41598-021-92909-2 & Clinical pharmacist interventions in elderly patients with mental disorders in primary care focused on psychotropics: a retrospective pre-post observational study. Ther Adv Psychopharmacol. 2021 Apr 22;11:20451253211011007. doi: 10.1177/20451253211011007. In both papers, clinical pharmacists did not have any genetic tests, but they recognized problems also based on pharmacokinetic knowledge (e.g., essential pharmacological and clinical experiences). As mentioned, they do not refer to PGx testing and this is was we wanted to focus on: especially in PGx a clinical pharmacist can be beneficial. Instead Stäuble CK, Lampert ML, Allemann S, Hatzinger M, Hersberger KE, Meyer Zu Schwabedissen HE, Imboden C, Mikoteit T. Pharmacist-guided pre-emptive pharmacogenetic testing in antidepressant therapy (PrePGx): study protocol for an open-label, randomized controlled trial. Trials. 2021 Dec 14;22(1):919. doi: 10.1186/s13063-021-05724-5. PMID: 34906208; PMCID: PMC8670138.

Was cited.

This collaboration led to fewer medication-related problems, and clinical pharmacists can recognize more problems (even those not recognized by psychiatrists themself).we did not analyze DRPs in this study. As this is in xenobiotics and not international journal of clinical pharmacy or similar, I tried not to focus to much on the beneficial cooperation of clinical pharmacists and psychiatrists.

LIMITATIONS

The authors should include more limitations, including sample size, type II, statistics and TDM. Type two error and sample size were addressed. How is TDM a limitation of our study?

FURTHER STUDIES

The authors should also focus on collaborative care, including clinical pharmacists' services (available in the U.S.).was added in this paragraph.

CONCLUSION

I suggest the authors remove references from Conclusions and focus more on their results, including limitations. Done

References: Recheck them because there are some mistakes (N#5 20 bolded etc..…). Ok, done

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear aythors.

I consider your research to be of a great interest. It defenetely matches aims and scooes of the journal. However, I ask for a major revision, Please, consider the following points that need your attention before the article could be accepterd for a further revision.

1. The writing of the manuscript must be improved. First of all careefully check the terminology, and explain the terms you use (like "divergent genotypes" and "genetic phenotypes". )

2. The methodology of the study is unclear. It needs a more detailed explanation oh how the key variables including the ones on histograms were calculated

3. What was the raw data wou dealt with?

4. "Activity scores for CYP2D6 and phenotypes are reported accordingly" - these data are not provided in the article

5. I encourafge you to present your results in the way that makes it possible to compare similar data from the other studies.

Generally, supplementary data are upload in the public database. It would facilitate understanding and usefulness of your research. I think alleles frequences would be greatly apreciated.

6. The reasoning of the study, aims and disccussion should be more cconsolidated and related to each other

I have identified a nubber of minor issues. I will provide the list in the case you decide to resubmit your paper.

Thank you and best regards.

Author Response

Reviewer 2

Comments and Suggestions for Authors

Dear aythors.

Thank you for your helpful comments for our manuscript. I hope we included all aspects that were suboptimal in our first version. Please find our answers in red behind the 6 points that you asked to be revised.

The writing of the manuscript must be improved. First of all careefully check the terminology, and explain the terms you use (like "divergent genotypes" and "genetic phenotypes". ) , the terminology was explained after the first occurrence in the paper.
The methodology of the study is unclear. It needs a more detailed explanation oh how the key variables including the ones on histograms were calculated Table two lists as genetic variants that counted as divergent.
What was the raw data wou dealt with? As stated on page 6, we used the patient file to retrieve the data. Excel sheet was used to gather the data.
"Activity scores for CYP2D6 and phenotypes are reported accordingly" - these data are not provided in the article sentence was cancelled, thank you for the remark.
I encourafge you to present your results in the way that makes it possible to compare similar data from the other studies. Table 3 shows all the numbers in two columns with a third column on the p value. Do you mean the result or discussion section?

Generally, supplementary data are upload in the public database. It would facilitate understanding and usefulness of your research. I think alleles frequences would be greatly apreciated. Uploading the raw data is possible.

The reasoning of the study, aims and disccussion should be more cconsolidated and related to each other was rewritten accordingly, each paragraph was changed, new studies (references) included. I hope this finds you appreciation.

I have identified a nubber of minor issues. I will provide the list in the case you decide to resubmit your paper. Thank you

Thank you and best regards.

