HLA Class I (A and B) Allele Polymorphism in a Moroccan Population Infected with Hepatitis C Virus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this report, Machraoui and coworkers exposed the outcome of a HLA class I genetic comparison between hepatitis C virus (HCV)-infected patients (n=40) and healthy controls (n=100) living in Southern Morocco around Marrakech. The authors used a high resolution (4 digits) genotyping method. The authors observed several significant differences including a protective role of HLA-A*02:01 that protects Moroccan individual against hepatitis C.

The paper is well-written and its presentation is pleasant.

However, there are different types of issues with it.

The first type concerns minor problems.

We do not know the clinical or demographical features of the healthy controls.

The size of the case group is very small (n=40).

The second type of problem is in my opinion more serious and pertains to the heuristic of science applied by the authors. I should say that most authors conducting genetic association studies in the domain of hepatitis have a vision of genetics that is rather crude.

The authors seem to consider hepatitis C as a genetic trait. Hence, by contrast with a genetic trait defined by alleles, Hepatitis C is not something that all human beings have. Patients have been exposed to the virus, first and did not get rid of it. Healthy controls recruited by the authors have never been exposed to the virus, thus, we do not know whether they are susceptible or not to this infection. Nevertheless, the authors are doing the assumption that controls represent the general population and although they were not exposed they are reflecting the genotype of non-susceptible individuals.

The problem is that we know that out of 100 persons exposed to HCV, 75 will develop a chronic infection and only 25 will spontaneously clear the virus. Thus, the proportion of susceptible individuals in a given population is three times larger than the proportion resistant ones. It is therefore difficult to find truly resistant persons in a population recruited without any criteria ie the method chosen by the authors.

A genuine control population will be persons who are anti-HCV(+)/HCV RNA(-) ie those who succeed wiping off the virus. There are not easy to find, though.

Another problem is linked to the structure of the Moroccan population that is a subtle mix of Berberic (majority), Arabic (in the cities), Sub-Saharan (in the south including Marrakech, where slaves were arriving until the beginning of the XXth century), and European ancestry (in the cities especially Tangiers in the North). It is known that HLA alleles differ significantly with geographical origin and ethnicity. The different ancestries are not evenly distributed both throughout Morocco and throughout social strata. Likewise, HCV is most probably not circulating evenly throughout the different component of the Moroccan society.

It is conspicuous that many factors irrelevant to the genetic susceptibility to hepatitis C might explain the genetic differences observed between cases and controls. Thus, it would have been more prudent either (i) to compare mitochondrial or Y-chromosome haplogroups in cases and controls before drawing strong conclusions about HLA-associated susceptibility to HCV, or (ii) to directly compare anti-HCV(+)/HCVRNA(+) ie susceptible individuals with anti-HCV(+)/HCVRNA(-) ie resistant individuals.

Author Response

1. Comment: We do not know the clinical or demographic features of the healthy controls.

Answer:
We have revised the Materials and Methods section to include detailed information on clinical and demographic characteristics of healthy controls. As potential organ or stem cell donors, controls were clinically healthy, with negative HCV, HBV, and HIV serologies. They were recruited from the same geographical region. Additionally, they had no history of chronic liver disease or known exposure to HCV or other bloodborne pathogens. This information was included in the revised manuscript (please see Lines77-79).

2. Comment: The size of the case group is very small (n=40).

Answer:

We acknowledge that the number of patients included in the study seems limited and may bias our results somewhat. However, it corresponds to the number of individuals collected over a two-year period, while ensuring that their clinical data are complete enough for a proper analysis, with the hope of enlarging the sample size for better inferences, We have explicitly stated this limitation in the Discussion section and emphasized the need for larger samle size and more diverse population to support our findings (please see Lines 289–290, 298–300).

Major Concerns

3. Comment: Healthy controls recruited by the authors have never been exposed to the virus; thus, we do not know whether they are susceptible or not to this infection. Controls are assumed to reflect the genotype of non-susceptible individuals, which is problematic.

Answer:
We do agree with this key limitation. In the revised manuscript, we have clarified that the control group represents a sample of the general population rather than a confirmed HCV-resistant individuals, especially as it is difficult to ensure rigorous follow-up of patients over a period that would allow assessment of viral clearance.

Additionally, we have rephrased our findings in the sense that they correspond to a distribution of allele frequencies rather than definitive associations with HCV susceptibility or resistance (please see Lines 272-273).

4. Comment: A genuine control population would include individuals who are anti-HCV(+)/HCV RNA(-), but these are not easy to find.

Answer:
Definitely, a comparison with anti-HCV(+)/HCV RNA(-) individuals would provide a more robust analysis of genetic resistance. However, such a cohort was unavailable for our study due to the challenges of recruiting these specific profils (essentially the time and testing required).

5. Comment: The structure of the Moroccan population, including ethnic diversity and uneven HCV circulation, could confound genetic associations.

Answer:
We fully agree that the genetic diversity of the Moroccan population and the uneven circulation of HCV could skew our results. Indeed, our study focused on a sub-population limited to southern Morocco. This would have reduced the ethnic heterogeneity and therefore the insensitivity to HCV variability that could be observed on a large national scale. On the other hand, this geographical and demographic constraint may improve the internal consistency of our sample and reduce the possibility of confounding variables possibly associated with higher ethnic and geographic diversity.

6. Comment: It would have been more prudent to compare mitochondrial or Y-chromosome haplogroups in cases and controls or to compare anti-HCV(+)/HCV RNA(+) individuals with anti-HCV(+)/HCV RNA(-) individuals.

