EFCAB4B (CRACR2A/Rab46) Genetic Variants Associated with COVID-19 Fatality

Round 1

Reviewer 1 Report

 

The present manuscript is an excellent attempt by the authors to report that genetic variation in EFCAB4B (CRACR2A/Rab46) is associated with COVID-19 fatality. Authors have demonstrated that three single nucleotide polymorphisms (SNPs) in the EFCAB4B gene cause missense variations in CRACR2A and Rab46, associated with COVID-19 fatality. It was found in the study that all three SNPs cause changes in amino acid residues that are highly conserved across species, thus indicating the importance of these residues in protein structure and function. The study further dives into the mechanism detailing the importance of critical functional domains: the EF-hand and coiled-coil domains. The study is meticulously designed and well-performed; the present manuscript is well-written.

 

Following are the specific comments to further strengthen the manuscript,

1.        From the demographic data, the study is on adult individuals, toward older age, 60+. Is there any such study on young individuals? And what are the outcomes?

2.        How about genetic variation EFCAB4B (CRACR2A/Rab46) impact young children? Is there any such study? Please discuss or provide expected results.

3.        What is the EFCAB4B (CRACR2A/Rab46) genetic variation percentage in the world population?

4.        How do we overcome this EFCAB4B (CRACR2A/Rab46) genetic variation-mediated COVID-19 fatality?

Author Response

We thank the reviewers for their comments, and we have revised and improved the manuscript accordingly. Please find the detailed point-by-point responses below.

Reviewer 1

Major comments

The present manuscript is an excellent attempt by the authors to report that genetic variation in EFCAB4B (CRACR2A/Rab46) is associated with COVID-19 fatality. Authors have demonstrated that three single nucleotide polymorphisms (SNPs) in the EFCAB4B gene cause missense variations in CRACR2A and Rab46, associated with COVID-19 fatality. It was found in the study that all three SNPs cause changes in amino acid residues that are highly conserved across species, thus indicating the importance of these residues in protein structure and function. The study further dives into the mechanism detailing the importance of critical functional domains: the EF-hand and coiled-coil domains. The study is meticulously designed and well-performed; the present manuscript is well-written.

 

Detail comments

Following are the specific comments to further strengthen the manuscript,

1. From the demographic data, the study is on adult individuals, toward older age, 60+. Is there any such study on young individuals? And what are the outcomes?

Authors’ response:

We have not yet undertaken studies on younger individuals as there is insufficient data available in the UK Biobank dated before the vaccination programme.

2. How about genetic variation EFCAB4B (CRACR2A/Rab46) impact young children? Is there any such study? Please discuss or provide expected results.

Authors’ response:

No studies have been undertaken specifically on a cohort of young children. A study investigating alcohol problems in young adults identified a genetic association with an intronic SNP in EFCAB4B (PMID: 25439982). We have published data on a patient who has bi-allelic mutations in EFCAB4B who developed late-onset immunodeficiencies (PMID: 34908525). This data suggests that EFCAB4B is important in stress responses and therefore explains why variants may have a pronounced effect in an aging population or in a disease such as COVID where the immune system is in overdrive.

3. What is the EFCAB4B (CRACR2A/Rab46) genetic variation percentage in the world population?

Authors’ response:

The frequency of the described EFCAB4B SNPs in the world population are shown under the MAF (ALFA) column in Table 2.

4. How do we overcome this EFCAB4B (CRACR2A/Rab46) genetic variation-mediated COVID-19 fatality?

Authors’ response:

Understanding the contribution of this gene to COVID fatality could provide diagnostics that allow identification of individuals who are most at risk. Advancements in genetic engineering such as in vivo CRISPR could one day be used therapeutically.

Reviewer 2 Report

Please provide a lucid hypothesis or research statement.

What were the selection criteria of the SNPs used in the analysis?

Why was regression analysis used in the current study, please provide the rationale as there are many other statistical analysis tools present.

Please provide the tables and figures to visualize the results.

Please provide the limitations of the current study in the discussion section.

Please update the references section and include more foundational studies on the roles of proteins and genetic epidemiological studies.

Please ensure consistent italics or bold SNP names or any other gene names.

Throughout the manuscript, please provide consistent statistical values.

In the introduction section, please provide the details of the association between COVID-19 and the EFCAB4B gene.

In the methodology section, please explain why these genes were analyzed in the current study.

If there is any possibility of bias in this genetic study, please state that it will not overshadow your results. It will rather improve the article. 

Please provide information on CRACR2A and Rab46 proteins and their corresponding roles in the discussion section.

Discuss the confounding factors and population stratification limitations in the current study.

While preparing the Figures or Tables, please include the identifiers of SNP, confidence intervals, odds ratios, p-values, and allele frequencies.

Author Response

We thank the reviewers for their comments, and we have revised and improved the manuscript accordingly. Please find the detailed point-by-point responses below.

Major comments

Please provide a lucid hypothesis or research statement.

