BIO FOr CARE: biomarkers of hypertrophic cardiomyopathy development and progression in carriers of Dutch founder truncating MYBPC3 variants—design and status

Hypertrophic cardiomyopathy (HCM) is characterised by hypertrophy of the ventricular wall not explained by abnormal loading conditions [1]. HCM is an important cause of sudden cardiac death (SCD) [2] and may also lead to end-stage heart failure and left ventricular outflow tract (LVOT) obstruction [3]. The prevalence of HCM has historically been estimated at 1:500 [4], making it the most common Mendelian heart disease, with more recent estimates as high as 1:200 [5]. HCM is typically inherited as an autosomal dominant disease and a likely pathogenic/pathogenic variant is found in approximately 50% of patients [6, 7]. The most commonly affected gene is MYBPC3, which encodes cardiac myosin-binding protein C (cMyBP-C), an important regulator of cardiomyocyte contraction [3].

Despite the association with life-threatening arrhythmia and end-stage heart failure, clinical severity in HCM is highly variable, with low overall mortality [8] and incomplete, age-dependent penetrance in carriers of pathogenic variants (G+) [9]. This highlights the need for risk stratification. Current guidelines advocate the use of the HCM Risk-SCD calculator to identify patients who may benefit from a prophylactic implantable cardioverter-defibrillator [1, 10]. However, prediction of SCD remains imperfect [11] and risk-prediction models of other aspects of HCM have not yet been established. Furthermore, underlying mechanisms contributing to disease progression, such as environmental and additional (epi)genetic factors, are still unclear.

Similarly, prediction of penetrance in G+ phenotype-negative (LVH-) individuals is limited. Risk factors and biomarkers found in exploratory studies, including electrocardiographic abnormalities and impaired diastolic function on echocardiography [12‐14], have not yet been validated in large, prospective cohorts. Without such data, these individuals undergo frequent cardiological screenings with a major impact on the health care system and costs.

The Dutch HCM population is relatively genetically homogeneous, with three MYBPC3 founder variants (c.2373dupG (p.Trp792fs), c.2827C > T (p.Arg943Ter) and c.2864_2865delCT (p.Pro955fs), each inherited from a distant common ancestor) accounting for up to 35% of Dutch HCM cases [15]. Another truncating MYBPC3 variant, c.3776delA (p.Gln1259fs), has been identified in multiple independently presenting HCM patients. These genetic variants result in truncated mRNA, absence of truncated cMyBP‑C protein and a reduction of functional cMyBP‑C (haploinsufficiency) [16], which impairs cardiomyocyte function [17], and have been shown to have a similar prognostic impact [18]. Together, they provide a unique opportunity to study effect modifiers free from confounding resulting from distinct genotypes.

In the BIO FOr CARe (Identification of BIOmarkers of hypertrophic cardiomyopathy development and progression in Dutch MYBPC3 FOunder variant CARriers) study we aim to investigate predictive factors and effect modifiers for penetrance and progression of HCM in the genetically homogeneous group of Dutch carriers of truncating MYBPC3 variants. Here we report the design and current status of this cohort and discuss ongoing and future studies.

A flowchart outlining subject inclusion and follow-up is presented in Fig. 2. A total of 250 G+ individuals were included in the study up to 1 January 2020. In 232 subjects the diagnosis could be ascertained. HCM was diagnosed in 169 subjects and DCM without prior diagnosis of HCM in 4 subjects. The remaining 59 subjects were G + LVH- at the time of inclusion. Baseline characteristics are presented in Tab. 1. A male predominance (66%) was observed among index patients, similar to previous studies [21, 22]. To date, blood samples have been obtained from 131 subjects. As shown in the Electronic Supplementary Material (Table S2), the baseline characteristics of subjects who have undergone blood collection and those who have not are similar.

Currently, two biomarker discovery studies are ongoing in the BIO FOr CARe cohort, both utilising metabolomics approaches. Previous studies have indicated a perturbed energy metabolism in HCM with an increased adenosine triphosphate utilisation and decreased energetic efficiency [16, 23]. Acylcarnitines are forms of fatty acids transported into mitochondria for energy production [24]. Changes in acylcarnitine concentrations have been shown to reflect a shift away from the fatty acid oxidation that is normally predominant in cardiac tissue, towards glucose utilisation in a MYBPC3 HCM mouse model treated with perhexiline (which blocks transportation of fatty acids into mitochondria) [25]. This shift to increased glucose utilisation is more generally observed in heart failure [26], in which increased levels of long-chain acylcarnitines have been associated with worse prognosis [27].

