Sudden unexpected death in asymptomatic infants due to PPA2 variants

Abstract Background Sudden death in children is a tragic event that often remains unexplained after comprehensive investigation. We report two asymptomatic siblings who died unexpectedly at approximately 1 year of age found to have biallelic (compound heterozygous) variants in PPA2. Methods The index case, parents, and sister were enrolled in the Sudden Unexplained Death in Childhood Registry and Research Collaborative, which included next‐generation genetic screening. Prior published cases of PPA2 variants, along with the known biology of PPA2, were also summarized. Results Whole exome sequencing in both siblings revealed biallelic rare missense variants in PPA2: c.182C > T (p.Ser61Phe) and c.380G > T (p.Arg127Leu). PPA2 encodes a mitochondrially located inorganic pyrophosphatase implicated in progressive and lethal cardiomyopathies. As a regulator and supplier of inorganic phosphate, PPA2 is central to phosphate metabolism. Biological roles include the following: mtDNA maintenance; oxidative phosphorylation and generation of ATP; reactive oxygen species homeostasis; mitochondrial membrane potential regulation; and possibly, retrograde signaling between mitochondria and nucleus. Conclusions Two healthy and asymptomatic sisters died unexpectedly at ages 12 and 10 months, and were diagnosed by molecular autopsy to carry biallelic variants in PPA2. Our cases add additional details to those reported thus far, and broaden the spectrum of clinical and molecular features of PPA2 variants.


| CASE HISTORIES
The index case was a 12-month-old Caucasian girl who was healthy and asymptomatic, with normal growth and development until 2 days before death. The prenatal history was unremarkable; mother smoked 10 cigarettes per day during pregnancy. The infant's only diagnosis was "bronchitis" at age 6 months. In the 2 days before death, she experienced fever, vomiting, diarrhea, and poor appetite. The day before her death, she was sleepy and was not especially active; she fell over a few times and looked like "her legs gave out." Throughout her last night alive, she had episodes of vomiting and diarrhea. During the last episode, she vomited twice and then seemed to gasp for air; mother felt her breathing change, her eyes rolled backward, and she stopped breathing. At autopsy, there were nonspecific features, which together with circumstances of a witnessed collapse, suggested a cardiac arrhythmia including: (a) contraction bands in cardiac muscle fibers, which while not pathological, suggested an acute cardiac event; (b) pleural and pericardial effusions, and ascites which may be secondary to resuscitation, but were considered more than is usually encountered; (c) prominent intra-alveolar hemorrhage and acute pulmonary edema which could be secondary to resuscitation, but was considered more striking than usual.
Family history revealed a sister who had died suddenly at 10 months of age, a death that was attributed to a cardiac arrhythmia (autopsy report noted that she was witnessed to have screamed, gasped for breath, and became rigid and blue) but whose death was certified as a sudden infant death syndrome (SIDS) case. This sibling had research genetic testing through the National Health Service: 169 genes were sequenced (38 genes associated with arrhythmia, with no pathogenic variants found). The mother had a history of episodic syncope of unknown etiology, not provoked by exercise or other factors, beginning at 13 years of age through her first pregnancy at 18 years of age.
The index case, parents, and sister were then enrolled in the SUDC Registry and Research Collaborative (SUDCRRC). To enroll in the SUDCRRC, parents consented to release deidentified data for research. The Foundation provided deidentified data for analysis. The New York University Institutional Review Board considered this study exempt. Testing included genetic screening at the Institute for Genomic Medicine (IGM) at Columbia University. Whole exome sequencing in both siblings revealed biallelic rare missense variants in PPA2: c.182C > T (p.Ser61Phe) and c.380G > T (p.Arg127Leu). PolyPhen-2 predicted these compound heterozygous variants to be "probably damaging": p.Ser61Phe score, 0.998 (sensitivity, 0.27; specificity, 0.99); and p.Arg127Leu score, 0.993 (sensitivity, 0.70; specificity, 0.97) (http://genet ics.bwh. harva rd.edu/pph2/, accessed 05/11/2019; Adzhubei et al., 2010). Neither variant is observed in the homozygous state in population databases, such as gnomAD (Lek et al., 2016). Prior cases of PPA2 variants are shown in the Table  1, in addition to our cases.

