Emil Ylikallio

Mechanisms of mitochondrial diseases

Abstract Mitochondria are essential organelles with multiple functions, the most well known being the production of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS). The mitochondrial diseases are defined by impairment of OXPHOS. They are a diverse group of diseases that can present in virtually any tissue in either adults or children. Here we review the main molecular mechanisms of mitochondrial diseases, as presently known. A number of disease-causing genetic defects, either in the nuclear genome or in the mitochondrias own genome, mitochondrial DNA (mtDNA), have been identified. The most classical genetic defect causing mitochondrial disease is a mutation in a gene encoding a structural OXPHOS subunit. However, mitochondrial diseases can also arise through impaired mtDNA maintenance, defects in mitochondrial translation factors, and various more indirect mechanisms. The putative consequences of mitochondrial dysfunction on a cellular level are discussed.

A Heterozygous Truncating Mutation in RRM2B Causes Autosomal-Dominant Progressive External Ophthalmoplegia with Multiple mtDNA Deletions

Autosomal-dominant progressive external ophthalmoplegia (adPEO) is a mitochondrial disorder that is characterized by accumulation of multiple mitochondrial DNA (mtDNA) deletions in postmitotic tissues. The disorder is heterogeneous, with five known nuclear disease genes that encode the proteins ANT1, Twinkle, POLG, POLG2, and OPA1. Defects in these proteins affect mtDNA maintenance, probably leading to stalled replication forks, consequent mtDNA deletion formation, and progressive respiratory chain deficiency. Here we present a large adPEO family with multiple mtDNA deletions, whose disease was not explained by mutations in any of the known adPEO loci. We mapped the disease locus in this family to chromosome 8q22.1-q23.3. The critical linkage region contained the RRM2B gene, which encodes the small subunit of the ribonucleotide reductase p53R2, which has previously been shown to be essential for the maintenance of mtDNA copy number. Mutation screening of RRM2B revealed a heterozygous nonsense mutation in exon 9 (c.979C-->T [p.R327X]) in all affected individuals that was absent in 380 control chromosomes. The same mutation was found to segregate in another adPEO family. The mutant mRNA escaped nonsense-mediated decay and resulted in a protein with truncation of 25 highly conserved C-terminal amino acids essential for the interaction with the ribonucleotide reductase subunit R1. We conclude that dominant-negative or gain-of-function mutations in RRM2B are a cause of multiple mtDNA deletions and adPEO.

High Mitochondrial DNA Copy Number Has Detrimental Effects in Mice

Mitochondrial DNA (mtDNA) is an essential multicopy genome, compacted into protein-DNA clusters called nucleoids. Maintaining an adequate mtDNA copy number is crucial for cellular viability. Loss of mtDNA results in severe human syndromes, whereas increased mtDNA copy number has been suggested to improve survival from myocardial infarction in mice and to be a promising therapeutic strategy for mitochondrial disease. The mechanisms that regulate mtDNA amount and organization are, however, not fully understood. Of the proteins required for mtDNA existence, only the mitochondrial helicase Twinkle and mitochondrial transcription factor A (TFAM) have been shown to increase mtDNA copy number in vivo, when expressed in physiological levels. Here we studied how Twinkle and TFAM affect mtDNA synthesis and nucleoid structure in mice. Using in vivo BrdU labeling, we show that Twinkle specifically regulates de novo mtDNA synthesis. Remarkably, high mtDNA copy number in mice is accompanied by nucleoid enlargement, which in turn correlates with defective transcription, age-related accumulation of mtDNA deletions and respiratory chain (RC) deficiency. Simultaneous overexpression of Twinkle and TFAM in bitransgenic mice has an additive effect on mtDNA copy number, increasing it up to 6-fold in skeletal muscle. Bitransgenic mice also exhibit further enlargement of nucleoids and aggravation of the RC defect. In conclusion, we show that Twinkle acts as a regulator of mtDNA replication initiation, and provide evidence that high mtDNA copy number and alteration of nucleoid architecture may be detrimental to mitochondrial function.

