Cell and mice studies suggest mitochondrial dysfunction may cause altered bone structure.

Hypothesis: Decreased mitochondrial energy production affects bone cell development and activity negatively.

Comparing humans with the mitochondrial DNA variant, m.3243A>G, pathogenic variants in POLG or TWNK genes to healthy controls, the aim is to evaluate the effect of mitochondrial dysfunction on: 1: bone-cell development and -activity in bone marrow stem cells and blood.

2: bone cell metabolism including glucose consumption. 3: bone structure assessed by electron microscopy and μCT scans of bone biopsies.

Intact mitochondrial activity including adequate energy supplies is vital for metabolic active tissues i.e. skeletal muscle, heart and brain. The human skeleton represent an additional highly metabolically active tissue; nevertheless the significance of the mitochondrial role in human skeletal bone health may be further investigated.

Bone remodelling constitutes the coupled and continuous regenerative process of bone degradation by bone resorbing cells osteoclasts (OC) followed by formation of bone matrix by bone forming osteoblasts (OB). Quantitative imbalance between resorption and formation results in skeletal disorders with low bone mass including osteoporosis, and its increased risk of fragility fractures.

Mitochondria generate cellular energy adenosine triphosphate (ATP) through oxidative phosphorylation process (OXPHOS) in the respiratory chain (RC) with a secondary production of the deleterious by-products free radicals i.e. reactive oxygen species (ROS). Notably, mitochondria hold their own DNA (m.DNA), and RC subunits are encoded by m.DNA and nuclear DNA (n.DNA) genes, respectively. With ageing, deleterious somatic m.DNA mutations accumulate in skeletal muscle and heart, and somatic m.DNA mutations as well as inherited m.DNA or n.DNA mutations may result in mitochondrial dysfunction with impaired ATP production and accumulation of ROS. m.DNA mutations may impair brain, skeletal-, and cardiac muscle function, but the effects on human bone cell metabolism and remodelling are unknown. A recent study of a cohort of young individuals indicates that mitochondrial diseases pose a risk for bone fragility fractures.

Preclinical studies suggest that ATP and ROS regulate bone metabolism. The m.DNA number and mitochondrial activity increase to support differentiation from human skeletal (mesenchymal) stem cells (hMSC) to mature bone forming OBs. Inhibition of mitochondrial activity or increase in ROS levels suppress OB differentiation. Similarly, OCs are rich in mitochondria. Human OC cultures demonstrate that energy supplies for OC differentiation from their progenitors is based on OXPHOS while OC resorption activity relies on glycolysis.

In addition, emerging evidence suggest that metabolic plasticity i.e. regulation of glycolysis, OXPHOS, and pyruvate levels, contribute to regulation of OB and OC differentiation.

Receptor activator of nuclear factor kappa-Beta ligand (RANKL) secreted by OBs activates OC resorption. In mice, RANKL stimulation of bone marrow OC progenitors increases intracellular levels of ROS, which stimulates OC differentiation and bone resorption in-vitro. Further, ROS inhibits the wingless-type (Wnt) signalling pathway with attenuation of osteoblastogenesis and decreased bone formation.

Furthermore, mice with mutations in the n.DNA encoded proof reading domain of m.DNA polymerase POLG (PolgA-/-) accumulate m.DNA mutations, and present with premature ageing phenotype including low bone mass. In addition, deficiency of the n.DNA encoded mitochondrial transcription factor (TFAM) causes ATP depletion, and mice with TFAM deficient OCs have increased OC activity and augmented bone resorption. Opposite, global loss of NADH (nicotinamide-adenine dinucleotide) ubiquinone oxidoreductase Fe-S protein 4 (NDUFS4) a subunit in RC complex 1 impairs bone resorption, and (ndufs4-/-) mice present with increased bone mineral density (BMD) and an apparent osteopetrosis bone phenotype.

The aim is to study bone cell phenotype in patients with rare mitochondrial disease Carriers of MT-TL1 m.3243A>G (MIM: 590050).The gene encodes the transcription factor tRNALeu(UUA/UUG) and m.3243A>G weakens the assembly of RC complex with a secondary impaired ATP production. The phenotype is, in part associated with the m.3243A>G mutation burden i.e. level of heteroplasmy (percentage of m.3243A>G/wildtype m.DNA). The study group also includes carriers of mutations in the nuclear encoded POLG (MIM: 174763) and TWNK (MIM: 606075).

Hypothesis: Impaired mitochondrial function affects human bone cell -differentiation, -metabolism, and -activity leading to impaired bone formation and bone fragility.

Aim: To determine if carriers of inherited mitochondrial mutations i.e. mitochondrial dysfunction, ATP depletion and secondary increase in ROS lead to change in:

1. In-vitro OB differentiation-rate, OB activity and bone formation.
2. In-vitro OC differentiation-, OC activity and higher overall bone resorption.
3. In-vivo changes in tissues level dynamics of bone formation and - resorption as examined in iliac crest bone biopsies.

Design, Participants and Methods: Cross-sectional case-control study including subjects (>18 years) carrying one of the following mutations:

1. MT-TL1 m.3243A>G
2. POLG mutation
3. TWNK

N=10 cases with each pathogenic genetic variant and equal number of controls (n=30) matched on sex, age and BMI.

Inclusion Criteria - cases:

• Genetic diagnosis with: MT-TL1 m.3243A>G, or POLG variant, het or TWNK variant, het, > 18 years
• Signed informed consent

Inclusion Criteria - controls:

• Healthy subjects matched on age and gender > 18 years
• Signed informed consent

Exclusion Criteria:

• Renal (creatinine > 90 µmol/l)
• Liver dysfunction (AST > 3 times the upper limit)
• Medical treatment influencing bone metabolism (oral corticosteroid <12 weeks, anti-osteoporosis treatment, sex steroids, anti-convulsants)
• Pregnancy
• Excessive consumption of alcohol
• Treatment with anticoagulants
• Pre-existing coagulopathy
• Allergy to lidocaine, morphine or diazepam.

Participants are masked with anonymized identifier (ID)