Proteome profiling of early gestational plasma reveals novel biomarkers of congenital heart disease

Abstract Prenatal diagnosis of congenital heart disease (CHD) relies primarily on fetal echocardiography conducted at mid‐gestational age—the sensitivity of which varies among centers and practitioners. An objective method for early diagnosis is needed. Here, we conducted a case–control study recruiting 103 pregnant women with healthy offspring and 104 cases with CHD offspring, including VSD (42/104), ASD (20/104), and other CHD phenotypes. Plasma was collected during the first trimester and proteomic analysis was performed. Principal component analysis revealed considerable differences between the controls and the CHDs. Among the significantly altered proteins, 25 upregulated proteins in CHDs were enriched in amino acid metabolism, extracellular matrix receptor, and actin skeleton regulation, whereas 49 downregulated proteins were enriched in carbohydrate metabolism, cardiac muscle contraction, and cardiomyopathy. The machine learning model reached an area under the curve of 0.964 and was highly accurate in recognizing CHDs. This study provides a highly valuable proteomics resource to better recognize the cause of CHD and has developed a reliable objective method for the early recognition of CHD, facilitating early intervention and better prognosis.

26th May 2023 1st Editorial Decision 26th May 2023 Dear Prof. Zhao, Thank you for the submission of your manuscript to EMBO Molecular Medicine.We have now received feedback from the three reviewers who agreed to evaluate your manuscript.As you will see from the reports below, all referees recognize potential interest of the study, but also raise serious concerns that should be addressed in a major revision.The referee #1 point about "another set of proteomics data from same cohort before or after pregnancy will be very helpful to rule out this possibility" should be addressed in discussion as a limitation of the study.If you would like to discuss further the points raised by the referees, I am available to do so via email or video.Let me know if you are interested in this option.
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Referee #1 (Remarks for Author): Yin et al describe an interesting study that they identified a set of plasma proteins, which can be used as biomarkers for pregnant women with CHD.The samples were collected from over 100 pregnant women in the first trimester and well matched with control pregnant women without CHD.The proteomic data were carefully analyzed and some results were further validated by WB.The interpretation of data was appropriate.However, the main concern is on the significance of their findings.The dysregulation of these maternal proteins might be a reflection of exacerbation of certain preclinical conditions of these women under pregnancy, a high stressed condition, regardless of the status (with or without CHD).The argument of control pregnant women without these protein dysregulation is invalid: it might be simply that the control cohort did not have these preclinical conditions.Therefore, another set of proteomics data from same cohort before or after pregnancy will be very helpful to rule out this possibility.Of course, it is very difficult to do that.

Referee #2 (Remarks for Author):
This study attempts to address the problems related to the lack of an accurate method for early detection of congenital heart disease.The findings are interesting and include the identification of new potential biomarkers that combined have a high discriminatory capacity.However, there are limitations ice the enthusiasm and recall a proper methodological approach.The two cohorts recruitment is not clearly illustrated.In particular, the first group consists of women in which the diagnosis was done posnatally, therefore this is in principle a retrospective study unless differently indicated.If this is the case, the criteria to select these particular cases should be mentioned to exclude any potential bias.It is unclear if the investigators were blind to the recruitment and whether the results of the first study was known to the researchers leading the second study.Although the identification of an association between modules and protein pattern is attractive, the authors did not perform any assessment of specificity and sensitivity according to the severity of the disease.This is clinically important as women may be left with an incomplete information of the severity of the disease.The study analyzeed the inter group variability.However, it would be important to assess the reproducibility of data within each case.Finally, the authors assessed the ROC analysis without correction for the prevalence of the disease.Their control group is quantitatively similar to the disease group, but the prevalence of CHD is much lower.Therefore, their conclusion is an overestimation of the predictive value.The discussion does not present any element on how this omic approach can be administered and what would be it practicality and cost-benefit.Referee #3 (Remarks for Author): The study by Ya-Nun Yin et al. investigates a relevant topic, trying to answer whether a specific protein expression profile can distinguish mothers of children with congenital heart disease (CHD) from healthy controls.The study design is simple but appropriate to address the research question.The findings are intriguing, as some of the de-regulated proteins belong to pathways that are known to be involved in the development of congenital anomalies (e.g.focal adhesion; actin cytoskeleton), while on the other hand some detected proteins are novel associations.The methodology is appropriate; however, some components are described very briefly.The identification of clusters is interesting, yet the adoption of machine learning appears overstretched, given the patient numbers.This should be discussed and better justified.Some of the proteins found to be modulated are involved in lipid metabolism.On one hand, this represents an intriguing finding as dysregulation of lipid metabolism has been suggested to be associated with congenital anomalies such as the bicuspid aorta or Down syndrome.On the other hand, these findings might reflect overfitting, as cases present significantly higher lipid values compared with controls in the study cohort.
The study involves mainly (if not almost exclusively) ASD and VSD CHD patients.This should be clearly stated in the first result paragraph and emphasized in the abstract as well as better presented in the discussion.Abbreviations of CHD types in Table 1 should be spelled in the note to the table.