Deficiency of the E3 ubiquitin ligase TRIM2 in early-onset axonal neuropathy

Inherited peripheral neuropathies are a heterogeneous group of disorders that can affect patients of all ages. Children with inherited neuropathy often develop severe disability, but the genetic causes of recessive early-onset axonal neuropathies are not fully known. We have taken a whole-exome sequencing approach to identify causative disease mutations in single patients with early-onset axonal neuropathy. Here, we report compound heterozygous mutations in the tripartite motif containing 2 (TRIM2) gene in a patient with childhood-onset axonal neuropathy, low weight and small muscle mass. We show that the patient fibroblasts are practically devoid of TRIM2, through mRNA and protein instability caused by the mutations. TRIM2 is an E3 ubiquitin ligase that ubiquitinates neurofilament light chain, a component of the intermediate filament in axons. Resembling the findings in our patients sural nerve biopsy, Trim2-gene trap mice showed axonopathy with accumulations of neurofilaments inside axons. Our results suggest that loss-of-function mutations in TRIM2 are a cause of axonal neuropathy, which we propose to develop as a consequence of axonal accumulation of neurofilaments, secondary to lack of its ubiquitination by TRIM2.

Selenoprotein biosynthesis defect causes progressive encephalopathy with elevated lactate

Objective: We aimed to decipher the molecular genetic basis of disease in a cohort of children with a uniform clinical presentation of neonatal irritability, spastic or dystonic quadriplegia, virtually absent psychomotor development, axonal neuropathy, and elevated blood/CSF lactate. Methods: We performed whole-exome sequencing of blood DNA from the index patients. Detected compound heterozygous mutations were confirmed by Sanger sequencing. Structural predictions and a bacterial activity assay were performed to evaluate the functional consequences of the mutations. Mass spectrometry, Western blotting, and protein oxidation detection were used to analyze the effects of selenoprotein deficiency. Results: Neuropathology indicated laminar necrosis and severe loss of myelin, with neuron loss and astrogliosis. In 3 families, we identified a missense (p.Thr325Ser) and a nonsense (p.Tyr429*) mutation in SEPSECS, encoding the O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase, which was previously associated with progressive cerebellocerebral atrophy. We show that the mutations do not completely abolish the activity of SEPSECS, but lead to decreased selenoprotein levels, with demonstrated increase in oxidative protein damage in the patient brain. Conclusions: These results extend the phenotypes caused by defective selenocysteine biosynthesis, and suggest SEPSECS as a candidate gene for progressive encephalopathies with lactate elevation.

Targeted next-generation sequencing reveals further genetic heterogeneity in axonal Charcot-Marie-Tooth neuropathy and a mutation in HSPB1.

Charcot–Marie–Tooth disease (CMT) is a group of hereditary peripheral neuropathies. The dominantly inherited axonal CMT2 displays striking genetic heterogeneity, with 17 presently known disease genes. The large number of candidate genes, combined with lack of genotype–phenotype correlations, has made genetic diagnosis in CMT2 time-consuming and costly. In Finland, 25% of dominant CMT2 is explained by either a GDAP1 founder mutation or private MFN2 mutations but the rest of the families have remained without molecular diagnosis. Whole-exome and genome sequencing are powerful techniques to find disease mutations for CMT patients but they require large amounts of sequencing to confidently exclude heterozygous variants in all candidate genes, and they generate a vast amount of irrelevant data for diagnostic needs. Here we tested a targeted next-generation sequencing approach to screen the CMT2 genes. In total, 15 unrelated patients from dominant CMT2 families from Finland, in whom MFN2 and GDAP1 mutations had been excluded, participated in the study. The targeted approach produced sufficient sequence coverage for 95% of the 309 targeted exons, the rest we excluded by Sanger sequencing. Unexpectedly, the screen revealed a disease mutation only in one family, in the HSPB1 gene. Thus, new disease genes underlie CMT2 in the remaining families, indicating further genetic heterogeneity. We conclude that targeted next-generation sequencing is an efficient tool for genetic screening in CMT2 that also aids in the selection of patients for genome-wide approaches.

