Mitochondrial cardiomyopathy and ventricular arrhythmias associated with biallelic variants in C1QBP

Abstract

Patients with biallelic mutations in the nuclear-encoded mitochondrial gene C1QBP/p32 have been described with syndromic features and autosomal recessive cardiomyopathy. We describe the clinical course in two siblings who developed cardiomyopathy and ventricular fibrillation in infancy. We provide genomic analysis and clinical-pathologic correlation. Both siblings had profound cardiac failure with ventricular arrhythmia. One child died suddenly. The second sibling survived resuscitation but required extracorporeal cardiopulmonary support and died shortly afterward. On cardiac autopsy, the left ventricle was hypertrophied in both children. Histological examination revealed prominent cardiomyocyte cytoplasmic clearing, and electron microscopy confirmed abnormal mitochondrial structure within cardiomyocytes. DNA sequencing revealed compound heterozygous variants in C1QBP (p.Thr40Asnfs*45 and p.Phe204Leu) in both children. Family segregation analysis demonstrated each variant was inherited from an unaffected, heterozygous parent. Inherited loss of C1QBP/p32 is associated with recessive cardiomyopathy, ventricular fibrillation, and sudden death in early life. Ultrastructural mitochondrial evaluation in the second child was similar to findings in engineered C1qbp-deficient mice. Rapid trio analysis can define rare biallelic variants in genes that may be implicated in sudden death and facilitate medical management and family planning.

1. INTRODUCTION

Mitochondrial disorders are characterized by multisystem involvement, often including cardiac disease along with skeletal muscle abnormalities, central and peripheral nervous system involvement, ophthalmologic abnormalities, sensorineural hearing loss, and disorders of the gastrointestinal tract, liver, and kidney. Cardiac involvement can be heterogeneous in presentation, with features ranging from arrhythmias and heart failure in infants to progressive coronary artery disease, venous disease, and myocardial dysfunction that develop in adulthood (Lev et al., 2004; Schiff et al., 2011). However, it is especially important to recognize mitochondrial cardiomyopathy in infancy to identify patients at high risk of rapid progression to a catastrophic cardiac outcome and to assess extracardiac involvement that may impact the decision to perform heart transplant (Weiner et al., 2020).

Complement component 1Q subcomponent-binding protein (C1QBP), also called mitochondrial p32, is a ubiquitously expressed mitochondrial protein, predominantly localized in the mitochondrial matrix (Yagi et al., 2012). Mitochondria are involved in energy production, fatty acid and amino acid oxidation, apoptosis, and other aspects of cellular metabolism. Cardiomyocytes have exceptionally high mitochondrial density to meet the high demand for ATP.

To date, five unrelated patients have been described with cardiac phenotypes related to C1QBP (Feichtinger et al., 2017; Zhang et al., 2020). Two patients had severe neonatal cardiac hypertrophy and death, while the others had later-onset cardiac findings associated with a more widespread syndrome including progressive external ophthalmoplegia. A minority of cases had liver, kidney, and central nervous system findings (Feichtinger et al., 2017). Three unrelated patients with biallelic C1QBP mutations had progressive external ophthalmoplegia and variable skeletal myopathy, without cardiac findings, suggesting that phenotypic expression is not uniform (Marchet et al., 2020; Zhang et al., 2020).

C1qbp-knockout mice exhibit a mid-gestational lethal phenotype associated with severe cardiac developmental defects (Yagi et al., 2012). Mice with inducible deletion of C1qbp at 15–20 weeks of age develop cardiomyopathy with contractile dysfunction, dilation, and fibrosis (Saito et al., 2017). Ultrastructural evaluation of C1qbp-deficient mouse cardiac tissue with electron microscopy demonstrated disordered alignment and enlargement of the mitochondria. The mitochondria had internal vacuoles, loss of cristae, and disordered cristae formation. Cells isolated from these hearts showed severe dysfunction of the respiratory chain because of impaired mitochondrial protein synthesis. The C1qbp-encoded p32 protein functions as an essential RNA-binding factor in mitochondrial transcription. Mouse, yeast, and patient-derived cell models show defects in ribosomal formation, mitochondrial protein assembly, and oxidative phosphorylation (Feichtinger et al., 2017; Hillman & Henry, 2019; Yagi et al., 2012).

In this case report, we describe two siblings with cardiac hypertrophy and cardiac arrest. Genetic testing with a cardiac gene panel was unrevealing but exome and genome sequencing in the siblings identified biallelic variants in C1QBP. Ultrastructural analysis of the heart demonstrated abnormal mitochondria with vacuolization, supporting C1QBP/p32-related pathology.