Point-by-point Responses to the Reviewers' comments
Referee #1 (Comments on Novelty/Model System for Author): they have chosen the right model to test their hypothesis: detection of plasma proteins possibly serving as biomarker for CHD.

Referee #1 (Remarks for Author):
Yin et al describe an interesting study that they identified a set of plasma proteins, which can be used as biomarkers for pregnant women with CHD.The samples were collected from over 100 pregnant women in the first trimester and well matched with control pregnant women without CHD.The proteomic data were carefully analyzed and some results were further validated by WB.The interpretation of data was appropriate.However, the main concern is on the significance of their findings.The dysregulation of these maternal proteins might be a reflection of exacerbation of certain preclinical conditions of these women under pregnancy, a high stressed condition, regardless of the status (with or without CHD).The argument of control pregnant women without these protein dysregulation is invalid: it might be simply that the control cohort did not have these preclinical conditions.Therefore, another set of proteomics data from same cohort before or after pregnancy will be very helpful to rule out this possibility.Of course, it is very difficult to do that.
Response: We appreciated the reviewer for the constructive comments.In our study, plasma samples of both case and control groups were collected from pregnant women during early pregnancy (10-12 weeks gestation), so we didn't detect the protein profile before pregnancy nor compare the difference before and after pregnancy.Pregnant women recruited in this study were confirmed not having obvious health problem nor severe metabolic disorders.However, there might be certain preclinical conditions 24th Aug 2023 1st Authors' Response to Reviewers undetected which can be exacerbated by pregnancy thus disturb the protein profile results.As what the reviewer recommended, another set of proteomics data from same cohort before or after pregnancy would be needed in future study to eliminate this possibility.We would like to further study it when suitable samples were available.
Following the reviewer's suggestions, we elaborated the exclusion criteria in the Materials and Methods part and discussed the limitation of this study in the Discussion part in our revised manuscript (lines 372-377, page 19; lines 325-329, page 17).

Referee #2 (Remarks for Author):
This study attempts to address the problems related to the lack of an accurate method for early detection of congenital heart disease.The findings are interesting and include the identification of new potential biomarkers that combined have a high discriminatory capacity.However, there are limitations ice the enthusiasm and recall a proper methodological approach.The two cohorts recruitment is not clearly illustrated.In particular, the first group consists of women in which the diagnosis was done posnatally, therefore this is in principle a retrospective study unless differently indicated.If this is the case, the criteria to select these particular cases should be mentioned to exclude any potential bias.
Response: Thank you for your comprehensive and insightful comments.We apologize for not clarifying the samples recruitment for two groups.In our study, the cases enrolled were the individuals confirmed having CHDs after birth who had echocardiographic evidence.Cases of isolated patent ductus arteriosus, patent foramen ovale, bicuspid aortic valve, coronary anomalies, as well as CHD that relate mainly to the vascular system were excluded from the present study.All patients with genetic syndromes or known chromosomal abnormalities (e.g., Down's syndrome, Holt-Oram syndrome, Alagille syndrome, DiGeorge syndrome, William syndrome, and Noonan syndrome) or family history of CHD in a first-degree relative (parent, sibling, or child) were excluded.Also, cases combined with other non-cardiovascular malformations, tumor or systematic diseases were not recruited.In our revised manuscript, we elaborated the exclusion criteria in the Materials and Methods part (lines 379-385, page 19).
It is unclear if the investigators were blind to the recruitment and whether the results of the first study was known to the researchers leading the second study.
Response: We apologize for not clarifying the process and the blinding of this research.
The recruitment, sample collection and the protein profiling of the group 1 from the Obstetrics & Gynecology Hospital of Fudan University and the group 2 from the International Peace Maternity and Child Health Hospital of the China Welfare Institute were conducted concurrently and independently.The investigators who conduct the following analysis and the building of machine leaning model were blind to the recruitment.The content above was supplemented into the revised Materials and Methods part (lines 367-372, 386-387, page 19-20).
Although the identification of an association between modules and protein pattern is attractive, the authors did not perform any assessment of specificity and sensitivity according to the severity of the disease.This is clinically important as women may be left with an incomplete information of the severity of the disease.

