Title: Analysis of the NUDT15 Gene and Metabolites of Azathioprine in Japanese Patients with
Inflammatory Bowel Disease
Version: 0 Date: 15 May 2023
Reviewer:  Prateek Bhatia
Reviewer's report:
The authors have tried to highlight the relationship between metabolites of Thiopurine and
different enzyme SNPs of NUDT15 and Clinical adverse reactions in a cohort of IBD cases.
These studies are very vital to understand the pharmacogenomic principles in relation to clinical
practice and guide important treatment and dose modification decisions.
However as reviewer I have many observations regarding the data which requires substantial
addresal for the manuscript to be re considered.
1. The authors have taken 49 IBD cases who had received 52 weeks of Thiopurine and
retrieved their biobank samples for analysis.  Though they mention different time point biobank
samples beng tested for intermediate metabolites, they do not highlight what was nature os
sample archived? I presume whole blood was freeze since RBX metabolites concentration were
measured . Please clarify on same and also detail whether freeze thawing and long term retrieval
of blood sample can affect thiopurine intermediate metabolite level measurement .?
2. How can authors get such a high frequency of heterozygotes for both ITPA and Nudt
15 ...nearly 31 or 32  out of 4 had some or other SNP including combine SNP of nudt15 in
6 cases. Please detail in statistical analysis whether this high allele frequency is in accordance
with the laws of allele distribution in population and whether authors have used hardy weinberg
correction for same.  This is very important to detail and clarify considering high frequency of
the SNPs.
3. Authors need to resort to terminology of polymorphism instead of mutation for all these
changes all throughout the manuscript.
4. It is known that 6TGN levels by HPLC have many drawbacks because it gives the total 6TGN
level and no information on the individual components of Mono, di or the most active and toxic
tri form . Rather many experrt groups now recommend resorting to measuring DNA-6TGN
levels by mass spec which specifically detects active DNA bound tri form. Please describe this
as limitation and discuss this as another  possible reason for not finding a correlation between
6TGN levels and NUDT15 SNPs.
5. Many relevant references in IBD  with nudt15 and itpa SNPs and systematic reviews as well as
recent study ftom india showing no relation  of itpa with Thiopurine toxicity in IBD are missing.
These are primary from the group in South Asia (india) led by Sharma V et al. Please add them
and discuss their relevant results in discussion.
Title: Analysis of the NUDT15 Gene and Metabolites of Azathioprine in Japanese Patients with
Inflammatory Bowel Disease
Version: 0 Date: 15 May 2023
Reviewer:  Prateek Bhatia
Reviewer's report:
The authors have tried to highlight the relationship between metabolites of Thiopurine and
different enzyme SNPs of NUDT15 and Clinical adverse reactions in a cohort of IBD cases.
These studies are very vital to understand the pharmacogenomic principles in relation to clinical
practice and guide important treatment and dose modification decisions.
However as reviewer I have many observations regarding the data which requires substantial
addresal for the manuscript to be re considered.
1. The authors have taken 49 IBD cases who had received 52 weeks of Thiopurine and
retrieved their biobank samples for analysis.  Though they mention different time point biobank
samples beng tested for intermediate metabolites, they do not highlight what was nature os
sample archived? I presume whole blood was freeze since RBX metabolites concentration were
measured . Please clarify on same and also detail whether freeze thawing and long term retrieval
of blood sample can affect thiopurine intermediate metabolite level measurement .?
2. How can authors get such a high frequency of heterozygotes for both ITPA and Nudt
15 ...nearly 31 or 32  out of 4 had some or other SNP including combine SNP of nudt15 in
6 cases. Please detail in statistical analysis whether this high allele frequency is in accordance
with the laws of allele distribution in population and whether authors have used hardy weinberg
correction for same.  This is very important to detail and clarify considering high frequency of
the SNPs.
3. Authors need to resort to terminology of polymorphism instead of mutation for all these
changes all throughout the manuscript.
4. It is known that 6TGN levels by HPLC have many drawbacks because it gives the total 6TGN
level and no information on the individual components of Mono, di or the most active and toxic
tri form . Rather many experrt groups now recommend resorting to measuring DNA-6TGN
levels by mass spec which specifically detects active DNA bound tri form. Please describe this
as limitation and discuss this as another  possible reason for not finding a correlation between
6TGN levels and NUDT15 SNPs.
5. Many relevant references in IBD  with nudt15 and itpa SNPs and systematic reviews as well as
recent study ftom india showing no relation  of itpa with Thiopurine toxicity in IBD are missing.
These are primary from the group in South Asia (india) led by Sharma V et al. Please add them
and discuss their relevant results in discussion.
Please review this paper.
user
Title: Analysis of the NUDT15 Gene and Metabolites of Azathioprine in Japanese Patients with
Inflammatory Bowel Disease
Version: 0 Date: 15 May 2023
Reviewer:  Prateek Bhatia
Reviewer's report:
The authors have tried to highlight the relationship between metabolites of Thiopurine and
different enzyme SNPs of NUDT15 and Clinical adverse reactions in a cohort of IBD cases.
These studies are very vital to understand the pharmacogenomic principles in relation to clinical
practice and guide important treatment and dose modification decisions.
However as reviewer I have many observations regarding the data which requires substantial
addresal for the manuscript to be re considered.
1. The authors have taken 49 IBD cases who had received 52 weeks of Thiopurine and
retrieved their biobank samples for analysis.  Though they mention different time point biobank
samples beng tested for intermediate metabolites, they do not highlight what was nature os
sample archived? I presume whole blood was freeze since RBX metabolites concentration were
measured . Please clarify on same and also detail whether freeze thawing and long term retrieval
of blood sample can affect thiopurine intermediate metabolite level measurement .?
2. How can authors get such a high frequency of heterozygotes for both ITPA and Nudt
15 ...nearly 31 or 32  out of 4 had some or other SNP including combine SNP of nudt15 in
6 cases. Please detail in statistical analysis whether this high allele frequency is in accordance
with the laws of allele distribution in population and whether authors have used hardy weinberg
correction for same.  This is very important to detail and clarify considering high frequency of
the SNPs.
3. Authors need to resort to terminology of polymorphism instead of mutation for all these
changes all throughout the manuscript.
4. It is known that 6TGN levels by HPLC have many drawbacks because it gives the total 6TGN
level and no information on the individual components of Mono, di or the most active and toxic
tri form . Rather many experrt groups now recommend resorting to measuring DNA-6TGN
levels by mass spec which specifically detects active DNA bound tri form. Please describe this
as limitation and discuss this as another  possible reason for not finding a correlation between
6TGN levels and NUDT15 SNPs.
5. Many relevant references in IBD  with nudt15 and itpa SNPs and systematic reviews as well as
recent study ftom india showing no relation  of itpa with Thiopurine toxicity in IBD are missing.
These are primary from the group in South Asia (india) led by Sharma V et al. Please add them
and discuss their relevant results in discussion.
assistant