2. CASE DESCRIPTION

2.1. Case 1

At the age of 7 months, a female child, born after a normal full-term pregnancy, presented with a 2–3 week history of intermittent vomiting. She was diagnosed with a presumed viral infection and sent home with instructions for supportive care. She became lethargic and was taken to a nearby emergency room, where her pulse was 126 beats per minute, blood pressure was 79/39 mmHg, and oxygen saturation was 75%. In the emergency room, she progressed to cardiopulmonary arrest with ventricular tachycardia, bradycardia, and then asystole. Resuscitation was attempted. A venous pH after obtaining a central line was 6.78 and the lactate was 90.4 mg/dl (normal 4–5 to 19.8 mg/dl). Return of spontaneous circulation could not be achieved. Her family history was negative for early death, cardiomyopathy, or heart rhythm disorders. Her mother and father obtained cardiac evaluations, including electrocardiograms and echocardiograms, which were normal.

2.2. Case 2

A second child by the same parents had a fetal ultrasound with normal heart structure and function at 20 weeks of gestation and an uncomplicated full-term delivery. On the first day of life, he was noted to be “dusky” with an oxygen saturation of 90% and a heart rate between 90 and 100 beats per minute. An echocardiogram was significant for right ventricular hypertrophy, mild qualitative left ventricular (LV) dysfunction, and a qualitatively mild concentric increase in the LV wall thickness. Supportive care was begun, including non-invasive supplemental oxygen, a nasogastric tube to avoid potential aspiration, and 36 h of intravenous antibiotics as standard prophylaxis against neonatal sepsis. Blood cultures were all negative and antibiotics were stopped. The mild decrease in cardiac function persisted on a repeat echocardiogram (LV ejection fraction 51%, normal 53%–74%). Due to his sister’s cardiac history and the mild cardiac dysfunction, he was transferred to our tertiary care center on day of life 3. Shortly after arrival, he became bradycardic and then developed coarse ventricular fibrillation (VF, Online Supplement, Figure S1). The coarse VF deteriorated into fine VF, followed by bradycardia with a slow ventricular escape. He was cannulated to a veno-arterial extracorporeal membrane oxygenator (ECMO) with 26 min from loss of spontaneous circulation to initiation of cannula flow. Temporary atrial and ventricular pacing leads were placed. Serum lactate peaked above 17 mmol/L (normal 0.5–2.2), returning to normal on the third day after cannulation. Electrolytes were normal with a potassium of 5.4 mmol/L (normal 4–5.9). An echocardiogram performed immediately after cannulation showed severely depressed biventricular function.

By 8 days post-arrest, he had recovered normal systolic function. ECMO support was removed, and he remained intubated, sedated, and fully paralyzed. Serum lactate and pyruvate measured 10 days after the arrest were normal (lactate 0.99 mM, pyruvate 0.07 mM, ratio 13; normal ratio = 10–20). Multiple attempts were made to reverse induced paralysis, but transient increases in spontaneous respiratory effort or skeletal muscle movement resulted in sinus bradycardia, desaturation, and hypotension, often requiring pacing using the temporary wires, despite normal LV systolic function and intact atrioventricular nodal conduction. His maximum LV mass, indexed to length, was 147 g/m (normal for age 19–39 g/m). Due to his end-stage heart failure and no extracardiac contraindications or confirmed mitochondrial disease at the time, he was listed as status 1A for cardiac transplantation. Despite escalating diuresis and ventilator settings, he developed progressive respiratory acidosis and his clinical condition deteriorated. The parents and the medical team decided to withdraw ventilatory support 27 days after presentation. Death occurred shortly afterward.

3. METHODS

Editorial Policies and Ethical Considerations: Appropriate human subjects approval was obtained from the Ann and Robert H. Lurie Children’s Hospital Institutional Review Board. Whole genome sequencing (WGS) was performed as detailed in the Online Supplement.

4. DIAGNOSTIC ASSESSMENT

4.1. Autopsy phenotype

4.1.1. Case 1

At autopsy, the heart weighed 90.7 g (upper 95% of normal for age: 45 g) (Schoppen et al., 2020). Grossly, the heart was globoid, with LV hypertrophy (Figure 1(d)). Microscopic examination of the heart showed scattered hypertrophic cardiac myocytes, many of which had cytoplasmic clearing (Figure 1(e), (f)). The myocytes stained negative for glycogen by periodic acid-Schiff stain with and without diastase. There was moderate subendocardial myocytolysis. Masson Trichrome and elastin histological stains demonstrated patchy, mild endocardial fibroelastosis within the left ventricle. No significant interstitial fibrosis was observed. CD3-positive T lymphocytes, CD68 positive macrophages, and a small number of CD20-positive B lymphocytes were scattered throughout the myocardium. Evidence for myocyte necrosis was absent.

