Identifying and replicating plasma proteins associated with hypertrophic cardiomyopathy severity in carriers of pathogenic MYBPC3 variants

This study identified and replicated 21 plasma proteins associated with hypertrophic cardiomyopathy severity in MYBPC3 variant carriers, prioritizing five druggable targets that offer potential for diagnosis, monitoring, and therapeutic intervention independent of genotype.

The Gist

The Heart's "Smoke Alarm" System: Finding Early Warning Signs for a Genetic Heart Condition

Imagine your heart is a high-performance engine. In some people, a specific genetic glitch (a typo in the instruction manual called the MYBPC3 gene) causes this engine to build its walls too thick. This condition is called Hypertrophic Cardiomyopathy (HCM).

The tricky part? This glitch doesn't affect everyone the same way. For some, the engine runs fine with no issues. For others, the walls get so thick the engine starts to struggle, leading to heart failure or sudden stops. Doctors currently have to wait until the engine is already making noise (symptoms) or the walls are visibly thick on a scan to know how bad it is. They lack a simple "dashboard light" to predict who will get sick and how fast.

This study is like a team of detectives trying to find those dashboard lights by looking at the "exhaust fumes" (proteins) floating in the blood.

The Investigation: Two Teams, One Goal

The researchers split their investigation into two phases, like a detective working a case in two different cities.

1. The "Family Tree" Clue (The Discovery Team)
First, they looked at a specific group of 144 people who all carry the same genetic typo (the MYBPC3 variant). Think of this as a family reunion where everyone has the same family heirloom, but some wear it as a necklace, some as a belt, and some don't wear it at all.

• The Group: They categorized these people into three groups:
  • Unaffected: The engine is fine.
  • Mildly Affected: The engine is a bit heavy, but running.
  • Severely Affected: The engine is struggling, walls are very thick, or symptoms are present.
• The Method: They took blood samples and ran a "protein sweep" (using a high-tech scanner called Olink) to see what chemical signals were different between the groups.
• The Result: They found 27 specific proteins that acted like smoke alarms. The levels of these proteins changed depending on how sick the person was.

2. The "General Population" Check (The Replication Team)
Finding 27 alarms in one family is great, but are they universal? To be sure, the researchers went to the UK Biobank, a massive database of over 50,000 regular people (not just the specific family).

• They checked if those same 27 proteins could predict heart issues in the general public.
• They looked at who developed heart disease, who had thick heart walls on MRI scans, and who had heart failure.
• The Result: 21 of the 27 proteins were confirmed! This means these aren't just quirks of one family; they are universal warning signs for heart trouble.

The "Top 5" VIPs: The Most Important Clues

From the 21 confirmed proteins, the researchers narrowed it down to the Top 5 VIPs (Very Important Proteins) that are the most promising for the future. They picked them because:

1. They are actually produced by the heart.
2. We already have drugs that can target them (or are testing them).

Here are the Top 5, explained with simple analogies:

1. NT-proBNP: The "Stress Siren."

   • What it is: A well-known signal the heart releases when it's under pressure.
   • The Analogy: Like a car's "Check Engine" light that turns on when the engine is overheating. It's already used in hospitals, but this study confirms it's a key player in predicting the severity of this specific genetic disease.

2. GDF-15: The "Damage Control Signal."

   • What it is: A protein released when cells are stressed or damaged.
   • The Analogy: Think of it as the "Fire Department" being called. The more fire (damage) there is, the more GDF-15 is in the blood. It tells us how much damage the heart is currently enduring.

3. FGF-23: The "Bone-Heart Messenger."

   • What it is: Usually known for regulating phosphate and bones, but it also talks to the heart.
   • The Analogy: Imagine a construction foreman who is supposed to manage the building site (bones) but is accidentally shouting orders to the engine room (heart), causing it to build too many walls. We have drugs that can silence this foreman (like burosumab), which might stop the heart from over-building.

4. ADM (Adrenomedullin): The "Traffic Cop."

   • What it is: A protein that helps blood vessels relax and widen.
   • The Analogy: If the heart walls are too thick, blood has a hard time flowing. ADM is like a traffic cop widening the lanes so blood can flow easier. If we can boost this signal, we might reduce the strain on the heart. There are new drugs in development to help this protein work better.