CHCHD10 variant p.(Gly66Val) causes axonal Charcot-Marie-Tooth disease

Objective: We describe the phenotype consistent with axonal Charcot-Marie-Tooth disease type 2 (CMT2) in 4 families with a c.197G>T (p.(Gly66Val)) variant in CHCHD10. Methods: We sequenced the CHCHD10 gene in a cohort of 107 families with CMT2 of unknown etiology. The patients were characterized by clinical examination and electroneuromyography. Muscle MRI and biopsy of the muscle or nerve were performed in selected cases. Neuropathologic autopsy was performed in 1 case. Results: The c.197G>T variant in CHCHD10 was found in 6 families, 4 of which included multiple individuals available for detailed clinical study. Variants in this gene have recently been associated with amyotrophic lateral sclerosis-frontotemporal dementia, mitochondrial myopathy, or spinal muscular atrophy Jokela type (SMAJ), but not with CMT2. Our patients had a late-onset distal axonal neuropathy with motor predominance, progressing to involve sensory nerves. Neurophysiologic and neuropathologic studies confirmed the diagnosis of sensorimotor axonal neuropathy with no loss of anterior horn neurons. Muscle biopsies showed occasional cytochrome c oxidase–negative fibers, combined with small amounts of mitochondrial DNA deletions. Conclusions: CHCHD10 c.197G>T (p.(Gly66Val)) is a cause of sensorimotor axonal neuropathy. This gene should be considered in patients presenting with a pure CMT2 phenotype, particularly when motor symptoms predominate.

Intrafamilial clinical variability in individuals carrying the CHCHD10 mutation Gly66Val

Mutations in the CHCHD10 gene, which encodes a mitochondrially targeted protein, have emerged as an important cause of motor neuron disease and frontotemporal lobar degeneration. The aim of this study was to assess the clinical variability in a large family carrying the p.Gly66Val mutation of the CHCHD10 gene. This mutation has recently been reported to cause late‐onset spinal muscular atrophy (SMAJ) or sensorimotor axonal Charcot–Marie–Tooth neuropathy (CMT2) in the Finnish population.

Dominant transmission of de novo KIF1A motor domain variant underlying pure spastic paraplegia

Variants in family 1 kinesin (KIF1A), which encodes a kinesin axonal motor protein, have been described to cause variable neurological manifestations. Recessive missense variants have led to spastic paraplegia, and recessive truncations to sensory and autonomic neuropathy. De novo missense variants cause developmental delay or intellectual disability, cerebellar atrophy and variable spasticity. We describe a family with father-to-son transmission of de novo variant in the KIF1A motor domain, in a phenotype of pure spastic paraplegia. Structural modeling of the predicted p.(Ser69Leu) amino acid change suggested that it impairs the stable binding of ATP to the KIF1A protein. Our study reports the first dominantly inherited KIF1A variant and expands the spectrum of phenotypes caused by heterozygous KIF1A motor domain variants to include pure spastic paraplegia. We conclude that KIF1A should be considered a candidate gene for hereditary paraplegias regardless of inheritance pattern.

The Overexpression of Twinkle Helicase Ameliorates the Progression of Cardiac Fibrosis and Heart Failure in Pressure Overload Model in Mice

Myocardial mitochondrial DNA (mtDNA) copy number decreases in heart failure. In post-myocardial infarction mice, increasing mtDNA copy number by overexpressing mitochondrial transcription factor attenuates mtDNA deficiency and ameliorates pathological remodeling thereby markedly improving survival. However, the functional significance of increased mtDNA copy number in hypertensive heart disease remains unknown. We addressed this question using transgenic mice that overexpress Twinkle helicase (Twinkle; Tg), the mtDNA helicase, and examined whether Twinkle overexpression protects the heart from left ventricular (LV) remodeling and failure after pressure overload created by transverse aortic constriction (TAC). Twinkle overexpression increased mtDNA copy number by 2.2±0.1-fold. Heart weight, LV diastolic volume and wall thickness were comparable between Tg and wild type littermates (WT) at 28 days after TAC operation. LV end-diastolic pressure increased in WT after TAC (8.6±2.8 mmHg), and this increase was attenuated in Tg (4.6±2.6 mmHg). Impaired LV fractional shortening after TAC operation was also suppressed in Tg, as measured by echocardiography (WT: 16.2±7.2% vs Tg: 20.7±6.2%). These LV functional improvements were accompanied by a decrease in interstitial fibrosis (WT: 10.6±1.1% vs Tg: 3.0±0.6%). In in vitro studies, overexpressing Twinkle using an adenovirus vector in cultured cardiac fibroblasts significantly suppressed mRNA of collagen 1a, collagen 3a and connective tissue growth factor, and angiotensin II-induced transforming growth factor β1 expression. The findings suggest that Twinkle overexpression prevents LV function deterioration. In conclusion, Twinkle overexpression increases mtDNA copy number and ameliorates the progression of LV fibrosis and heart failure in a mouse pressure overload model. Increasing mtDNA copy number by Twinkle overexpression could be a novel therapeutic strategy for hypertensive heart disease.