Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease characterized by left ventricular hypertrophy in the absence of abnormal loading conditions, affecting 0.16 to 0.29% of the general adult population (1). HCM is thought to be the most common cause of sudden cardiac death (SCD) in adolescents and young adults and is a major cause of heart failure, stroke, and ventricular arrhythmia (2). Given that approximately 30 to 60% of HCM is explained by pathogenic genetic variants in genes encoding proteins of the cardiac sarcomere, it was traditionally regarded as a monogenic disorder with an autosomal dominant pattern of inheritance (1). The missing causal gene of the remaining HCM cases remains largely unsolved.

ALPK3 codes for alpha-protein kinase 3 and is involved in the differentiation and maturation of cardiomyocytes in mice (3). It was first reported in pediatric cardiomyopathy that patients with biallelic truncating genetic variants in ALPK3 (ALPK3tv) displayed a phenotype of dilated cardiomyopathy (DCM) in utero, at birth, or early childhood, and often evolved to an HCM phenotype over time (4, 5). Subsequent studies emphasized the involvement of heterozygous truncating variants of ALPK3 in HCM, where co-segregation of a heterozygous ALPK3tv with HCM was reported in a family and enrichment of ALPK3tv in HCM was observed in cardiomyopathy cohorts compared with the Genome Aggregation Database (gnomAD) population (5, 6). More recently, Lopes et al. (7) demonstrated that heterozygous ALPK3tv are disease-causing variants with autosomal dominant inheritance in ∼1.5% of HCM cases in an international cohort. In addition, a high proportion of implantable cardioverter–defibrillator (ICD) referrals and fibrosis was also observed in ALPK3tv carriers in this study. However, little is known about the role of rare deleterious missense variants in ALPK3 in HCM. Whether ALPK3tv is also enriched in HCM patients in the East Asian population has not been closely examined. The prevalence of ALPK3tv carriers in East Asians and whether these cases are associated with the risk for SCD remains to be ascertained.

Thus, we compared the frequency of rare deleterious variants in ALPK3 in our HCM cohort with whole-exome sequencing to that of the gnomAD control population using the gene burden test. These were missense and truncating variants. The association was established using an independent cohort. Moreover, these heterozygous ALPK3 variant carriers were compared with non-carriers at baseline and follow-up to identify the clinical features of ALPK3 carriers.

The study cohort included 793 patients diagnosed with HCM, with a mean age of 52.64 ± 14.13 years at enrollment; 551 (69.5%) were male; all individuals were of East Asian descent (Supplementary Figure 1). Clinical information are shown in Table 1.

In this study, we performed exome sequencing of ALPK3 in a study cohort comprised of 793 HCM cases with East Asian ancestry and identified a batch of rare variants in ALPK3. Gene-based burden tests suggested that both missense and truncating variants in ALPK3 were enriched in HCM compared to gnomAD controls. Replication of these findings in another independent cohort with 419 HCM cases supported this association. Further baseline comparisons between carriers and sarcomere negatives indicated that ALPK3 variants were associated with more severe hypertrophy in IVS and apex. TWAS using published GWAS and GTEx data, found evidence to support the causal effects of ALPK3 expression on the odds of HCM.

Early studies focused more on biallelic ALPK3tv, which has been demonstrated to be associated with severe, childhood-onset cardiomyopathy and musculoskeletal abnormalities. Most ALPK3tv homozygotes die in the early years of life (4, 18–20). In our study and validation cohort, all ALPK3 variant carriers were heterozygous, and none of them displayed an additional dysmorphic phenotype or had raised serum creatine kinase apart from adult-onset HCM. This is in accordance with a more recent publication, where no apparent musculoskeletal involvement, except for HCM, was found in a Thai family with a heterozygous ALPK3tv (6). Consistent with the data reported by Lopes et al. (7), heterozygous ALPK3tv was also enriched in our cohorts made up of East Asians. However, the prevalence of ALPK3tv observed in our cohort was much lower than that observed by Lopes et al. (0.58% vs. 1.56%, P = 0.03), especially compared to the South Asian population subgroup (0.58% vs. 5.68%, P = 6.89e-4). Another notable observation is that none of the ALPK3tv identified in our study was previously reported, together indicating a population-specific prevalence and distribution across the transcript of ALPK3tv.