| DISCUSSION
We identified two sisters with biallelic variants in PPA2, which encodes a mitochondrially located inorganic pyrophosphatase (PPase) that is implicated in progressive and lethal cardiomyopathies. The combination of both the p.Ser61Phe and p.Arg127Leu variants in our compound heterozygotes has not been reported before; each of these variants was identified separately in two sudden death cases from two earlier case series (Guimier et al., 2016;Kennedy et al., 2016). Our cases represent the severe range of a clinical spectrum that exhibits sudden death in infancy with minimal prodromic symptoms, and further underscore the importance of PPA2 variants in sudden, unexpected death and in cardiac biology.
Initially reported in yeast, PPA2 (MIM: 609988, GenBank: NM_176869.2) codes for an inorganic PPase, localized to the mitochondrial matrix; the protein product of its counterpart PPA1 localizes to the cytosol (Lundin, Baltscheffsky, & Ronne, 1991;Lundin, Deopujari, Lichko, Silva, & Baltscheffsky, 1992). Alternate transcriptional splice variants, encoding different isoforms, have been characterized. The protein encoded by PPA2 is highly similar to other members of the inorganic pyrophosphatase family, and contains the signature sequence essential for PPase catalytic activity. PPases catalyze the hydrolysis of pyrophosphate to inorganic phosphate, essential for cellular phosphate metabolism and required in many processes including the synthesis of DNA, RNA, proteins, polysaccharides, and lipids, as well as energy metabolism (Guimier et al., 2016). PPA2 is highly conserved across species (https ://www.ncbi.nlm.nih.gov/gene/27068 , accessed 12/25/18).
Because PPA2 functions within the mitochondrial matrix, variants in this nuclear-encoded protein cause mitochondrial diseases. In 2016, two separate case series described biallelic PPA2 variants in several pedigrees exhibiting sudden death (Guimier et al., 2016;Kennedy et al., 2016). Moreover, a large Finnish cardiomyopathy registry of severe childhood cardiomyopathy (KidCMP) recently included two cases (index case + brother, out of 66) with a PPA2 biallelic variant who died at 8 and 5 months of age, respectively. Given that the incidence of "severe" childhood cardiomyopathy is ~0.50 per 100,000 population under the age of 10-15 years, the estimated the incidence of cardiomyopathy due to PPA2 biallelic variants would be approximately 0.01 per 100,000 (1 per 10,000,000) Patients are identified/grouped according to the family ("F", family; "P", patient); ages at death are abbreviated as "d" (days), "m" (months), and "y" (years). References: "G16" (Guimier et al., 2016)  N/A Heart tissue Western blotting: In the cardiac autopsy sample of P7, PPA2, and complex I subunit levels were decreased as was the expression of the mitochondrial marker proteins, suggestive of a more general reduction of mitochondrial number possibly due to changes in tissue composition.
c.500C > T (p.Pro167Leu); c.500C > T (p.Pro167Leu) N/A Fibroblasts Seahorse XF data: Basal respiration and oligomycin-inhibited OCR was similar in affected individuals (P5, P6, and P7) but after the addition of the mitochondrial uncoupler FCCP, affected individuals exhibited a higher activity than controls. The reserve respiratory capacity was twice as high in PPA2deficient fibroblasts compared to controls. pediatric population (Vasilescu et al., 2018). Another estimate of the cumulative heterozygous carrier frequency of likely pathogenic PPA2 variants is 0.0024; that is prevalence for compound heterozygous or homozygous pathogenic PPA2 mutations of 0.58 per 100,000 (1 in 170,000; Kennedy et al., 2016). Notably, while most variants associated with sudden death in childhood are inherited in autosomal dominant fashion, PPA2 variants exhibit an autosomal recessive pattern. There is therefore a 25% recurrence risk for families. Many of the infants reported in these three series demonstrated a viral prodrome (often, a viral gastroenteritis) with pallor within days of their deaths. Our two siblings were asymptomatic except for a viral illness shortly before death in one; their deaths were sudden and unexpected. The initial genetic panel screening did not show any definite or likely pathogenic variants, although several variants of unknown clinical significance were identified. With the advent of next-generation sequencing, molecular autopsies are now shedding insights into causes of sudden, unexpected death in infancy and childhood. Most causative variants, including our patients, occurred as biallelic variants or compound heterozygotes; only in one family (#2 in reference) was a homozygous variant (c.500C > T; p.Pro167Leu) reported. Neither of the individual variants in our cases (p.Ser61Phe; p.Arg127Leu) have been found in homozygous form in gnomAD (accessed 03/01/2019).
Biallelic PPA2 variants display a spectrum of severity (Table 1). Personal histories have included seizures and lactic acidosis typical of mitochondrial diseases, and cardiac arrhythmia. Provocative factors associated with sudden death have included acute viral gastroenteritis in young children, and small amounts of alcohol (Family 1, Kennedy et al., 2016). Age of death has ranged from early infancy to adolescence. Autopsy findings have frequently demonstrated myocarditis, cardiac inflammation, and cardiac fibrosis, which our cases did not demonstrably exhibit.
None of the cases previously reported (Guimier et al., 2016;Kennedy et al., 2016;Vasilescu et al., 2018) harbored both the p.Ser61Phe and p.Arg127Leu variants. The p.Ser-61Phe variant (associated with the additional allelic variant p.Gln294Pro, in family F1; Guimier et al., 2016) was associated with sudden death in early toddler age (14-15 months) and histological evidence of myocarditis. Kennedy et al. (2016) described a toddler (in family F4) carrying p.[Ar-g127Leu];[Glu172Lys] biallelic variants in whom mild left ventricular hypertrophy was diagnosed at age 10 months, associated with seizures; death occurred at 2 years of age, with histological evidence of extensive left ventricular fibrosis and acute myocardial ischemia.

