Review for 10.1186/s12876-023-03081-y (2023)

Title: Exploration of the causal effects of leukocyte telomere length and four gastrointestinal
diseases: A two-sample bidirectional Mendelian randomization study
Version: 2 Date: 24 Oct 2023
Reviewer's report:
The authors performed a very interesting study based on the application of bidirectional
Mendelian randomization (MR) study to investigate the potential causal effect of leukocyte
telomere length (LTL) on four different outcomes (irritable bowel syndrome (IBS),
gastroesophageal reflux disease (GERD), peptic ulcers disease (PUD), and non-alcoholic fatty
liver disease (NAFLD)), and the potential causal effect of such variables as exposure on LTL.
The MR approach is an innovative methodology which has been well exploited by the authors
for this scope. The aims of the study are clearly stated, and the methodology is easy to follow.
However, the results of the statistical analysis do not support the conclusions of the authors,
which probably misinterpreted their results. Indeed, the results of the inverse-variance weighted
(IVW) method, which was used by the authors as the reference method to infer causality,
should be confirmed by the results of the other MR methodologies (for more information, see:
https://doi.org/10.12688/wellcomeopenres.15555.2). This is due to the fact that the IVW method
assumes all instrumental variables to be valid, but this is hardly true. The authors reported a
statistically significant effect of LTL on IBS (OR of 1.002, 95% CI 1.002-1.004, and p=0.040),
and of LTL on GERD (OR of 0.890, 95% CI 0.800-0.991, and p=0.033) using the IVW method.
However, none of the other methods supported a causal effect of LTL on IBS or LTL on GERD,
as they reported in the manuscript. This clearly indicates that there is no evidence to support
the existence of a causal effect of LTL on such traits. Despite this, the authors concluded that
“The shortening of LTL predicts a low risk of IBS and a high risk of GERD”, contradicting
their own results based on the weighted median, weighted mode and Egger methods. I strongly
recommend the authors to re-elaborate their “Conclusion” and “Discussion” sections.
I have some minor comments and suggestions that I hope could help the authors to increase the
reliability of their results, the quality of their analyses, and more generally the clarity of this
MR-based manuscript. Such points are reported below.
- “MR-presso” should be written as “MR-PRESSO”.
- P-values and significance thresholds are often reported in a different format from scientific
notation. They should be changed accordingly. For example, “5e-7” should be changed to
“5x10-7”.
- I suggest including some more up-to-date MR methods in the analysis, such as the
contamination mixture, the MR-mix or the MR-RAPS approach, which are more suitable and
better performing.
- I suggest including a forest plot for the effect of each single instrumental variable, which is a
complementary analysis to the leave-one-out method. This could help to give additional clarity
and information on the data.
- In the “Introduction” section the authors state “Telomeres are complex of …., consisting of a
repeating base sequence “TTAGGG” and telomeric protein”. The sentence should be corrected
to: “Telomeres are complex of …., consisting of a repeating base sequence “TTAGGG” and
telomeric proteins”.
- In the “Introduction” section the authors state “.., and the telomere length of white blood cells
in blood has a good correlation with the expression of telomere length of other tissues”. Maybe
the authors meant “.., and the telomere length of white blood cells in blood has a good correlation
with the telomere length of other tissues”.
- The term “race”, used throughout the manuscript, should be corrected to “ethnicity”, “ethnic
background” or “genetic ancestry”.
- In the “Introduction” section the authors state “Due to observational causal research results…,
and it is difficult to clarify wo is the primary pathogenesis”. The first part of this sentence is not
clear and should be improved to allow the reader to better understand the concept the authors are
expressing, which is essential to justify the use of MR. In addition, “who is” should be changed
to “which is”.
- When appearing for the first time, the term SNP should be put between brackets “()” and the
long definition of single nucleotide polymorphism should be reported.
- In the “Introduction” section the authors state “… in order to genomically elucidate the
correlation…”. Instead of “genomically”, the word “genetically” should be used. In addition,
the term “correlation” is not appropriate in a MR setting (the same has been repeated in other
parts of the manuscript).
- The final paragraph of the “Introduction” section is basically repeated twice using slightly
different words.
- In the “Study Design” paragraph of the “Methods and Materials” section the sentence
“The two-sample Mendelian analysis….” Should be corrected to “The two-sample Mendelian
randomization analysis….”.
- In the “Data source and Instrumental Variable Selection for GWAS” paragraph of the “Methods
and Materials” section, a brief but complete description of the studies from which the SNPs were
extracted should be given. It should not be secondarily relegated in the text of the figure 1. This
is essential to give the reader a clear understanding of the data source. In addition, it should be
stated whether the same set of covariates were used in the different GWASs for the different
traits analyzed, as this may be a source of bias, and therefore the authors should address it.
- The terms “SNPs” and “instrumental variables” are often used as synonymous in the
manuscript. Despite this may be a good approximation depending on the context, the definition
of the term “instrumental variable” is never specified within the text.