Missense genetic variants in sarcomere genes account for the majority of HCM cases, which may alter protein structure and function by changing the amino acid composition of the encoded protein (2). For this reason, it is necessary to evaluate the effect of missense variants in ALPK3 on HCM, whereas very little has been described in previous studies on this issue due to challenges in determining the pathogenicity of missense variants. Here, we attempted to identify rare deleterious missense variants using the in silico computational methods REVEL and VEST3. The two methods have been demonstrated to be effective with the best performance in predicting the pathogenicity of missense variants compared with other commonly used methods (11). Therefore, we additionally compared rare missense variants predicted as deleterious by both methods to minimize the risk of false positives between HCM cases and controls. Although these rare deleterious missense variants were no longer significantly enriched in the HCM validation cohort due to the limited number of carriers (Supplementary Table 5), the overall prevalence in the meta-analysis of the discovery and validation cohorts remained significantly higher relative to controls (4/1212 vs. 1/4523, P = 8.26e-3) with an OR of 14.96. The combination of these findings provides further support for the involvement of missense variants in ALPK3 in HCM, although the causal effect remains to be confirmed.

It was observed that a high proportion of ALPK3tv carriers were judged to be at high risk of SCD by Lopes et al.; however, no arrhythmic events were observed to verify this correlation. In this study, we only observed 1 single incidence of SCD among the 27 ALPK3 carriers that were followed, which was underpowered to draw any conclusion. Therefore, the link between ALPK3 and SCD needs to be ascertained in larger cohorts. Given the greater maximum LV wall thickness in ALPK3 variant carriers, it is reasonable to assume that ALPK3 variants partially contribute to SCD through this risk factor. Therefore, it is clearly of urgent importance to focus research on the arrhythmogenic role of ALPK3 variants. Preclinical evaluations in such patients are critical, particularly with respect to their capacity for early life-threatening arrhythmia. More aggressive strategies such as family screening and early arrhythmia monitoring should be recommended, as these patients may benefit from the primary prevention of SCD.

Recent GWAS have identified a locus near ALPK3 that is significantly associated with HCM and DCM with opposite effects (16). Considering the haploinsufficiency caused by ALPK3tv, which serves as the main mechanism causing HCM, it could conceivably be hypothesized that a reduced dosage of the ALPK3 transcript is causally associated with the risk of developing HCM. Our TWAS results supported this speculation. It is interesting to note that global alteration of LV traits associated with decreased ALPK3 expression highly coincides with the representative morphological characteristics of HCM, including decreased LV volume, increased LV wall thickness and mass, and enhanced LV contractility. So we further speculated that any variant that could negatively alter the expression of the ALPK3 protein may potentially contribute to HCM relevant phenotype, whether they are rare or common. On the contrary, it is unlikely that ALPK3tv could cause adult DCM, owing to the results that reduced ALPK3 expression in left ventricle was associated with decreased LV volume (LVEDV, LVEDVi, LVESV, LVESVi) and increased LV contractility (LVEF, global strain in radial and circumferential directions). The observations in our DCM cohort also supported this point of view, where the frequency of ALPK3tv in DCM was similar to that in gnomAD controls.

This study identified that heterozygous rare variants in ALPK3, both missense and truncating variants, were associated with an autosomal dominant HCM in East Asia via the demonstration of an enrichment of these variants in HCM cases. A considerable amount of work needs to be done to establish the causative role of ALPK3 missense variants in HCM, as well as the arrhythmogenic effect of these variants.

The data presented in the study are deposited in the https://zenodo.org/record/6580063#.Yo4G__n1u2U, accession number doi: 10.5281/zenodo.6580063.

The studies involving human participants were reviewed and approved by the Ethics Committee of Tongji Hospital, Wuhan, China. The patients/participants provided their written informed consent to participate in this study.

JD: conceptualization, formal analysis, investigation, methodology, visualization, and writing – original draft. KL and MH: data curation and investigation. YS: conceptualization, data curation, methodology, supervision, writing, review, and editing. HL: investigation. ZL and PC: conceptualization and methodology. HWa, DWu, YC, LX, and HWe: data curation. RL, LP, and TY: investigation. YW: project administration and supervision. DWa: conceptualization, funding acquisition, project administration, resources, supervision, writing, review, and editing. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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