Mitochondrial DNA heteroplasmy distinguishes disease manifestation in PINK1/PRKN-linked Parkinson's disease

Abstract

Biallelic mutations in PINK1/PRKN cause recessive Parkinson's disease. Given the established role of PINK1/Parkin in regulating mitochondrial dynamics, we explored mitochondrial DNA integrity and inflammation as disease modifiers in carriers of mutations in these genes. Mitochondrial DNA integrity was investigated in a large collection of biallelic (n = 84) and monoallelic (n = 170) carriers of PINK1/PRKN mutations, idiopathic Parkinson's disease patients (n = 67) and controls (n = 90). In addition, we studied global gene expression and serum cytokine levels in a subset. Affected and unaffected PINK1/PRKN monoallelic mutation carriers can be distinguished by heteroplasmic mitochondrial DNA variant load (area under the curve = 0.83, CI 0.74-0.93). Biallelic PINK1/PRKN mutation carriers harbour more heteroplasmic mitochondrial DNA variants in blood (P = 0.0006, Z = 3.63) compared to monoallelic mutation carriers. This enrichment was confirmed in induced pluripotent stem cell-derived (controls, n = 3; biallelic PRKN mutation carriers, n = 4) and post-mortem (control, n = 1; biallelic PRKN mutation carrier, n = 1) midbrain neurons. Last, the heteroplasmic mitochondrial DNA variant load correlated with IL6 levels in PINK1/PRKN mutation carriers (r = 0.57, P = 0.0074). PINK1/PRKN mutations predispose individuals to mitochondrial DNA variant accumulation in a dose- and disease-dependent manner.

Keywords: PINK1; PRKN; modifiers; mtDNA heteroplasmy; penetrance.

Figure 1

Overview of study. Flow chart showing individuals incorporated in this study, biospecimen, rationale and techniques. NGS = next-generation sequencing; NM = neuromelanin; TGS = third-generation sequencing.

Figure 2

Disease manifestation and PRKN/PINK1 status are associated with number of heteroplasmic mtDNA variants in blood-derived DNA. (A) The sensitivity and specificity of the heteroplasmic mtDNA variants (HF > 5%) as a biological marker plus age at examination were assessed with ROC analysis. The AUC indicated good discrimination for affected (n = 29) versus unaffected (n = 109) monoallelic PRKN/PINK1 mutation carriers (AUC = 0.83, CI 0.74–0.93). (B) Scatter plot showing the number of heteroplasmic mtDNA variants for affected (n = 29) versus unaffected (n = 109) monoallelic PRKN/PINK1 mutation carriers. Mann–Whitney U-test was performed. (C and D) Scatter plots showing number heteroplasmic mtDNA variants (HF > 5%) for (C) idiopathic PD (iPD, n = 54) versus PINK1/PRKN/PD+ (n = 78); (D) iPD (n = 54) versus PINK1+/PRKN+/PD+ (n = 29) versus PINK1++/PRKN++/PD+ (n = 49) and (E) controls (n = 67) versus iPD (n = 54) versus all monoallelic mutation carriers (n = 150) versus all biallelic mutation carriers (n = 52). Mann–Whitney U-test was performed for C and Kruskal–Wallis tests for D and E are shown in bold. Post hoc analyses are not bolded. Bars indicate means and 95%CI. PINK1 = monoallelic and biallelic PINK1 mutations; PRKN = monoallelic and biallelic PRKN mutations; PINK1+ = PINK1 monoallelic, PINK1++ = PINK1 biallelic mutations; PRKN+ = PRKN monoallelic, PRKN++ = PRKN biallelic mutations; PD+ = patient with Parkinson’s disease; all = individuals in study with or without Parkinson’s disease.

Figure 3

Analysis of mtDNA major arc deletions and mtDNA transcription-associated 7S DNA by real-time PCR in blood-derived DNA. Simultaneous real-time PCR quantification of the mtDNA genes ND4 relative to ND1 or detection of 7S DNA relative to ND1. Scatter plots showing ND4:ND1 ratios for (A) patients with idiopathic PD (iPD, n = 50) versus patients with PD due to monoallelic (n = 25) or biallelic (n = 57) mutations in PINK1 or PRKN. (B) Controls (n = 65) versus iPD (n = 50) versus all PINK1 or PRKN monoallelic mutation carriers (n = 130) versus all PINK1 or PRKN biallelic mutation carriers (n = 57). Scatter plot showing 7S DNA:ND1 ratios for (C) patients with iPD (n = 49) versus patients with PD due to monoallelic (n = 25) or biallelic (n = 57) mutations in PINK1 or PRKN. (D) Controls (n = 65) versus iPD patients (n = 49) versus all monoallelic PINK1/PRKN mutation carriers (n = 131) versus all PINK1 or PRKN biallelic mutation carriers (n = 57). Kruskal–Wallis tests were performed and in bold. Post hoc analyses are not bolded. Bars indicate means and 95% CI; PINK1+ = PINK1 monoallelic; PINK1++ = PINK1 biallelic mutations; PRKN+ =: PRKN monoallelic; PRKN++ = PRKN biallelic mutations; PD+ = patient with Parkinson’s disease.

Figure 4

Heteroplasmic mtDNA variants are associated with PRKN mutation carriers in iPSC-derived and post-mortem neurons and inflammation. Scatter plot showing number of heteroplasmic mtDNA variants in iPSC-derived midbrain neurons from three to four differentiations of controls (n = 4) and biallelic PRKN-PD patients (n = 4) performed with (A) Illumina short-read sequencing at >5%, (B) Nanopore long-read sequencing at >5%, (C) Nanopore long-read sequencing at >2% and (D) Nanopore long-read sequencing at >1%. T-tests were performed. PRKN++ = PRKN biallelic mutations; PD+ = patients with Parkinson’s disease. (E) Line plot showing number heteroplasmic mtDNA variants for control- versus PRKN++/PD+-derived nigral laser-microdissected neuromelonin (NM)-positive neurons and non-nigral midbrain tissue at >5%, >2% and >1%. (F) Correlation of serum IL6 and total mtDNA variant load in PINK1 or PRKN monoallelic (n = 14) and biallelic (n = 6) mutation carriers. Bars indicate means and 95% CI.