- Sample overlap is an issue in MR studies, which is known to lead to bias in the causal estimates.
As the authors used summary statistics for LTL, IBS, and PUD from UKBB, they should address
this issue and discuss it in the limitations of the study.
- In the “Instrumental Variable Selection” paragraph of the “Methods and Materials” section,
the sentence “In the reverse MR analysis, due to potential…, while the remaining conditions
were the same as in the forward analysis” is not clear.
- The two paragraphs “Data source and Instrumental Variable Selection for GWAS” and
“Instrumental Variable Selection” of the “Methods and Material” section have almost the same
title.
- In the “Instrumental Variable Selection” paragraph of the “Methods and Materials” section,
the authors state “Additionally, palindrome SNPs with intermediate allele frequencies were
removed…”. What do the authors mean by “intermediate allele frequencies”? Which is the
range? In addition, the term “palindrome SNPs” should be corrected to “palindromic SNPs”.
- How was the F-statistic calculated?
- To remove the SNPs associated with potential confounders, the authors searched on
Phenoscanner for potential associations. However, this may not be enough. I suggest the authors
to also perform a scan using GWAS Catalog (https://www.ebi.ac.uk/gwas/home), as GWAS
Catalog and Phenoscanner do not contain completely overlapping data.
- It is not clear whether the authors used proxies for missing SNPs, and which was the r2
threshold they used in that case.
- In the “MR Analysis” paragraph of the “Methods and Materials” section, the authors state
“…, while other methods can estimate causal effects if they are invalid”. It seems that the other
methods have all the same assumptions. I suggest to briefly specify the assumptions of the other
methods.
- In the “Sensitivity Analysis” paragraph of the “Methods and Materials” section, the term “MR-
Rgger” should be corrected to “MR-Egger”.
- The authors used the classical MR methods for estimating causality. However, I suggest
including some additional analysis and more up-to-date methods, like the contamination
mixture, the MR-RAPS, or the MR-Mix methods (for more information, see: DOI: 10.1002/
gepi.22295, and https://doi.org/10.1007/s40471-017-0128-6). Moreover, the inclusion of a
forest plot showing the causal effect of each single instrumental variable could be extremely
useful, as it is complementary to the leave-one-out analysis that the authors performed.
- Figure 2 is not cited within the main text of the manuscript. Moreover, the description of figure
3 states that it is a forest plot, but the results are presented as a table, rather than a plot. Indeed,
they only reported the values of the beta coefficients, the standard errors and the p-values. The
authors may include a table instead or alternatively report the real forest plot.
- Figure 4 is a screenshot of a table. Why did not the authors simply report a table?
- In the “Causal Effects of Digestive System Diseases on LTL” paragraph of the “Results”
section, the authors state “The results still indicated a significant association between LTL and
IBS and GERD (Table S3)”. The authors should specify that this is only true for the IVW method,
as none of the other methods supported a statistically significant association between LTL and
IBS, or LTL and GERD. This clearly indicates that the results of the IVW method are not reliable.
- In the “Conclusion” section, the authors state “The shortening of LTL predicts a low risk of IBS
and a high risk of GERD”. In the light of all the results and of sensitivity analyses, this conclusion
is wrong. None of the alternative MR methods supported a causal association. The IVW method
alone is not sufficient to establish a causal relationship between two variables. As its assumptions
are hardly satisfied in real biological data, the IVW method alone is not a reliable method to
define causality. That’s exactly why alternative analyses must be carried out and must be taken
into account. The authors should put emphasis on all results obtained, including sensitivity
analyses and alternative MR methods, and not just focus on the IVW method. Additionally, the
IVW result for the association between LTL and IBS estimated an extremely small effect size,
with an OR of 1.002 (95% CI 1.000-1.004), which is completely irrelevant from a clinical point
of view. Therefore, I strongly invite the authors to modify this part of their manuscript.
- In the “Discussion” section, the authors stated that their study suggests or provides evidence
for a causal relationship. This is wrong for the same reasons explained above.
- In the “Discussion” section the term “psychological SNPs” is not correct. Maybe the authors
meant “SNPs associated with psychological traits”.
- In the “Discussion” section the authors reported important information in relation to the IBS
population (“… we only included IBS that did not show diarrhea”): such information may
be given in the “Materials and Methods” section, as it helps the reader in understanding the
characteristics of the population under study.
- The linkage disequilibrium pattern in the Finnish population is not exactly the same of the other
European populations. This may be an issue which should addressed in the “Discussion” section.
- The authors should perform a statistical power calculation to verify whether they were able to
observe a causal association. In this way, they could understand whether they found evidence
for the absence of an effect or absence of evidence.
- It may be useful to add table descriptions or captions for supplementary tables.
- To allow for reproducibility, the authors should compile the STROBE-MR checklist (https://
www.strobe-mr.org/), and include it as supplementary data.