| Biology of PPA2
The yeast homologue of the mitochondrial PPA2 is required for mitochondrial DNA maintenance and yeast cells lacking the enzyme exhibit mitochondrial DNA depletion. However, a small study of 13 patients with mitochondrial depletion syndromes found no pathogenic mutations identified in the PPA2 of these patients and therefore, investigators did not believe PPA2 mutations are a common cause of mitochondrial diseases in humans (Curbo et al., 2006). Three recent studies investigated patients' cells or introduced mutations into yeast as a model organism to gain further mechanistic insights into the biology of PPA2 (Guimier et al., 2016;Kennedy et al., 2016;Vasilescu et al., 2018;see  Note: "OCR", oxygen consumption rate. References: "G16" (Guimier et al., 2016); "K16" (Kennedy et al., 2016); "V18" (Vasilescu et al., 2018) be deleterious. Molecular modeling of PPA2 variants predicted compromised stability of the protein; unfavorable steric, or electrostatic interactions leading to destabilization/misfolding of hPPA2; and/or impairment of enzymatic function due to disruption of hydrogen bonds. Cellular data from various cells and tissues (fibroblasts, heart, skeletal muscle) showed decreases in complex I, complex IV, and possibly decreased mitochondrial number, as well as alterations in FCCP-stimulated oxygen consumption rates, but even the same variant could yield different results in different patients. Recombinant modeling in E. coli suggested that different variants would lead to somewhat different residual protein activities, while PPA2Δ (knockout) yeast exhibited lower antioxidant concentrations, mtDNA depletion, lower oxygen consumption (with reduced complexes III and IV) and reduced ATP synthesis, growth defects, and an inability to maintain the mitochondrial membrane potential. A new study investigating the effects of PPA2 deletion on yeast longevity also implicated a role in retrograde (mitochondrial-to-nucleus) signaling (Muid, Kimyon, Rez, Karakaya, & Koc, 2019). The biological functions of PPA2 are summarized in Figure 1.
Despite what we know of the biology of PPA2, admittedly we do not have a true understanding of how the cellular deficits lead to organ-level dysfunctions that most likely leads to death, cardiomyopathy, lethal arrhythmias, or predeliction to a rapidly fatal myocarditis. Nevertheless, we would expect the numerous interactions of PPA2 with other mitochondrial and cellular proteins (Figure 1b) as curated in BioGRID (Oughtred et al., 2019;Stark et al., 2006) to lead to wide-ranging effects of PPA2 deficiency.
In summary, we describe two sisters, approximately 1 year of age, who died suddenly and were diagnosed by molecular autopsy to carry biallelic variants in PPA2. This gene encodes a mitochondrially located inorganic pyrophosphatase and is implicated in progressive and lethal cardiomyopathies. Our cases add additional details to those reported thus far, and broaden the spectrum of clinical and molecular features of PPA2 variants.

ACKNOWLEDGMENTS
This work was supported by the SUDC Foundation and Finding A Cure for Epilepsy and Seizures (FACES). We thank the Medical Examiner and Coroner offices for their support in referrals to the SUDCRRC. We are indebted to the bereaved parents who courageously participated in this study to help us better understand sudden unexplained death