5. NCAM1: The "Glue Glitch."

   • What it is: A protein that helps heart cells stick together.
   • The Analogy: Heart cells are like bricks in a wall. NCAM1 is the mortar holding them together. In this disease, the mortar gets messy, causing the wall to become unstable and the electrical signals (the spark plugs) to misfire. Drugs targeting this are currently being tested for cancer, but they might be "repurposed" to fix the heart's mortar.

Why This Matters: The "Repurposing" Revolution

The most exciting part of this paper is the Drug Repurposing angle.

Imagine you have a key that opens a door in a house you don't live in. Instead of making a whole new key, you realize that key fits a door in your house too.

• Three of the Top 5 proteins (FGF-23, ADM, and NCAM1) are already being targeted by drugs for other diseases (like bone disorders, heart failure, or cancer).
• This study suggests we might be able to take those existing drugs and use them to treat HCM patients, potentially speeding up treatment by years.

The Bottom Line

This study is a major step forward. It moved from "guessing" who will get sick to having a chemical checklist (the 21 proteins) that can:

1. Detect the disease earlier, even before symptoms appear.
2. Monitor how fast the disease is progressing.
3. Target the disease with drugs we might already have in the pharmacy.

By listening to the heart's "exhaust fumes," doctors might soon be able to fix the engine before it breaks down completely.

Technical Summary

1. Problem Statement

Hypertrophic Cardiomyopathy (HCM) is a clinically heterogeneous disease characterized by left ventricular hypertrophy (LVH) not explained by loading conditions. While pathogenic variants in the MYBPC3 gene account for approximately 40% of genetic HCM cases, the disease exhibits incomplete penetrance and highly variable severity (ranging from asymptomatic carriers to severe heart failure or sudden cardiac arrest).

• Current Gap: There is a lack of robust plasma biomarkers that can distinguish disease onset, monitor progression, and identify druggable pathways independent of genotype. Existing studies often focus on distinguishing HCM from hypertensive heart disease rather than stratifying severity within a specific genetic cohort.
• Objective: To identify plasma protein biomarkers associated with HCM severity in MYBPC3 carriers, replicate these findings in a general population, and prioritize targets for drug repurposing and disease monitoring.

2. Methodology

The study employed a discovery-replication-prioritization framework using two distinct cohorts.

A. Discovery Cohort (BIO FOr CARe Study)

• Population: 144 carriers of pathogenic MYBPC3 founder variants (truncating mutations causing haploinsufficiency).
• Phenotyping: Participants were classified into three severity groups based on clinical criteria:
  • Unaffected: No HCM diagnostic criteria met.
  • Mildly Affected: Met HCM criteria but lacked severe features (e.g., LV wall thickness <20mm, no obstruction, no heart failure).
  • Severely Affected: Met criteria for severe disease (e.g., LV wall thickness ≥20mm, heart failure, malignant arrhythmias).
• Proteomics: Plasma levels of 268 proteins were measured using Olink Target 96 panels (Cardiovascular II, III, and Cardiometabolic).
• Statistical Analysis: Random Forest machine learning models were used to identify feature importance. Two contrast strategies were applied:
  1. One-vs-one (e.g., Mild vs. Unaffected).
  2. All affected vs. Unaffected.
  • Hyperparameters were tuned via grid search; feature importance was ranked by the Gini index.

B. Replication Cohort (UK Biobank)

• Population: Up to 52,560 participants with Olink proteomics data; a subset of 6,492 had Cardiac MRI (CMR) data.
• Outcomes:
  • Time-to-onset of HCM (67 cases) and Cardiomyopathy (CMP) (156 cases) using Cox proportional hazards models.
  • Cross-sectional association with Left Ventricular Maximum Wall Thickness (LVMWT) via linear regression.
• Quality Control: Proteins with >25% values below the limit of detection were excluded. Missing data was imputed using K-nearest neighbors.