FIGURE 1.

Gross and microscopic features of cardiomyopathy in case 1 (d, e, f) and case 2 (g, h, i). (a) Macroscopic examination dissected on the lateral left border along the base-to-apex plane. The mitral valve (MV) is at the top and normal or mildly hypertrophied left ventricular (LV) myocardium is shown. (b) Normal microscopic appearance of the myocardium with no significant cytoplasmic vacuolization. (c) Normal microscopic appearance, high power. (d) Case 1. Macroscopic examination of the opened left ventricle along the base-to-apex plane on the left lateral cardiac side shows the mitral valve at the top and marked left ventricular hypertrophy. (e) Microscopic section shows prominent cardiomyocyte cytoplasmic clearing. (f) Microscopic section, high power. (g) Case 2. Macroscopic examination of the left ventricle, cut in cross-section just below the papillary attachments shows marked concentric left ventricular hypertrophy. (h) Case 2. Microscopic section shows marked cardiomyocyte cytoplasmic vacuolization. (i) Case 2. Microscopic section, high power. All histologic sections were fixed with hematoxylin & eosin stain and shown at x200 magnification. All gross pathology and microscopic sections in (a-c) are from the same unrelated, age-matched control

4.1.2. Case 2

The heart weighed 50 g at autopsy (upper 95% of normal for age: 47 g) (Schoppen et al., 2020). Grossly, there was concentric LV hypertrophy (Figure 1(g)) with a LV wall thickness of 1.1 cm (expected for age: 0.59 cm, SD 0.15 cm) (Schulz et al., 1962). The right ventricle was also thickened at 0.9 cm (expected for age: 0.26 cm, SD 0.07 cm) (Schulz et al., 1962). Microscopically, there was marked cardiomyocyte cytoplasmic vacuolization (Figure 1(h), (i)), with no intracytoplasmic glycogen or fat deposits as demonstrated by periodic acid-Schiff stain with and without diastase and Oil red O stain, respectively. Mild individual cardiomyocyte hypertrophy and mild patchy architectural disarray were also present. There was mild, focal biventricular endocardial fibroelastosis and minimal interstitial fibrosis noted on trichrome stain. There was no evidence of interstitial inflammation.

Cardiac ultrastructural analysis with transmission electron microscopy was consistent with a mitochondrial myopathy (Figure 2). The preservation of myocytes was not optimal, potentially due to post-mortem collection of the sample. In areas with normal-appearing contractile units, mitochondrial alignment was disordered and mitochondrial size was variable. Internal vacuoles were present with loss of cristae, disordered cristae formation, and crystalloid globular inclusion bodies. There was no evidence of excessive glycogen, or lipid, or inflammation. These findings were similar to the ultrastructural mitochondrial changes observed in mice with inducible deficiency of C1qbp (Saito et al., 2017).

FIGURE 2.

Electron microscopy demonstrating abnormal mitochondria. (a). Electron micrograph of an unrelated case with normal appearance of the mitochondria. (b). Electron micrograph from Case # 2 shows disordered mitochondrial alignment and variable sizes of the mitochondria. Internal vacuoles were present with loss of cristae and disordered cristae formation with several crystalloid globular inclusion bodies (examples marked with arrows)

4.2. Genotype

4.2.1. Case 1

A post-mortem genetic test was performed (combined arrhythmia, cardiomyopathy, and autosomal recessive syndromic pediatric cardiomyopathy panels, 136 genes, Invitae, San Francisco CA). Two variants of uncertain significance were reported: AGL c.2930G>A, p.Arg977Gln (heterozygous, VUS) and MYLK2 c.558G>A, silent (heterozygous, VUS). Neither variant adequately explained the clinical phenotype and the family was counseled that the results were not diagnostic.

The family provided post-mortem DNA for research-based WGS. The initial analysis identified a heterozygous frameshift insertion in C1QBP (chromosome 17, position 5342275G>GT, c.118dupA, p.Thr40Asnfs*45). The allele frequency in gnomAD was 3.2 × 10⁻⁵. It was classified as a variant of uncertain significance following ACMG guidelines (Richards et al., 2015).

