J Neurol 263 (2188-2195)

Introduction to Mitochondrial Disorders

• Mitochondrial disorders are caused by mutations in mitochondrial DNA (mtDNA) or nuclear genes impacting mitochondrial function.

• Mutations can lead to deficiencies in oxidative phosphorylation (OXPHOS), crucial for energy production.

• Ataxia is a common symptom of mitochondrial disorders, often associated with specific mutations in MT-ATP6.

Case Study Overview

• Objective: Investigate a novel mutation (m.8561C>G) in MT-ATP6/8 affecting two adult siblings.

• Symptoms Present:

  • Cerebellar ataxia

  • Peripheral neuropathy

  • Diabetes mellitus

  • Sensorineural hearing impairment

  • Hypergonadotropic hypogonadism

Mutation Discovery

• The m.8561C>G mutation alters amino acids in both MT-ATP6 and MT-ATP8, correlating with disease severity.

• Heteroplasmy (presence of more than one type of mitochondrial DNA) observed in family members.

• The mutation leads to impaired assembly and decreased ATP production of complex V (ATP synthase).

Molecular Investigations

• Patient Profiles:

  • Patient II-5:

    • Diagnosed with hypergonadotropic hypogonadism and diabetes.

    • Symptoms: Ataxic gait, absent tendon reflexes, cerebellar atrophy, etc.

  • Patient II-4:

    • Experienced coordination issues since childhood; later diagnosed with sensorineural hearing impairment.

• Genetic testing on both patients ruled out other conditions, leading to mtDNA analysis.

Genetic Analysis Techniques

• Sequencing of mtDNA was performed using PCR amplification.

• Specific attention to the m.8561C>G mutation revealed its presence in affected individuals but absence in controls.

• Sequencing Details: Use of conformation-sensitive gel electrophoresis and sequencing for thorough analysis.

Pathogenicity Evaluation

• Impact of m.8561C>G Mutation:

  • Protein Substitution:

    • p.Pro12Arg in subunit a of ATP synthase

    • p.Pro66Ala in A6L subunit

  • Mutations predicted both neutral and pathogenic effects, suggesting joint influence on disease.

Biochemical Characteristics

• ATP Production:

  • Patient myoblasts displayed lower ATP levels compared to controls.

  • Decreased ATP found during mitochondrial uncoupling experiments (FCCP treatment).

• Complex V Analysis:

  • Similarities in the quantity of OXPHOS subunit proteins between patients and controls.

  • Detected increased amounts of assembly intermediates of complex V and correlated decrease in overall ATP production.

Clinical Phenotype Correlation

• Clinical manifestations aligned with mitochondrial dysfunction signs:

  • Ataxia, diabetes, and hearing impairment were prominent symptoms in both siblings.

  • Hypergonadotropic hypogonadism noted, previously underreported in mitochondrial disorders.

Conclusion and Implications

• The study identifies a novel mutation in the MT-ATP6/8 overlapping region, supporting associations with specific mitochondrial disease phenotypes.

• Highlights the importance of acknowledging gonadal dysfunction in patient assessments for mitochondrial diseases.

• Recommendations for screening family members due to potential genetic transmission.

Acknowledgements

• Technical support acknowledged, with funding from several research foundations.

• Compliance with ethical standards and informed consent from all participants in the study.

Introduction to Mitochondrial Disorders

Mitochondrial disorders are a group of genetic conditions caused by mutations in mitochondrial DNA (mtDNA) or nuclear genes, which can significantly impact mitochondrial function. Mitochondria, often referred to as the powerhouses of the cell, are crucial for energy production through a process known as oxidative phosphorylation (OXPHOS). Mutations in these genetic materials can disrupt this process, leading to various clinical manifestations.

Ataxia, characterized by a lack of voluntary coordination of muscle movements, is a common symptom of mitochondrial disorders and is often associated with specific mutations in the MT-ATP6 gene, which is pivotal for ATP production.