Response:
We appreciated the reviewer for the constructive comments.Here in the revised manuscript, we calculated the specificity and sensitivity of our model, which were added in revised Fig 5B , 5E and 5H.In our centers, most critical CHD cases were detected during early or mid-gestation by echocardiogram and aborted according to parents will, leaving no chance for genetic study or postnatal phenotype confirmation.As we only recruited cases who had a complete physical examination, echocardiogram and genetic study by neonatologists and pediatric cardiologists after birth, cases studied were mainly mild VSD and ASD, leaving insufficient data to conduct stratified analysis to assess the specificity and sensitivity according to the severity of the disease, which is certainly important to the clinical settings.This limitation is further elaborated in the revised Discussion part (lines 330-338, page 17).
The study analyzed the inter group variability.However, it would be important to assess the reproducibility of data within each case.

Response:
We appreciate the reviewer's comment.According to the reviewer's comment, we further assess the reproducibility of data within each case.In this study, we randomly selected 20 samples (10 CHD and 10 control) from 207 samples to analyze the abundance of correlated proteins across the whole measuring range.The results showed that the average correlation value of quantified protein signals between individual replicates in the healthy control group was 0.978, with a range of 0.96-0.99.Similarly, the average correlation value in the CHD group was 0.965, with a range of 0.93-0.99.However, the average correlation value between CHD and healthy control was 0.939 (Figure EV1A).In addition, to further illustrate the good reproducibility of data within each case, we picked 5 well-characterized plasma proteins and found that the quantification was highly reproducible in 103 healthy control and 104 CHD cases (Figure EV1B) (lines 118-127, page 7; lines 812-816, pages 37-38).B Reproducibility of the FOT of six proteins in 207 samples.FOT was defined as the iBAQ of a protein divided by the total iBAQ of all identified proteins within a sample.
Finally, the authors assessed the ROC analysis without correction for the prevalence of the disease.Their control group is quantitatively similar to the disease group, but the prevalence of CHD is much lower.Therefore, their conclusion is an overestimation of the predictive value.
Response: We appreciated the reviewer for the constructive comments.We agree with the reviewer and acknowledge that considering the low prevalence of CHD in the general population, the ROC analysis might overestimate the value of this diagnostic approach.
We emphasized this limitation in the revised Discussion part and stated that this approach should be applied with caution before verified in larger scale trials (lines 338-342, page 17).

The discussion does not present any element on how this omic approach can be
administered and what would be it practicality and cost-benefit.

Response:
We appreciated the reviewer for the constructive comments.Only 2 μL plasma from pregnant women at early gestation would allow the administration of this omic approach and guide mom-to-be with high risk of having CHD offspring to receive more intensive follow-up and prepare for prenatal intervention, which certainly helps to the targeted use of medical resources.In the revised manuscript, we elaborated the expectant administration and benefit of this omic approach in the Discussion part (lines 349-353, page 18).

13th Sep 2023 1st Revision -Editorial Decision
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(Reagents and ToolsTable, Materials and Methods, Figures, Data Availability Section) Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) (

In which section is the information available?
Table, Materials and Methods, Figures, Data Availability Section) (Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)Studies involving human participants: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.

granting approval and reference number for
Table, Materials and Methods, Figures, Data Availability Section) Could your study fall under dual use research restrictions?Please check biosecurity documents and list of select agents and toxins (CDC): https://www.selectagents.gov/sat/list.htmNot Applicable If you used a select agent, is the security level of the lab appropriate and reported in the manuscript?Not Applicable If a study is subject to dual use research of concern regulations, is the name of the authority the regulatory approval provided in the manuscript?

and III randomized controlled trials
Table, Materials and Methods, Figures, Data Availability Section) State if relevant guidelines or checklists (e.g., ICMJE, MIBBI, ARRIVE, PRISMA) have been followed or provided.Not Applicable For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right).See author guidelines, under 'Reporting Guidelines'.Please confirm you have followed these guidelines., please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission.See author guidelines, under 'Reporting Guidelines'.Please confirm you have submitted this list.Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) (