In the BIO FOr CARe study, acylcarnitines were determined using targeted metabolomics in 121 plasma samples, together with 27 samples collected prior to genetic testing as part of the UNRAVEL biobank [28], including 12 genotype-negative relatives not eligible for inclusion in BIO FOr CARe. To further study changes in the metabolome associated with HCM, untargeted metabolomics using a previously published direct-infusion high-resolution mass spectrometry platform [29] was performed in a nested case-control study, consisting of 30 subjects with a severe phenotype and 30 age- and sex-matched subjects with a mild or no phenotype, as well as 10 age- and sex-matched genotype-negative family members from the UNRAVEL biobank.

Additionally, an exploratory proteomics study using the Olink Cardiovascular III multiplex immunoassay (for more information, visit: https://​www.​olink.​com/​products/​cvd-iii-panel/​) is being designed. Furthermore, the BIO FOr CARe infrastructure is currently being used for observational cohort studies involving other HCM genotypes and for gene-expression studies in induced pluripotent stem-cell-derived cardiomyocytes, and will be used for potential future genome-wide association studies/polygenic risk score analyses.

Standardisation of blood collection allows for future biomarker studies requiring plasma, serum or RNA. By repeating blood collection at subsequent time points, changes in biomarker concentrations can be validated over time and across progressive stages of the phenotype. The current biomarker studies are still limited to a cross-sectional design. However, longitudinal assessment of biomarkers will become possible over time, as disease onset and progression may occur in carriers during follow-up.

The prognostic utility of biomarkers in HCM is also being studied in several other studies, such as the HCMR—Novel Markers of Prognosis in Hypertrophic Cardiomyopathy (HCMR) study (ClinicalTrials.gov Identifier: NCT01915615) [30], the Predictive Factors and Consequences of Myocardial Fibrosis in Hypertrophic Cardiomyopathy (HCM) study (NCT02922517), the Cardiac Biomarkers in Pediatric Cardiomyopathy (PCM Biomarkers) study (NCT01873976), and the An Integrative-‘Omics’ Study of Cardiomyopathy Patients for Diagnosis and Prognosis in China (AOCC) study (NCT03076580). Our study distinguishes itself by its genotype-driven study population, including carriers of pathogenic variants regardless of their phenotype, and its longitudinal design. The inclusion of G + LVH- individuals is made possible by the widespread availability of genetic screening to family members (covered by the national basic healthcare plan), which has led to the identification of many G + LVH-family members [7]. This provides a unique opportunity to study biomarkers and their prognostic utility at subsequent stages of disease progression. Furthermore, the high prevalence of MYBPC3 founder variants in the Dutch HCM population allows analyses free from confounding resulting from genetic heterogeneity [15].

Additionally, the ancillary REDCap registry offers a user-friendly, sustainable platform for clinical data collection using standardised definitions. Similar to the Netherlands Arrhythmogenic Cardiomyopathy Registry [31], this may serve as a foundation for future observational studies, involving carriers of other variants and other genetic cardiomyopathies. A wide range of data is collected from the subject’s first presentation onwards, enabling both cross-sectional and longitudinal studies as well as hypothesis-driven and hypothesis-generating approaches. New variables can easily be added to accommodate studies focussing on specific disease aspects.

Both data and samples are available to external researchers through submission of an application to our data access board, which consists of investigators from each participating centre. So far, inclusions have focussed on the genetically homogeneous cohort of pathogenic truncating MYBPC3 variants. Although the prognosis of HCM patients carrying founder variants in MYBPC3 has been shown to be similar to other G + HCM [18, 21], findings in our current cohort will have to be validated in the general HCM population. An amendment to the BIO FOr CARe protocol to include all HCM genotypes has recently been accepted. External validation may be performed in large multi-national cohorts such as the HCMR study [30]. Potenzial effect modifiers may be further studied in myocardial samples collected from septal myectomies or during heart transplantation.

A limitation of the current study design is the risk of referral bias, as blood collection is limited to academic hospitals, which may hinder patients who are under cardiological follow-up elsewhere and do not live nearby. This likely contributed to the higher than anticipated number of subjects fulfilling the primary outcome at baseline (50%). Extending blood collection to regional hospitals would ameliorate the potential bias. A second limitation is that clinical data were not collected prospectively, resulting in missing data. A further limitation to this study is that event rates are low in HCM [8]. As a result, a large sample size and long follow-up duration are required for prospective and longitudinal studies, for which embedding in a sustainable infrastructure and long-term funding are essential. Finally, as the subjects in this study were ascertained as index patients or through their relatedness to one, results may not be generalisable to carriers of MYBPC3 variants in the general population. Instead, population-based studies are required to investigate the prognosis of carriers in the general population.