Biallelic PTPMT1 variants disrupt cardiolipin metabolism and lead to a neurodevelopmental syndrome

Abstract

Primary mitochondrial diseases (PMDs) are among the most common inherited neurological disorders. They are caused by pathogenic variants in mitochondrial or nuclear DNA that disrupt mitochondrial structure and/or function, leading to impaired oxidative phosphorylation (OXPHOS). One emerging subcategory of PMDs involves defective phospholipid metabolism. Cardiolipin, the signature phospholipid of mitochondria, resides primarily in the inner mitochondrial membrane, where it is biosynthesized and remodelled via multiple enzymes and is fundamental to several aspects of mitochondrial biology. Genes that contribute to cardiolipin biosynthesis have recently been linked with PMD. However, the pathophysiological mechanisms that underpin human cardiolipin-related PMDs are not fully characterized. Here, we report six individuals, from three independent families, harbouring biallelic variants in PTPMT1, a mitochondrial tyrosine phosphatase required for de novo cardiolipin biosynthesis. All patients presented with a complex, neonatal/infantile onset neurological and neurodevelopmental syndrome comprising developmental delay, microcephaly, facial dysmorphism, epilepsy, spasticity, cerebellar ataxia and nystagmus, sensorineural hearing loss, optic atrophy and bulbar dysfunction. Brain MRI revealed a variable combination of corpus callosum thinning, cerebellar atrophy and white matter changes. Using patient-derived fibroblasts and skeletal muscle tissue, combined with cellular rescue experiments, we characterized the molecular defects associated with mutant PTPMT1 and confirmed the downstream pathogenic effects that loss of PTPMT1 has on mitochondrial structure and function. To further characterize the functional role of PTPMT1 in cardiolipin homeostasis, we created a ptpmt1 knockout zebrafish. This model had abnormalities in body size, developmental alterations, decreased total cardiolipin levels and OXPHOS deficiency. Together, these data indicate that loss of PTPMT1 function is associated with a new autosomal recessive PMD caused by impaired cardiolipin metabolism, highlighting the contribution of aberrant cardiolipin metabolism towards human disease and emphasizing the importance of normal cardiolipin homeostasis during neurodevelopment.

Keywords: cardiolipin; mitochondria; mitochondrial dynamics; neurodevelopmental syndrome; primary mitochondrial disease.

Figure 1

Family pedigrees and details of PTPMT1 variants. (A) Pedigrees of the three unrelated families showing consanguinity (double horizontal lines). Affected subjects (Subjects S1–S6) are represented by filled circles for females and filled squares for males. Arrow indicates proband (Subject S5) of Family 3. (B) Chromatograms from Sanger sequencing of PTPMT1 (NM_175732.3) genotypes for probands, parents and siblings of Families 1–3.

Figure 2

PTPMT1 variants and gene structure. (A) Schematic representation of the PTPMT1 gene showing the localization of variants c.65A>C and c.255G>C and their effects on protein sequence. PTPMT1 protein sequences alignment, indicating identical and highly conserved residues. (B) RNA-sequencing analysis of the PTPMT1 c.255G>C splicing variant. Schematics of normal PTPMT1 splicing (top) and the three aberrant splicing events associated with the c.255G>C variant.

Figure 3

MRI sections of the brain of the three probands harbouring biallelic PTMT1 variants. (A) T1 sequence, sagittal view. Evidence of thin corpus callosum (arrow), atrophy of pons and medulla (star). (B) T2 sequence, axial view. No white or grey matter changes were observed. (C) T1 sequence, sagittal view, showing hypoplasia of corpus callosum (arrow), cerebellar and brainstem atrophy (squares), and mega cisterna magna anomaly (asterisk). (D) T1 sequence, axial view, showing supratentorial white matter signal hyperintensity in keeping with hypomyelination. (E) T2 sequence, sagittal view, showing diffuse cortical atrophy, and cerebellar atrophy (arrow). (F) T2 sequence, axial view, showing areas of hypomyelination.

