Title: An Alternative Splicing Signature in Human Crohn’s Disease
Version: 2 Date: 28 May 2021
Reviewer's report:
Considering the prevalence of alternative splicing (AS) (about 95% of human genes), it is not
surprising that aberrations of this process are involved in different human disease. In line with
previous observations, Daowei Li and colleagues reported a number of alternative splicing (AS)
alterations in Crohn’s disease (CD), an inflammatory disorder that affects the gastrointestinal
tract.
In this work, focused on the re-analysis of a public mRNA-seq data, Daowei Li  and colleagues
identified 2980 AS events differently regulated between CD patients and normal samples.
Interestingly, in these identified alternatively splicing genes the most enriched pathways are
Lysine degradation and Sphingolipid metabolism. In parallel, these authors also performed an
RNA-seq experiment by using RNA extract from colon tissues from four CD patients and four
controls.
I think that this is a very preliminary work and additional experiments are required in order to
make this manuscript suitable for publication in BMC Gastroenterology. Thus, my decision is
Major revision.
Major comments
1) These authors identified several AS events differently regulated between CD patients and
normal samples. This story would become more interesting if the authors could predict the effect
of selected AS events on the structure/function of the resulting proteins.
2) What is the overlap between genes that change in AS and genes that change in expression
levels? The authors must verify that some AS events could influence the activation of the AS-
NMD program and determine changes the expression level of the resulting mRNA.
3) The identified changes in AS (between CD patients and normal samples) correlate with the
changes of expression levels of some AS regulators. These authors should also determine if the
identified AS events are enriched for binding sites for these AS regulators; this could be helpful
to identify direct pre-mRNA targets.
4) Figure 3 (panel A-C) and Figure 7 (panel B) are very difficult to understand for a person
who is not an expert in splicing field. It is important to show in the diagrams only the part of
the gene that undergoes AS and the result of the splicing reaction that differs between healthy
and CD patients.
5) In Figure 7 C: these authors showed that the expression of exon1 of STAT3 and IRF1 genes
were both significantly upregulated in the CD group. Moreover, total mRNA expression levels
of STAT3 and IRF1 were also increased in CD vs normal. Thus, exon1 upregulation could be
due to transcription and not to AS process. Thus, authors must perform an RT-PCR reaction with
primers that map on exons upstream and downstream exon 1 and run the reaction on agarose gel
(in this way they can see simultaneously the inclusion/exclusion of exon 1 in a single reaction
if this exon is regulated by AS).
6) In figure 7C: they showed STAT3 and IRF1 analyses in CTR1 and CD1 samples. Is this
abbreviation (with number 1) because only one patient and only one control was analyzed? Also,
are SEM and pvalue calculated for different patients or are they derived from technical replicates
of PCR?
7) These authors have already extracted RNA from healthy and CD samples; thus, some of these
AS events must be validated by using RT-PCR and agarose gel.
8) In general, Figure legends must be written better because they are very difficult to understand
for a person who is not an expert in splicing or bioinformatics analysis.
9) Include a new table showing which genes are in common between their RNA-seq experiment
and analysis of public mRNA-seq data.
Minor point:
1) In many parts of the manuscript the acronym AS is used to indicate "alternative splicing",
whereas in the other sections it is written in extenso. Change consistently.
2) In the text, replace “qRT-PCR” with “RT-qPCR”
3) In the Discussion replace “Dysregulation of mRNA splicing” with Dysregulation of pre-
mRNA splicing”.
4) The manuscript should be read by a native speaker
Title: An Alternative Splicing Signature in Human Crohn’s Disease
Version: 2 Date: 28 May 2021
Reviewer's report:
Considering the prevalence of alternative splicing (AS) (about 95% of human genes), it is not
surprising that aberrations of this process are involved in different human disease. In line with
previous observations, Daowei Li and colleagues reported a number of alternative splicing (AS)
alterations in Crohn’s disease (CD), an inflammatory disorder that affects the gastrointestinal
tract.
In this work, focused on the re-analysis of a public mRNA-seq data, Daowei Li  and colleagues
identified 2980 AS events differently regulated between CD patients and normal samples.
Interestingly, in these identified alternatively splicing genes the most enriched pathways are
Lysine degradation and Sphingolipid metabolism. In parallel, these authors also performed an
RNA-seq experiment by using RNA extract from colon tissues from four CD patients and four
controls.
I think that this is a very preliminary work and additional experiments are required in order to
make this manuscript suitable for publication in BMC Gastroenterology. Thus, my decision is
Major revision.
Major comments
1) These authors identified several AS events differently regulated between CD patients and
normal samples. This story would become more interesting if the authors could predict the effect
of selected AS events on the structure/function of the resulting proteins.
2) What is the overlap between genes that change in AS and genes that change in expression
levels? The authors must verify that some AS events could influence the activation of the AS-
NMD program and determine changes the expression level of the resulting mRNA.
3) The identified changes in AS (between CD patients and normal samples) correlate with the
changes of expression levels of some AS regulators. These authors should also determine if the
identified AS events are enriched for binding sites for these AS regulators; this could be helpful
to identify direct pre-mRNA targets.