4.2.2. Case 2

DNA from Case 1, Case 2, and both parents were sent for rapid whole exome sequencing analysis (GeneDx, Gaithersburg MD, 13 days turnaround-time). Both siblings had compound heterozygous variants in C1QBP. Variant 1 was C1QBP c.118dupA, p.Thr40Asnfs*45, which had previously been identified in Case 1 by research-based WGS. Variant 2 was C1QBP c.612C>G, p.Phe204Leu (gnomAD frequency 2.4 × 10⁻⁵; deleterious by PP2 score, 81%, and the Sorting Intolerant from Tolerant (SIFT) algorithm, 79%; high evolutionary conservation of the residue). Each parent was heterozygous for one variant (maternal c.118dupA, paternal c.612C>G). Thus, each sibling inherited the C1QBP alleles in trans, consistent with an autosomal recessive disease pattern. Mitochondrial DNA (mtDNA) testing detected no pathogenic sequence changes, and the C1QBP p.Phe204Leu variant was present in the Case 1 WGS data, re-analyzed in context of the new family segregation data.

5. DISCUSSION

We present two siblings with cardiomyopathy, plus sudden death or ventricular fibrillation, with biallelic variants in C1QBP, inherited from heterozygous parents with normal cardiac function.

The two siblings shared the same macroscopic and histologic cardiac abnormalities including striking concentric LV hypertrophy and microscopic cardiomyocyte cytoplasmic vacuolization. Both patients exhibited scattered individual hypertrophic cardiomyocytes as well as mild endocardial fibroelastosis with minimal interstitial fibrosis. Similar endocardial fibroelastosis has been seen in Barth syndrome, which is due to abnormal mitochondrial function caused by mutations in the tafazzin gene (Barth et al., 1999). While the mild inflammatory infiltrate present in Case 1 may have been secondary to a viral infection, as suspected clinically pre-mortem, it is insufficient for a pathologic diagnosis of active myocarditis given the lack of damaged myocytes.

Case 2 had abnormal mitochondrial ultrastructural imaging in the setting of biallelic C1QBP variants. The clusters of variable-sized mitochondria with abnormal cristae architecture associated with crystalloid globular inclusion bodies is consistent with the previously published ultrastructural findings in mice with engineered C1qbp deficiency (Saito et al., 2017). The cases described here provide further delineation of the cardiomyopathy and arrhythmia phenotypes associated with C1QBP mutation and the second case supports ultrastructural mitochondrial findings previously noted only in mouse models.

From a clinical perspective, the process of genomic analysis in this family was also important. Neither C1QBP variant was identified with standard genetic screening of candidate genes. The tested gene panel included common arrhythmia and cardiomyopathy genes, plus well-recognized autosomal recessive genes associated with pediatric diseases. Recently, systemic re-evaluations of panels intended to diagnose common arrhythmia syndromes such as congenital long QT syndrome have questioned the value of including genes without monogenic genotype–phenotype correlation and without multiple lines of evidence supporting pathogenicity (Adler et al., 2020; Giudicessi et al., 2020; Ingles & Semsarian, 2020). While more stringent gene panel curation is one strategy to avoid identifying variants of uncertain significance which have limited clinical utility, the limitation of a narrow cardiac gene panel is that it excludes the discovery of potentially causative variants in rare, syndromic, or poorly described genes. Because cardiomyopathy associated with syndromes may manifest in severe form before other clinical features develop, it is important to consider mitochondrial syndromes in infants presenting with cardiomyopathy or sudden cardiac death. Furthermore, mitochondrial diseases with multiorgan failure are considered contraindications to cardiac transplantation, highlighting the importance of identifying these etiologies in all potential candidates.

The two cases in this manuscript also illustrate the difficulty and complexity of performing and interpreting post-mortem genomic sequencing. Genomic analysis of a single proband typically uncovers dozens or hundreds of single nucleotide polymorphisms in genes potentially related to cardiac function (Golbus et al., 2014). Furthermore, the arrangement of two or more variants in a single gene often cannot easily be identified as cis or trans without DNA from another family member. Once the family could be considered as a four-person unit, including both unaffected parents and both affected children, it was possible to identify potentially causative biallelic variants in C1QBP. The current study emphasizes the strength of a familial approach, using genomic analysis, rather than cardiac panels.

This case report illustrates an important association between the biallelic C1QBP variants and the clinical outcome. The parents offered a written perspective on the importance of genomic data for their family (Online Supplement).

The recessive inheritance of biallelic variants in C1QBP, combined with cardiac pathology, histology, and electron microscopy ultrastructural analysis support a mitochondrial etiology of disease. These data underscore the importance and utility of genomic diagnosis in early life for prognosis and family planning.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request. Sequence data will be submitted to dbGAP.