C. Prioritization and Annotation

Proteins were scored based on:

1. Discovery: Ranking in the top 10 features for severity comparisons.
2. Replication: Significant association with HCM, CMP, or LVMWT in the UKB.
3. Annotation: Cardiac tissue overexpression (Human Protein Atlas), pathway enrichment (Reactome), and druggability (ChEMBL database: approved drugs or compounds in clinical trials).

• Selection Criteria: Proteins required a prioritization score ≥5 (including at least one replication point) to be considered "prioritized."

3. Key Results

A. Discovery Phase

• 27 proteins were identified as top features associated with HCM severity in MYBPC3 carriers.
• NT-proBNP, GDF-15, and ACE2 were consistently important across all severity comparisons (indicating onset).
• Cystatin C and NCAM1 distinguished affected/mildly affected from unaffected.
• MERTK, ADM, REN, and FGF-23 were critical for distinguishing severe cases from unaffected.

B. Replication Phase

• 21 of the 27 proteins were successfully replicated in the UK Biobank, showing associations with incident HCM, CMP, or increased LVMWT.
• Five proteins met the strict prioritization criteria (Score ≥5):
  1. NT-proBNP: Strongly associated with HCM and CMP onset.
  2. GDF-15: Associated with increased risk of HCM/CMP and larger LVMWT.
  3. FGF-23: Associated with higher LVMWT and increased HCM risk.
  4. ADM (Adrenomedullin): Associated with higher LVMWT and increased HCM/CMP risk.
  5. NCAM1: Associated with HCM/CMP risk (notably, it showed a risk-decreasing effect direction in some contexts compared to others).

C. Annotation and Druggability

• Cardiac Expression: All five prioritized proteins are expressed in the heart; NT-proBNP, FGF-23, and NCAM1 are overexpressed in cardiac tissue relative to other tissues.
• Pathway Enrichment: FGF-23 and NCAM1 are involved in the Ras-MAPK pathway. ADM is involved in G-protein alpha (s) signaling (cAMP production).
• Drug Repurposing Potential:
  • ADM: Targeted by enibarcimab (in development for Heart Failure).
  • FGF-23: Inhibited by burosumab (approved for X-linked hypophosphatemia).
  • GDF-15: Targeted by visugromab and ponsegromab (Phase 2 for HF and cancer/cachexia).
  • NCAM1: Targeted by antibodies in clinical development for cancer.
  • Note: NT-proBNP is a standard biomarker but not a direct drug target in this context.

D. Pleiotropic Associations

The five prioritized proteins were further tested against major HCM endpoints in the UKB:

• Heart Failure (HF): All five proteins were associated.
• Dilated Cardiomyopathy (DCM), Sudden Cardiac Arrest (SCA), Ventricular Arrhythmias (VA): Four of the five proteins (excluding NCAM1 for some outcomes) showed significant associations, suggesting these markers reflect broad mechanisms of cardiac remodeling and electrical instability.

4. Significance and Conclusion

• Genotype-Independent Biomarkers: By replicating findings in the unselected UK Biobank (which includes non-MYBPC3 and non-genetic HCM), the study demonstrates that these proteins (NT-proBNP, GDF-15, FGF-23, ADM, NCAM1) are associated with HCM severity independent of the specific genetic cause.
• Clinical Utility: These proteins offer a multi-modal approach for:
  • Early Detection: Identifying at-risk variant carriers before overt hypertrophy.
  • Risk Stratification: Monitoring disease progression from mild to severe stages.
  • Therapeutic Targets: Highlighting four proteins (GDF-15, FGF-23, ADM, NCAM1) with existing or developmental pharmacological interventions, providing immediate opportunities for drug repurposing in HCM.
• Mechanistic Insight: The findings implicate pathways beyond sarcomeric function, specifically the Ras-MAPK pathway and vasodilatory/RAAS pathways, suggesting that HCM progression involves complex remodeling processes that can be targeted pharmacologically.

Limitations: The discovery cohort was genetically homogeneous (truncating MYBPC3 only), and replication relied on ICD codes which may miss early-stage disease. However, the consistency across cohorts strengthens the validity of the prioritized biomarkers.