Case Study Overview

Objective

The primary objective of the study is to investigate a novel mutation (m.8561C>G) located in the overlapping region of the MT-ATP6/8 genes, which is believed to impact mitochondrial function, particularly in two adult siblings who exhibit a range of symptoms.

Symptoms Present

The affected siblings presented with a spectrum of symptoms including:

• Cerebellar Ataxia: Impaired balance and coordination due to dysfunction of the cerebellum.

• Peripheral Neuropathy: Damage to the peripheral nerves, leading to weakness and numbness.

• Diabetes Mellitus: A metabolic disorder characterized by high blood sugar levels.

• Sensorineural Hearing Impairment: A type of hearing loss resulting from damage to the inner ear or the auditory nerve.

• Hypergonadotropic Hypogonadism: A condition characterized by high levels of gonadotropins in the presence of low sex hormone levels.

Mutation Discovery

The m.8561C>G mutation results in amino acid substitutions in both MT-ATP6 and MT-ATP8 genes, which correlate positively with the severity of mitochondrial disease manifestations observed in patients. The study also noted heteroplasmy, indicating the presence of more than one type of mitochondrial DNA within family members, which can influence the phenotype.

The mutation is implicated in impairing the assembly and function of Complex V (ATP synthase), leading to a decreased ATP production crucial for cellular energy.

Molecular Investigations

Patient Profiles

Patient II-5

• Background: Diagnosed with hypergonadotropic hypogonadism and diabetes mellitus at an adult age.

• Symptoms: Ataxic gait, absent tendon reflexes, significant cerebellar atrophy observed through neuroimaging.

Patient II-4

• Background: Reported development of coordination issues since childhood; later diagnosed with sensorineural hearing impairment at an early age.

Genetic testing on both patients ruled out other conditions, leading to a thorough mtDNA analysis to pinpoint the underlying mutation.

Genetic Analysis Techniques

Sequencing of mtDNA was performed using Polymerase Chain Reaction (PCR) amplification followed by conformation-sensitive gel electrophoresis. This detailed methodology ensured the identification of the m.8561C>G mutation, with its presence confirmed in affected individuals and absence in healthy controls.

Pathogenicity Evaluation

Impact of m.8561C>G Mutation

The mutation leads to significant protein substitutions in essential mitochondrial proteins:

• p.Pro12Arg in subunit a of ATP synthase

• p.Pro66Ala in A6L subunit

These mutations were predicted to have both neutral and pathogenic effects, suggesting a complex interplay that may influence the overall disease pathology.

Biochemical Characteristics

ATP Production

Patient-derived myoblasts displayed notably lower ATP levels compared to healthy controls, indicating compromised energy production capabilities. Further mitochondrial uncoupling experiments using FCCP treatment showed a marked decrease in ATP production in patient samples.

Complex V Analysis

Comparative analyses of OXPHOS subunit proteins exhibited similarities between patients and controls, although increased levels of assembly intermediates for complex V were detected, correlating with the decreased ATP production observed.

Clinical Phenotype Correlation

Clinical manifestations observed during the study were in alignment with signs indicative of mitochondrial dysfunction. Notably, symptoms of ataxia, diabetes, and hearing impairment were consistently present in both siblings, with hypergonadotropic hypogonadism being previously underreported in relation to mitochondrial disorders.

Conclusion and Implications

The findings from this study highlight the significance of the novel mutation in the MT-ATP6/8 overlapping region, establishing a clear association with specific mitochondrial disease phenotypes. Additionally, it underscores the necessity of including gonadal dysfunction assessments in patients diagnosed with mitochondrial diseases.

Recommendations also include further genetic screening of family members due to the potential hereditary nature of these mitochondrial abnormalities.

Acknowledgements

Technical support for the study was acknowledged, complemented by funding from several research foundations, with all ethical standards adhered to and informed consent obtained from all participants involved in the study for compliance purposes.