Answer:
We appreciate the reviewer’s thoughtful suggestions for improving the study design. Unfortunately, our current dataset does not include mitochondrial or Y-chromosome haplogroups, nor were we able to recruit an anti-HCV(+)/HCV RNA(-) cohort. These are important research perspectives.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Correlating different HLA allele polymorphisms with susceptibility to various infections, or disease progression is not a novel approach, but the data generated from those research can help with understanding the mechanisms of pathogenesis. The manuscript: "HLA class I (A and B) allele polymorphism in a Moroccan population infected with Hepatitis C Virus" does offer an insight into distribution of HLA class I allele polymorphisms in general versus HCV infected population, however, the HCV study group is to miscellaneous to draw any conclusions other than mere polymorphism distribution. The introduction is somewhat short and I advise the authors to extend it a bit. Materials and methods do explain the experiment in detail, but the inclusion/exclusion criteria for the HCV study group is a bit vague. Was the only criteria that the patients were HCV positive? Presentation of the results works well, but I'm not sure the conclusions drawn from it are correct, especially considering small sample size. Assuming that statistically significant differences imply that certain allele forms are related to clearing the virus, it would be obligatory to present evidence that some of the study participants from the general population were, at one point in time HCV infected. To conclude, the manuscript is decently written, but the study design, as well as conclusions have flaws. Small sample size and insufficient inclusion/exclusion criteria make every conclusion drawn questionable. It would be better to do a comparison with two or more groups of HCV patients, divided for the severity of their symptoms than compare it to the general population. However, if the editor decides to publish the manuscript in this form I will not object.

Author Response

1. Comment: Correlating different HLA allele polymorphisms with susceptibility to various infections or disease progression is not a novel approach, but the data generated from those researches can help with understanding the mechanisms of pathogenesis.

Answer:
Absolutely. Our study has probably the merit of providing the first insight into the distribution of HLA A and B alleles in HCV-infected individuals from a Moroccan population, which has not previously been reported in the literature. The main contribution of our study lies in adding region-specific data to the growing body of research on HLA polymorphism and its potential role in HCV pathogenesis and outcome.

2. Comment: The introduction is somewhat short, and I advise the authors to extend it a bit.

Answer:
We have expanded the introduction to provide additional context about the role of HLA polymorphisms in infectious diseases, with a particular focus on HCV pathogenesis (please see Lines 36–44, 60–70).

3. Comment: The inclusion/exclusion criteria for the HCV study group are a bit vague. Was the only criterion that the patients were HCV positive?

Answer:
We have given the necessary details on the inclusion and exclusion criteria used in the study in Material and Methods section (please see Lines 77–89).

4. Comment: The small sample size and insufficient inclusion/exclusion criteria make every conclusion drawn questionable.

Answer :

We acknowledge that the number of patients included in the study is relatively limited and may affect the conclusions to be drawn. However, it corresponds to the number of individuals collected over a two-year period, while ensuring that their clinical data are complete enough for a proper analysis, with the hope of enlarging the sample size for better inferences, We have explicitly stated this limitation in the Discussion section and emphasized the need for larger sample size and more diverse population to support our findings.

5. Comment: It would be better to compare two or more groups of HCV patients divided by the severity of their symptoms rather than compare it to the general population.

Answer:
Following your recommendation, we have reanalyzed the data by dividing the HCV-infected population into two groups based on the treatment response and disease severity: (i). The first group included 26 patients with chronic HCV infection, confirmed by high viral load and fibroscan score between F3 and F4, assessed at 2 intervals (3 and 6 months after treatment). (ii) The second group consisted of 14 patients who were deemed to have successfully cleared the virus. This was evidenced by 3 and 6-month post treatment assessments, showing a significant decrease of viral loads, indicative of a sustained virologic response, and minimal liver damage, with fibroscan scores between F0 and F1.

These complementary analysis results are now included in the revised Results section. In addition, these findings are discussed in detail in the revised Discussion section, with a focus on the implications of HLA allele polymorphisms in disease progression and highlight on the differences observed between the severity groups.

Additionally, we have updated the conclusions to reflect these new findings and the potential for further research in larger, well-stratified cohorts.

6. Comment: Assuming that statistically significant differences imply that certain allele forms are related to clearing the virus, it would be obligatory to present evidence that some of the study participants from the general population were, at one point, HCV infected.

Answer:
We agree that differentiating between susceptibility to HCV infection and the ability to clear the virus is critical for interpreting our findings. In our study, the general population group consisted of clinically healthy individuals who were potential organ or stem cell donors. These participants were confirmed as negative for HCV, HBV, and HIV, with no history of chronic liver disease or known exposure to HCV or other bloodborne pathogens. However, as these individuals have not been tested for HCV antibodies, we cannot confirm whether any of them may have been previously exposed to HCV and successfully cleared the virus.

To address this limitation, we have mentioned that our findings showed statistically significant differences in allele frequencies between HCV patients and the general population, suggesting a possible protective effect of certain HLA alleles. Whereas, we have refrained from making definitive claims about the association of these alleles with resistance to HCV infection or the ability to clear the virus. This clarification has been added to the Discussion section (please see Lines 291–294).

Additionally, we have explicitly acknowledged the limitation of not having a confirmed anti-HCV(+)/HCV RNA(-) subgroup within the controls. Therefore, future studies should consider these aspects to better distinguish between susceptibility and clearance mechanisms.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I consider that the authors understand my point of view and I hope they agree with me at least partly.

They did their best to reply with honesty to my criticisms. I consider that the changes brought will enable the reader to have a more faithful idea of the situation of HLA-lined susceptibility to hepatitis C virus infection