Figure 4

Cardiolipin content and acyl chain composition in patient-derived tissues. (A) The 72:8 cardiolipin (CL) molecular species in three healthy controls (CTRs) and Subject S1 (S1) skeletal muscle determined by mass spectrometry-based lipidome analysis. (B) Total cardiolipin levels measured in dry blood spots from 11 CTRs, Subjects S1 and S5. (C) Total cardiolipin abundance measured in primary fibroblasts from four CTRs, Subjects S1, S2 and S5. Error bars represent standard deviation; n = 3–8, where each data-point represents an independent biological sample.

Figure 5

Mitochondrial morphology in patient-derived fibroblasts. (A) Relative expression of PTPMT1 mRNA in primary fibroblasts analysed by quantitative PCR. No decrease was observed in Subject S1 (S1), whereas Subject S2 (S2) showed a severe loss of PTPMT1 mRNA. Error bars represent standard error of the mean; n = 5, where each data-point represents an independent biological sample. (B) Western blot detecting the PTPMT1 protein expression in primary fibroblasts from three healthy controls (CTRs), Subjects S1 and S2. An asterisk indicates a non-specific signal. β-Actin was used as a loading control. (C) Representative confocal images of mitochondrial morphology from three healthy CTRs, Subjects S1 and S2. Mitochondria were labelled with an anti-TOMM20 antibody. Scale bar = 10 μm. (D) Quantification of mitochondrial morphology related to C. (E and F) Quantification of mitochondrial length (E) and area (F), per region of interest (ROI) (225 µm²) from D. Data are expressed as mean ± standard error of the mean (SEM) (A) or standard deviation (D–F); n = 3 independent experiments. D: Two-way ANOVA, Tukey’s multiple comparison test; E and F: unpaired Mann–Whitney U-test (two-tailed). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

PTPMT1 expression and mitochondrial morphology in complemented patient-derived fibroblasts. (A) Representative confocal images of mitochondrial morphology and PTPMT1 expression in a healthy control (CTR1), Subject S1 (S1) and Subject S2 (S2) before and after transduction with pLenti6.3/V5-DEST-GFP (empty vector, EV) or pLenti6.3/V5-DEST vector expressing wild-type PTPMT1. Mitochondria and PTPMT1 were labelled with anti-TOMM20 and anti-PTPMT1 antibodies, respectively. Scale bar = 10 μm. (B) Quantification of mitochondrial morphology related to A. (C and D) Quantification of mitochondrial length (C) and area (D) per region of interest (ROI) (225 µm²) from B. Data are expressed as mean ± standard deviation; n = 4 independent experiments. B: Two-way ANOVA, Tukey’s multiple comparison test; C and D: unpaired Mann–Whitney U-test (two-tailed). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Morphological, cardiolipin content, and oxidative phosphorylation complex analysis of ptpmt1 knockout zebrafish. (A) Representative image of ptpmt1^+/+ (CTR) and ptpmt^Δ4bp/Δ4bp. (B) Quantification of zebrafish body length. (C) Quantification of zebrafish head area. (D) Total cardiolipin (CL) levels measured by mass spectrometry-based lipidome analysis. (E) Western blot analysis of blue-native polyacrylamide gels loaded with 23.8 μg of protein extracted from a pool of two CTRs and 10 ptpmt^Δ4bp/Δ4bp zebrafish samples. (F) In-gel activity of Complex (Cx) I in a polyacrylamide blue-native gel loaded with 35.7 μg of protein extracted from a pool of two CTRs and 10 ptpmt^Δ4bp/Δ4bp zebrafish samples. (G) Spectrophotometric enzyme activity of Complex IV in CTRs and 10 ptpmt^Δ4bp/Δ4bp zebrafish samples. (H) In-gel activity of Complex V in a polyacrylamide clear-native gel loaded with 11.4 μg of protein extracted from a pool of two CTRs and 10 ptpmt^Δ4bp/Δ4bp zebrafish samples. (I) Citrate synthase activity measured in CTRs and ptpmt^Δ4bp/Δ4bp. All measures were performed at 19 days post fertilization (dpf). The mutant allele is referred to as ptpmt1^Δ4bp. Control groups (CTRs) were obtained by pooling together ptpmt1^+/+ and ptpmt^+/Δ4bp samples. Error bars represent standard error of the mean, n = 3–71, where each data-point represents an independent biological sample. Significance values are shown for t-test compared to CTRs.