Reviewer 2

Comments and Suggestions for Authors

Dear aythors.

The writing of the manuscript must be improved. First of all careefully check the terminology, and explain the terms you use (like "divergent genotypes" and "genetic phenotypes". ) , the terminology was explained after the first occurrence in the paper.
The methodology of the study is unclear. It needs a more detailed explanation oh how the key variables including the ones on histograms were calculated Table two lists as genetic variants that counted as divergent.
What was the raw data wou dealt with? As stated on page 6, we used the patient file to retrieve the data. Excel sheet was used to gather the data.
"Activity scores for CYP2D6 and phenotypes are reported accordingly" - these data are not provided in the article sentence was cancelled, thank you for the remark.
I encourafge you to present your results in the way that makes it possible to compare similar data from the other studies. Table 3 shows all the numbers in two columns with a third column on the p value. Do you mean the result or discussion section?

The reasoning of the study, aims and disccussion should be more cconsolidated and related to each other was rewritten accordingly, each paragraph was changed, new studies (references) included. I hope this finds you appreciation.

I have identified a nubber of minor issues. I will provide the list in the case you decide to resubmit your paper. Thank you

Thank you and best regards.

Author Response File: Author Response.pdf

Reviewer 3 Report

I think that this work gives a valuable contribution for the field, since the existence of this type of clinical studies is of utmost importance to improve the health strategies and policies. Clinical studies together with in vitro models, can elucidate the mode of action of drugs in individuals with divergent genetics, improving the treatment towards personalized medicine.

It is my opinion that the manuscript should be accepted for publication, after including some minor revisions.

See attched file.

Best Regards

Comments for author File: Comments.pdf

Author Response

Reply to reviewer 3: (answers in red)

The present manuscript describes a systematic DNA Real Time-PCR analysis of biological samples

of a group of hospitalized psychiatric patients, to investigate the presence of divergent genetic

variants in a diverse panel of drug metabolizing enzymes genes. Besides the limitations, the

results are outstanding, since 100% of individuals show at least 2 genetic polymorphisms

present. I must admit that I am not completely surprised with the results, since its is known that

polymorphism in drug metabolizing enzymes plays a central role in personalized medicine, and

in individual toxicological outcome.

I think that this work gives a valuable contribution for the field, once the existence of this type

of clinical studies is of utmost importance to improve the health strategies and policies. Clinical

studies together with in vitro models, can elucidate the mode of action of drugs in individuals

with divergent genetics, improving the treatment towards personalized medicine.

It is my opinion that the manuscript should be accepted for publication, after including some

minor revisions.

Minor issues:

In general, this 3rd version of manuscript already has been improved significantly in comparison

with the 1st and 2nd versions. However, I recommend further spelling check and typos check.

As for example:

1) First page, first paragraph of introduction must be rephrased.

The authors version:

“Tolerability and efficacy of a drug are depending on the concentration of the drug, the

serum of the central circulatory system serum or other compartments of the body like the

cerebrospinal fluid, according to general principles of pharmacology.”

Reviewer suggestion:

“According to the general principles of pharmacology, tolerability and efficacy of a drug

depend on its concentration in the serum of the central…” Done accordingly, thank you!

2) Third page, second line of “2.Aim”, the word “divergent” is repeated. Done, thank you!

Comments and suggestions:

a) Concerning the Discussion section:

When the authors compare their own results with the Danish cohort, several valuable

explanations are presented to justify the differences found between the two works.

However, it is my opinion that this kind of differences are expectable since the plethora

of genetic backgrounds in drug metabolizing enzymes is rather the rule than exception.

As so, it is urgent that health systems and regulators consider the concept of

personalized medicine. We included this in the conclusion section. In this context, the authors focus too much in trying to explain

that those variations in results are due to differences in methodologies, rather than

arising from the differences of genetic backgrounds between populations. This should

also be discussed and included, to reinforce the necessity of a vaster PGx to be used in

future. Thank you for this hint, we included a short section in the discussion about the genetic backgrounds.

To emphasize this point of view, and other pharmacological/toxicological differences

found, the authors can use one or both of the following references:

Sim, S.C., M. Kacevska, and M. Ingelman-Sundberg, Pharmacogenomics of drug

metabolizing enzymes: a recent update on clinical implications and endogenous

effects. Pharmacogenomics J, 2013. 13(1): p. 1-11. Thank you fort he advice, I added this study in the references and cite in the discusion.