Authors’ response:

After a description of the mechanisms that contribute to COVID-19 severity and describing the function of CRACR2A and Rab46 (the protein products of the EFCAB4B gene) we stated the hypothesis in the introduction as ‘we hypothesise that genetic variations in EFCAB4B may be instrumental in a subset of COVID-19 fatalities’.

What were the selection criteria of the SNPs used in the analysis?

Authors’ response:

We described in the methods that ‘filtering was carried out using --geno 0.1, --maf 0.01, and --hwe 0.000001 for variants’ and ‘only the variants residing on EFCAB4B gene have been retained according to start coordinate (3714799) and end coordinate (3874985) on human chromosome 12 based on the genome assembly version GRCh37’.

Why was regression analysis used in the current study, please provide the rationale as there are many other statistical analysis tools present.

Authors’ response:

Logistic regression is a commonly used approach in the genome-wide association studies to investigate the association between a genotype and a binary outcome. In this study, we used this approach to analyse three groups of binary outcomes: 1) Non-fatal vs negative; 2) Fatal vs negative; and 3) Fatal vs non-fatal.

Please provide the tables and figures to visualize the results.

Authors’ response:

All figures (Figure1-6) and tables (Table 1-4) are included in the manuscript.

Please provide the limitations of the current study in the discussion section.

Authors’ response:

In the discussion we have provided the limitations of the current study as follows:

“We recognise that a potential limitation of our study is the relatively small size of the COVID-19 data set, especially as the higher p values observed here could be due to the small sampling size in cohort 3. Analysis of additional data as they become available will be important for deeper understanding and exploration of EFCAB4B, especially in different populations and ethnicities.”

“We also need to be aware of the limitations of the study because the reported data are from a relatively small sample size in an aged population prior to the rollout of a vaccination programme in the UK. Moreover, there may be population differences in the allele frequencies, and due to the number of samples, we had to restrict our analysis to those who self-identified as British. The obtained results would require further validation in larger cohorts with different ethnicities and geographical regions.”

Please update the references section and include more foundational studies on the roles of proteins and genetic epidemiological studies.

Authors’ response:

We have included the key references (reference 17-25) on the roles of the gene/protein.

Please ensure consistent italics or bold SNP names or any other gene names.

Authors’ response:

We have gone through the manuscript and amended any errors.

Throughout the manuscript, please provide consistent statistical values.

Authors’ response:

In this study we have provided p-values for Table 1, and odds ratios (OR) and p-values for Table 3 and Table 4.

Detail comments

In the introduction section, please provide the details of the association between COVID-19 and the EFCAB4B gene.

Authors’ response:

In the introduction section, we have described two studies on the relevance of changes in CRACR2A protein expression to the recovery phase of the COVID-19 infection and long COVID (reference 24 and 25).

In the methodology section, please explain why these genes were analyzed in the current study.

Authors’ response:

There is only one gene analysed in this study – EFCAB4B – and the reasoning is explained in the introduction section where we describe how the function of the proteins encoded by EFCAB4B (Rab46 and CRACR2A) could contribute to the mechanisms underlying COVID-19 severity.

If there is any possibility of bias in this genetic study, please state that it will not overshadow your results. It will rather improve the article. 

Authors’ response:

To account for the potential effect of various factors, we have incorporated a number of covariates in the logistic regression model, including sex, age, the first ten principal components and six comorbidities.

Please provide information on CRACR2A and Rab46 proteins and their corresponding roles in the discussion section.

Authors’ response:

We have described the roles of Rab46 in endothelial cell degranulation and CRACR2A in immune cell calcium signalling in the introduction section, which are two key features of COVID-19.

In the discussion section we have indicated how these roles could contribute to COVID-19 as follows:

“We know that in endothelial cells, Rab46 acts as a brake in response to certain stimuli to prevent total release of WPB contents. Therefore, we could predict that inhibition of this function would lead to an increase in the release of pro-thrombotic and pro-inflammatory cargo thus leading to some of the clinical features observed in severe cases of COVID-19. In T-cells Rab46 is important for T-cell signalling, moreover we have shown that a patient who has biallelic mutations in the gene EFCAB4B (double mutation in allele1: R144G and E300*; allele 2 E278D), so that they have no longer express full length Rab46 or CRACR2A, exhibits reduced cytokine expression due to decreased calcium influx and JNK signalling. This suggests that overexpression or over-activation of Rab46 in T-cells could lead to the increase in cytokine release as observed in patients with cytokine storm reactions to COVID-19.”

Discuss the confounding factors and population stratification limitations in the current study.

Authors’ response:

We have added sex, age, the first ten principal components and six comorbidities as covariates in the logistic regression analysis to account for the potential confounding factors and population stratification.

While preparing the Figures or Tables, please include the identifiers of SNP, confidence intervals, odds ratios, p-values, and allele frequencies.

Authors’ response:

These are included in tables (Table 1-4) wherever appropriate.