Fig 2: Distribution of PPA2 variants and conservation of affected amino acids.(a) Genomic structure and location of all known disease-associated variants within PPA2 (GenBank NM_176869.2) encoding the mitochondrial inorganic pyrophosphatase. Green-filled boxes represent coding exons 1 to 12. Numbers above the gene’s schematic indicate the position of complementary DNA (cDNA) variants. Red numbers indicate the novel variants reported in this manuscript. Translated protein (Genbank NP_789845.1) is represented below the gene as spliced green boxes with black borders. (b) Phylogenetic conservation of amino acids affected by PPA2 missense variants is shown by multiple sequence alignment performed with the Clustal omega algorithm. Numbers reflect amino acid position. Red numbers indicate amino acids affected by novel variants reported in this paper. e exon, aa amino acids, nt nucleotides, UTR untranslated region.

Fig 3: Enzyme activity of recombinant PPA2 variants.(a) The percentage of activity of the recombinant PPA2 relative to recombinant wild-type PPA2 at 37 °C is indicated at different pyrophosphate concentrations along the x-axis. At least three replicates were performed for all conditions. Two PPA2 variants (Arg84Gln and Val243Leu), which are found in homozygosity in the gnomAD database, are shown in shades of green and serve as positive controls. Error bars show the standard error of the mean (SEM). (b) Enzyme activities of the recombinant proteins were determined at different temperatures (25 °C, 37 °C, 50 °C) with a pyrophosphate concentration of 0.2 mmol/l. Three replicates were carried out per recombinant enzyme, per temperature. The two PPA2 variants in shades of green serve as positive controls. Error bars show the SEM.

Fig 4: Pedigree structures and survival curves.(a) Twenty families with biallelic PPA2 variants. Variants are marked in red text. Age at death (or current age) is listed under the genotype. d days, m months, pn prenatal diagnosis, NT not tested. Symbols: triangle (miscarriage), diamond (sex unknown). (b) Kaplan–Meier survival curves. Kaplan–Meier curves displaying survival data from five previous studies and this cohort. Black and colored marks indicate living individuals (preceded by horizontal lines) or deceased individuals (steps down). Larger steps indicate more individuals who died at a given age. The curve in red summarizes the 34 individuals from this study (six surviving; 28 deceased). Two prenatal individuals of family 6 are not included in this figure because the pregnancies were terminated.

Fig 5: Cardiac Fibrosis in PPA2 Deficiency(A) Affected individual P3, post mortem section through left ventricle showing a virtually circumferential lamina of scarring in midmyocardium with focal subendocardial involvement. Fibrosis is marked by arrows.(B and C) Low-power (B) (bar equals 1 mm) and high-power (C) (bar equals 25 µm) microscopy of the posterior freewall of the left ventricle showing prominent midmyocardial loose fibrosis in P3 (hematoxylin and eosin staining).(D) Cardiac MRI showing prominent midmyocardial fibrosis in affected individual P4 (at 25 years of age), marked by arrows.