Zanger, U.M. and M. Schwab, Cytochrome P450 enzymes in drug metabolism:

regulation of gene expression, enzyme activities, and impact of genetic variation.

Pharmacol Ther, 2013. 138(1): p. 103-41. We integrated it in the discussion section.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors accepted all of my recommendations, so I suggest an acceptance.

Author Response

Thank you for reviewing the article a second time. We highly appreciate it.

Reviewer 2 Report

«Divergent genotypes» is the most frequently used term in your study. In particular, It can be found in the title and in the abstract (6 times). This term is quite common in evolutionary sciences, but not in applied genetics, That’s why I asked you to clarify the meaning of the term in the present paper. In your answer you say that « the terminology was explained after the first occurrence in the paper». However, the only explanation I can now find in the revised version was the two words in parenthesis in the Aims section:

«examines the frequencies of divergent (non‐wild type) genotypes in pharmacokinetic genes»

This explanation is now even more confusing, but still not that much as the second term I asked you to explain ("genetic phenotypes"). This received no attention at all in the revised version.

Many other issues were not properly addressed as well. However, I still do believe that you can present an interesting data. That’s why I recommend the following:

1,. Clarify and simplify the paper. Saying that «the research compares allele frequencies of high impact variants of the genes involved in individual response to harmacotherapy..» sounds nice and meaningful to me. And then do not stray far from this idea in the article.

2. Ask a fundamental geneticist to revise the text .

3. Please upload the row data (since you say it is possible).

4. Specify the method that was used to identify the alleles in patients DNA.

I apologize for the directive tone. That is the way I found to express my opinion in a constructive manner.

Author Response

Reviewer two, second review:

Dear reviewer,

Thank you for the second review of our article. We applied some changes to the manuscript and hope it finds your appreciation. Supplement is uploaded as requested.

«Divergent genotypes» is the most frequently used term in your study. In particular, It can be found in the title and in the abstract (6 times). This term is quite common in evolutionary sciences, but not in applied genetics, That’s why I asked you to clarify the meaning of the term in the present paper. In your answer you say that « the terminology was explained after the first occurrence in the paper». However, the only explanation I can now find in the revised version was the two words in parenthesis in the Aims section:

«examines the frequencies of divergent (non‐wild type) genotypes in pharmacokinetic genes»

This explanation is now even more confusing, but still not that much as the second term I asked you to explain ("genetic phenotypes"). This received no attention at all in the revised version. Divergent genotypes are defined in Table 2. Additionally it was added at the first occurence in the article what is meant by the term: “(i.e. divergent from the common phenotypes considered normal, e.g. extensive metabolizer and normal metabolizer“ was added. The term „divergent“ is used as in the publication of Luneburg (Danish cohort that we compared our cohort to), but is also used in other publication of applied genetics recently to avoid terms like „non-normal“ metabolizers.

The genetic phenotype term is used to clarify that the phenotype (as measured by therapeutic drug monitoring) can be in discordance, e.g. by phenoconversion effects. However, the terminology was changed to „genotype“ in the entire article.

Many other issues were not properly addressed as well. However, I still do believe that you can present an interesting data. That’s why I recommend the following:

This is in discordance with the other reviewer who wanted more aspects to be covered and I do not know how to address it in a way that both reviewers agree. However, I tried to cancel part about the harmonization of guidelines, harmonization of panels and aspects of the integration of PGx results into the patient file in the discussion section. I hope that reviewer 1 will still appreciate the manuscript.

Ask a fundamental geneticist to revise the text . What exact aspect needs revision? The panel is provided in the supplement 1. The 12 genes´ definition of “divergent” is given in table 1. Our manuscript in regards to genotypes is in the same format as Lunenburg´s (our comparison cohort), so that the comparison was even possible. I assume that the reviewer one was a geneticist, he had no further remarks on the manuscript after revision 1.
Please upload the row data (since you say it is possible). Supplement 2 was added.
Specify the method that was used to identify the alleles in patients DNA. It is a commercial test that we used. The testing panel (all the rs numbers) were uploaded in the supplemental material. The laboratories name and test name were given in the text. Laboratory method was added in method section. If of interest, here is the whole information (but too long for the manuscript):

DNA extraction

DNA is extracted from collected human buccal swab samples using a manual or automated DNA extraction protocol. The manual extraction protocol uses a QIAamp-DSP-DNA Blood-Mini Kit or a QIAamp DNA-Blood- Mini Kit. For the automated DNA extraction protocol the Qiagen BioRobot 9604 and the QIAamp 96 DNA Blood BioRobot Kit are used. Both manual and automated processes generate high-quality DNA that is suitable for PCR amplification.