4) Figure 3 (panel A-C) and Figure 7 (panel B) are very difficult to understand for a person
who is not an expert in splicing field. It is important to show in the diagrams only the part of
the gene that undergoes AS and the result of the splicing reaction that differs between healthy
and CD patients.
5) In Figure 7 C: these authors showed that the expression of exon1 of STAT3 and IRF1 genes
were both significantly upregulated in the CD group. Moreover, total mRNA expression levels
of STAT3 and IRF1 were also increased in CD vs normal. Thus, exon1 upregulation could be
due to transcription and not to AS process. Thus, authors must perform an RT-PCR reaction with
primers that map on exons upstream and downstream exon 1 and run the reaction on agarose gel
(in this way they can see simultaneously the inclusion/exclusion of exon 1 in a single reaction
if this exon is regulated by AS).
6) In figure 7C: they showed STAT3 and IRF1 analyses in CTR1 and CD1 samples. Is this
abbreviation (with number 1) because only one patient and only one control was analyzed? Also,
are SEM and pvalue calculated for different patients or are they derived from technical replicates
of PCR?
7) These authors have already extracted RNA from healthy and CD samples; thus, some of these
AS events must be validated by using RT-PCR and agarose gel.
8) In general, Figure legends must be written better because they are very difficult to understand
for a person who is not an expert in splicing or bioinformatics analysis.
9) Include a new table showing which genes are in common between their RNA-seq experiment
and analysis of public mRNA-seq data.
Minor point:
1) In many parts of the manuscript the acronym AS is used to indicate "alternative splicing",
whereas in the other sections it is written in extenso. Change consistently.
2) In the text, replace “qRT-PCR” with “RT-qPCR”
3) In the Discussion replace “Dysregulation of mRNA splicing” with Dysregulation of pre-
mRNA splicing”.
4) The manuscript should be read by a native speaker
Please review this paper.
user
Title: An Alternative Splicing Signature in Human Crohn’s Disease
Version: 2 Date: 28 May 2021
Reviewer's report:
Considering the prevalence of alternative splicing (AS) (about 95% of human genes), it is not
surprising that aberrations of this process are involved in different human disease. In line with
previous observations, Daowei Li and colleagues reported a number of alternative splicing (AS)
alterations in Crohn’s disease (CD), an inflammatory disorder that affects the gastrointestinal
tract.
In this work, focused on the re-analysis of a public mRNA-seq data, Daowei Li  and colleagues
identified 2980 AS events differently regulated between CD patients and normal samples.
Interestingly, in these identified alternatively splicing genes the most enriched pathways are
Lysine degradation and Sphingolipid metabolism. In parallel, these authors also performed an
RNA-seq experiment by using RNA extract from colon tissues from four CD patients and four
controls.
I think that this is a very preliminary work and additional experiments are required in order to
make this manuscript suitable for publication in BMC Gastroenterology. Thus, my decision is
Major revision.
Major comments
1) These authors identified several AS events differently regulated between CD patients and
normal samples. This story would become more interesting if the authors could predict the effect
of selected AS events on the structure/function of the resulting proteins.
2) What is the overlap between genes that change in AS and genes that change in expression
levels? The authors must verify that some AS events could influence the activation of the AS-
NMD program and determine changes the expression level of the resulting mRNA.
3) The identified changes in AS (between CD patients and normal samples) correlate with the
changes of expression levels of some AS regulators. These authors should also determine if the
identified AS events are enriched for binding sites for these AS regulators; this could be helpful
to identify direct pre-mRNA targets.
4) Figure 3 (panel A-C) and Figure 7 (panel B) are very difficult to understand for a person
who is not an expert in splicing field. It is important to show in the diagrams only the part of
the gene that undergoes AS and the result of the splicing reaction that differs between healthy
and CD patients.
5) In Figure 7 C: these authors showed that the expression of exon1 of STAT3 and IRF1 genes
were both significantly upregulated in the CD group. Moreover, total mRNA expression levels
of STAT3 and IRF1 were also increased in CD vs normal. Thus, exon1 upregulation could be
due to transcription and not to AS process. Thus, authors must perform an RT-PCR reaction with
primers that map on exons upstream and downstream exon 1 and run the reaction on agarose gel
(in this way they can see simultaneously the inclusion/exclusion of exon 1 in a single reaction
if this exon is regulated by AS).
6) In figure 7C: they showed STAT3 and IRF1 analyses in CTR1 and CD1 samples. Is this
abbreviation (with number 1) because only one patient and only one control was analyzed? Also,
are SEM and pvalue calculated for different patients or are they derived from technical replicates
of PCR?
7) These authors have already extracted RNA from healthy and CD samples; thus, some of these
AS events must be validated by using RT-PCR and agarose gel.
8) In general, Figure legends must be written better because they are very difficult to understand
for a person who is not an expert in splicing or bioinformatics analysis.
9) Include a new table showing which genes are in common between their RNA-seq experiment
and analysis of public mRNA-seq data.
Minor point:
1) In many parts of the manuscript the acronym AS is used to indicate "alternative splicing",
whereas in the other sections it is written in extenso. Change consistently.
2) In the text, replace “qRT-PCR” with “RT-qPCR”
3) In the Discussion replace “Dysregulation of mRNA splicing” with Dysregulation of pre-
mRNA splicing”.
4) The manuscript should be read by a native speaker
assistant