Integrating Genetic, Clinical, and Histopathological Data for Definitive Diagnosis of PRKAG2-Related Disease

Abstract

Background: PRKAG2-related disease is an autosomal dominant disorder caused by pathogenic variants in the PRKAG2 gene, leading to glycogen accumulation in cardiomyocytes. It is characterized by left ventricular hypertrophy (LVH), ventricular pre-excitation, and conduction disease. Due to the rarity of the condition and the frequent occurrence of private variants, functional or pathological testing is required for definitive pathogenicity classification. Case Presentation: We describe a 22-year-old male referred for evaluation after experiencing exertional dyspnea and a syncopal episode. Family history revealed sudden cardiac deaths and conduction disease requiring pacemaker implantation. The patient exhibited mild LVH on imaging, conduction abnormalities on electrophysiological study, and a heterozygous PRKAG2 variant (c.1643C>T; p.Ser548Leu), classified as likely pathogenic according to ACMG guidelines. Cascade screening identified the variant in three family members, one of whom exhibited a positive phenotype. Endomyocardial biopsy revealed glycogen accumulation, providing histopathological confirmation of PRKAG2-related disease. Conclusions: This case underscores the importance of integrating genetic, clinical, and histopathological data in variant interpretation. Endomyocardial biopsy can provide definitive evidence to reclassify a PRKAG2 variant as pathogenic, thereby guiding management and family screening.

1. Introduction

PRKAG2-related disease (OMIM #602743) is an autosomal dominant disorder caused by pathogenic variants in the PRKAG2 gene, which encodes the γ2 regulatory subunit of AMP-activated protein kinase (AMPK) [1]. AMPK is a central regulator of cellular energy homeostasis, sensing AMP/ATP ratios and modulating metabolic pathways. Variants in PRKAG2 impair AMPK function, leading to glycogen accumulation within cardiomyocytes and a characteristic triad of left ventricular hypertrophy (LVH), ventricular pre-excitation, and conduction disease [1].

Its clinical presentation can mimic other genetic cardiomyopathies with diverse molecular aetiologies, including sarcomeric gene variants (e.g., MYH7, MYBPC3), cytoskeletal defects (e.g., DES), and metabolic storage disorders (e.g., LAMP2, GLA) [2]. This phenotypic overlap underscores the need for precise molecular diagnosis to guide clinical management and family screening.

Given the rarity of PRKAG2-related disease and the frequent occurrence of private variants, variant classification can be challenging [3]. Variants are designated as likely pathogenic when genetic and phenotypic evidence supports causality but lacks confirmation. Functional or histopathological data can provide the additional evidence needed for reclassification. We describe a case in which the integration of genetic, clinical, and histopathological data allowed reclassification of a PRKAG2 variant from likely pathogenic to pathogenic. Furthermore, the purpose of this report is not only to establish pathogenicity but also to critically assess the relationship between the genetic variant and the patient’s phenotype.

This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of our institution.

2. Case Summary

The proband was the third of four children born to non-consanguineous parents. At age 22, he was referred for evaluation after several weeks of exertional dyspnoea and one episode of syncope.

2.1. Family History

The family history was notable for sudden death (maternal aunt at age 16, grandmother at age 46) and conduction disease. The proband’s mother had a pacemaker implantation at age 46 for third-degree atrioventricular block (AVB). One brother had LVH and required a pacemaker for advanced AVB.

2.2. ECG

The electrocardiogram showed normal atrioventricular conduction, right bundle branch block, and early repolarization in the inferior leads.

2.3. Imaging

Echocardiography showed increased left ventricular wall thickness (maximum 13 mm), while cardiac magnetic resonance showed mild LVH with preserved systolic function and normal right ventricular volumes and systolic function (Figure 1). Late gadolinium enhancement showed no fibrosis or scarring.

2.4. Electrophysiological Study

The electrophysiological study demonstrated prolonged sinus node recovery time and prolonged atrioventricular nodal conduction time, without inducible sustained arrhythmias.