DNA quantification

Extracted DNA is quantified and normalized using an automated robotic platform and spectrometer (Molecular Devices Spectra Max 190 Spektrometer). DNA is stored at 4°C until the specimen is ready for the PCR amplification step of the SNP or CNV analysis.

SNP determination with real-time PCR via TaqMan Assays

Determination of SNPs is performed by real-time PCR using automated Life Technologies QuantStudio 12k flex. TaqMan^® GTXpress™ Master Mix is used in combination with the IVD PCR primer/probe panel (see TaqMan^® SNP Genotyping Assay, User guide, 2014). The IVD product consists of PCR primer/probe combinations (assays) to specifically detect variants of ABCB1, ABCG2, ADRB1, ADRB2, COMT, COQ2, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, DPYD, GNB3, GSTP1, HLA-A, HLA-B, HMGCR, HTR2A, IL28B, ITPA, MTRNR1, NAT2, OPRM1, SLC19A1, SLCO1B1, TPMT, and VKORC1. For each SNP, a PCR reaction is performed with the diluted DNA of the sample, the master mix and the specific assay. Each assay contains two PCR primers and two fluorescence-labeled probes. The probes are short DNA molecules that specifically bind to the sequences of the wild type or the mutant. Performance of this genotyping technique has been validated according to the methods described by Welch et al (2008)

This “classical” PCR is used for analysis of < 12 samples to be analyzed and is being used for confirmation of ambiguous results from SNP determination with real-time PCR via array (see description below) as needed.

SNP determination with real-time PCR via array

The IVD PCR primer/probe panel can also be analyzed on a TaqMan® OpenArray® Genotyping Plate (see TaqMan® OpenArray® Genotyping, Getting Started Guide, Rev. E, 2016). For the array approach, all 94 TaqMan^® SNP Genotyping Assays are pre-loaded and dried down in the through-holes of the array. Real-time PCR in OpenArray format is performed on the same Life Technologies QuantStudio 12k flex as the classical PCR, the device is only configured with a different block/heated cover and TaqMan® OpenArray™ Genotyping Master Mix is used instead of TaqMan^® GTXpress™ Master Mix. Prior to the real-time PCR, a multiplexed short PCR-reaction is necessary for preamplification of the 94 loci of interest (see TaqMan® OpenArray® Genotyping, Sample Preamplification Guide, Rev. A.0, 2015).

The array variant for SNP determination is generally used in cases where > 11 samples should be analyzed in parallel.

CNV determination with real-time PCR

For the determination of the CNV by a PCR reaction diluted DNA of the sample is mixed with a master mix (TaqMan^® Genotyping Master Mix), a reference assay (TaqMan^® Copy Number Reference Assay TERT) and the assay specific for analysis of CYP2D6 CNVs. The assay also contains two PCR primers, but only one probe that binds specifically to the sequence of the gene.

Data analysis

The SNP assay analysis is performed independently by two scientists. One scientist is using the device (QuantStudio 12k Flex) software which performs an automatic allele pre-calling based on the typical cluster formation in the allelic discrimination plot (VIC vs. FAM) which is confirmed by the scientist. For data points that cannot be called by this method the scientist compares the amplification plots with reference images and assigns manual calls. The second scientist uses the TaqMan Genotyper software which also performs an automatic allele pre-calling which are confirmed by the scientist. TaqMan Genotyper software furthermore allows for parallel evaluation of multiple PCR runs and loading of reference panels to improve the analysis.

Ambiguous results from the two independently performed analyses are not accepted and cause further control and/or repeat actions.

The TaqMan Genotyper software is developed by the device manufacturer and provided free of charge as off-the-shelf software.

The Copy Caller Software performs an analysis algorithm that determines the number of copiesof the target sequence in each test genomic DNA sample and assesses the quality of the results. The TaqMan Genotyper software and Caller Copy software are developed by the device manufacturer and provided free of charge as off-the-shelf software.

I apologize for the directive tone. That is the way I found to express my opinion in a constructive manner.

Round 3

Reviewer 2 Report

Comments for author File: Comments.pdf

Author Response

Reviwer 2, review 3

«Divergent genotypes» is the most frequently used term in your study. In particular, It can be found in

the title and in the abstract (6 times). This term is quite common in evolutionary sciences, but not in

applied genetics, That’s why I asked you to clarify the meaning of the term in the present paper. In your

answer you say that « the terminology was explained after the first occurrence in the paper». However,

the only explanation I can now find in the revised version was the two words in parenthesis in the Aims

section:

«examines the frequencies of divergent (non‐wild type) genotypes in pharmacokinetic genes»

This explanation is now even more confusing, but still not that much as the second term I asked you to

explain ("genetic phenotypes"). This received no attention at all in the revised version. Divergent

genotypes are defined in Table 2. Additionally it was added at the first occurence in the article what is

meant by the term: “(i.e. divergent from the common phenotypes considered normal, e.g. extensive

metabolizer and normal metabolizer“ was added. The term „divergent“ is used as in the publication of

Luneburg (Danish cohort that we compared our cohort to), but is also used in other publication of

applied genetics recently to avoid terms like „non‐normal“ metabolizers.

In Luneburg publication the term «divergent phenotypes» (not «genotypes») was explained explicitly

and clarified, including it the abstract. You could also give a detailed explanation in your article, and a

brief one in the abstract. The terminology was changed to phenotype in the article and explanations given in the abstract and the main body using the same explanation as Luneburg in her article. Also, a table with the phenotypes that lead to a „divergent“ phenotype is given. But in my opinion that it will still be confusing for many readers. I’d

recommend a linguist advice cause the issue is probably related to some language differences.

The article actually deals with allele frequencies. I don’t see the reason to avoid this term. It would make

your data more comparable with other studies thus providing much more interest for the readers. As this is a clinical view that we use, allel frequencies are indeed to confusing. The raw data includes only phenotypes.

The genetic phenotype term is used to clarify that the phenotype (as measured by therapeutic drug

monitoring) can be in discordance, e.g. by phenoconversion effects. However, the terminology was

changed to „genotype“ in the entire article. There must have been a huge misunderstanding. I changed genotype to phenotype in the article. However, I do not find the expression „genetic phenotype“ in the article.

Many other issues were not properly addressed as well. However, I still do believe that you can present

an interesting data. That’s why I recommend the following:

1,. Clarify and simplify the paper. Saying that «the research compares allele frequencies of high impact

variants of the genes involved in individual response to harmacotherapy..» sounds nice and meaningful

to me. And then do not stray far from this idea in the article.

This is in discordance with the other reviewer who wanted more aspects to be covered and I do not

know how to address it in a way that both reviewers agree. However, I tried to cancel part about the

harmonization of guidelines, harmonization of panels and aspects of the integration of PGx results into

the patient file in the discussion section. I hope that reviewer 1 will still appreciate the manuscript.

Ask a fundamental geneticist to revise the text . What exact aspect needs revision? The panel is

provided in the supplement 1. The 12 genes´ definition of “divergent” is given in table 1. Our manuscript

in regards to genotypes is in the same format as Lunenburg´s (our comparison cohort), so that the

comparison was even possible. I assume that the reviewer one was a geneticist, he had no further

remarks on the manuscript after revision 1.

Are you sure that comparing allele frequencies obtained by different methods (high‐density DNA array

vs PCE) and from distinct DNA source is absolutely correct? The mistake rates for these methods might

be quite different., and probably it needs a comparison within the differences in allele frequency that

were considered statistically significant. I´m not an expert in the laboratory issued and the article wants not to compare laboratory methods, but show a clinical view point. Many clinicians still believe, that genetic polymprhisms are very rare, however we could sho that they are very common indeed. If there are such huge discepancies by the method in the phenotype results, this would be of high interst for another article. As a clinican, I trust a commercial test that is regulated by the drug agencies.

Please upload the raw data (since you say it is possible). Supplement 2 was added.

Raw data generally implies allele frequencies like in the article you are mentioning. This data is not available, but only phenotypes.

Specify the method that was used to identify the alleles in patients DNA. It is a commercial test that we

used. The testing panel (all the rs numbers) were uploaded in the supplemental material. The

laboratories name and test name were given in the text. Laboratory method was added in method

section. If of interest, here is the whole information (but too long for the manuscript):

These methods are crucial for the replicability of your results, And please see two previous comments. The infomration is confidential and the commercial test provider did not allow publication.

You can also notice the description provided below says that some discrepancies can appear demanding

an attendance of two experts.

Fully addressing these issues is absolutely necessary for you article to be considered for the further

publication. Another reviewer was invited to review the manuscript and only had minor remarks.

Author Response File: Author Response.docx