2.5. Genetic Analysis

Given the clinical presentation and family history, genetic counselling was performed, and next-generation sequencing (NGS) was conducted using a 325-gene cardiomyopathy panel (a full list of included genes is provided in the Supplemental Material). A blood sample was collected in an EDTA tube from the patient. Genomic DNA was then extracted from this sample using the Maxwell 16 instrument (Promega, Madison, WI, USA), ensuring high-quality DNA suitable for sequencing. DNA quality and concentration were assessed using a Nanodrop spectrophotometer.

The molecular testing involved analysing the selected target genes through an NGS-based procedure. Sequence data generated were processed and analysed using Alyssa software (Agilent, Santa Clara, CA, USA), which aligns the sequences to the human reference genome. This alignment enables the identification of genomic variants, which are subsequently prioritized by a custom bioinformatic pipeline designed to highlight pathogenic or potentially pathogenic mutations. The minor allele frequency (MAF) threshold was set at 5% using the Illumina Variant Interpreter Software to filter out common benign variants.

A heterozygous PRKAG2 variant (c.1643C>T; p.Ser548Leu; NM_016203.4; chr7-151257645 G>A) was identified. According to the American College of Medical Genetics and Genomics (ACMG) guidelines [4], the variant met criteria PM2 (moderate), PM5 (moderate), PP3 (moderate), and PP5 (supporting) and was classified as likely pathogenic. No other clinically relevant variants were detected.

2.6. Cascade Screening

Cascade screening revealed the same variant in one brother and two daughters of the proband (Figure 1). The brother had a positive cardiac phenotype (i.e., LVH); both daughters were phenotypically negative at the time of testing. Other family members were unavailable for testing.

2.7. Endomyocardial Biopsy and Histopathology

A multidisciplinary review concluded that additional evidence was required for definitive pathogenicity classification. Although the genetic result was consistent with a PRKAG2-related disease, the proband’s mild LVH and absence of overt pre-excitation were considered borderline for the syndrome. Given this incomplete fulfillment of the classical phenotype, the multidisciplinary team concluded that histopathological confirmation was required to establish disease expression. As per ACMG recommendations, functional or histopathological confirmation can fulfill PS3 criteria (strong) for pathogenicity. Endomyocardial biopsy demonstrated glycogen accumulation within cardiomyocyte vacuoles (Figure 1), confirming a glycogen storage phenotype consistent with PRKAG2-related disease. In this case, while functional cell-based assays were not performed, histopathological evidence served as functional corroboration. Based on these findings, the variant was reclassified from likely pathogenic to pathogenic.

3. Discussion

This case illustrates the stepwise process of reclassifying a PRKAG2 variant from likely pathogenic to pathogenic by integrating genetic, clinical, and histopathological data. Beyond variant classification, the main clinical question concerned the degree to which the PRKAG2 finding accounted for the patient’s phenotype.

The p.Ser548Leu variant has been previously reported in a patient with hypertrophic cardiomyopathy (maximal left ventricular wall thickness 18 mm) and high-degree atrioventricular block presenting with syncope [5]. In our case, the proband showed only mild LVH and no overt signs of glycogen storage disease, underlining the need for a comprehensive diagnostic approach that includes histopathology when the phenotype is subtle. Although the electrophysiological study demonstrated conduction abnormalities, this evidence alone was not considered sufficient to fulfill PS3 criteria under ACMG guidelines. In our opinion, histopathological confirmation remains the strongest form of functional evidence for PRKAG2-related disease in this context.

While the use of multi-gene panels is the standard of care in patients affected by cardiomyopathies [6], interpretation of novel or private variants can be difficult. The ACMG framework provides criteria for the assignment of pathogenicity to variants, which can help physicians in their interpretations [4]. These criteria are based on multiple parameters, including clinical data from patients and families, bioinformatic analysis that utilizes population data, and the interpretation of established data repositories and clinical databases.

The role of clinicians in variant interpretation is essential since many of the ACMG criteria are based on clinical and phenotypic data, as well as family segregation studies [7]. Many PRKAG2 variants are initially reported as VUS. A large multicentre study found that among 90 patients diagnosed with PRKAG2-related disease, a pathogenic or likely pathogenic variant was detected in only 28 cases (31%) [3].

Given the phenotypic overlap of PRKAG2-related cardiomyopathy with other conditions, clinicians should consider EMB in select cases where genetic findings remain short of full pathogenic criteria. Although invasive, EMB can provide evidence for pathogenicity and influence patient management and family screening.