User:Eteceterally/Mitochondrial Genetic Disorders

Brain MRIs of stroke-like lesions typical of MELAS, a mitochondrial disorder.

Mitochondrial genetic disorders are chronic inherited diseases characterized by defective mitochondria. The inheritance of mitochondrial disorders mainly follows an autosomal recessive pattern, with other variations such as maternal uniparental inheritance, X-linked recessive inheritance and autosomal dominant inheritance.

Mitochondrial dysfunction results in insufficient production of energy for normal bodily functions, with debilitating symptoms ranging from muscle weakness to learning disabilities. Patients with mitochondrial genetic disorder suffer from continuous disease development and worsening symptoms over time.

A variety of tests are used to diagnose mitochondrial disorders, including but not limited to: lactate and pyruvate level measurements, DNA testing, tissue testing etc. Current technology provides limited support in the areas of treatment and management, in vitro fertilization (IVF) mitochondria transfer and gene therapy treatment are the most effective solutions available to date.

Inheritance

Maternal Inheritance

Harmful mutations from within the mitochondrial genome are solely inherited from the mother. Mitochondria are heteroplasmic, meaning there are multiple variations of mitochondrial genomes available in human tissue. In healthy individuals, harmful mutations in a small fraction of mitochondria are compensated by the abundance of functional mitochondria. Essentially, the human body can tolerate up to a certain level of mitochondrial dysfunction. A person suffers from maternally inherited mitochondrial disorder when the level of dysfunctional mitochondria passes that threshold.

The process by which maternal mitochondria are inherited are structured to limit the amount of defective mitochondria that gets passed on to children. During the formation of a zygote, mitochondria are segregated to allow only a small portion of mitochondria to be shared with progeny. As the zygote reaches its multicellular stage (up to 128 cells), only around 3 cells will constitute the formation of the offspring, while the rest are needed to form the extraembryonic tissue for the developing fetus. This process of mitochondrial selection creates a bottleneck sampling effect, the probability of a large portion of dysfunctional mitochondria getting inherited is very small. As such, mitochondrial diseases are quite rare, with an incidence of only 1 out of 5000 people.

Other forms of Inheritance

Mutations in mitochondrial DNA (mtDNA) are not solely responsible for all forms of mitochondrial dysfunction. The mitochondria imports many proteins coded for by nuclear DNA (nDNA), defect-causing mutations in these proteins are also common. Conditions caused by mutations in nDNA are inherited through either autosomal recessive, autosomal dominant, or X-linked patterns.

When the disease is inherited in an autosomal recessive fashion, possession of two copies of the mutated gene would trigger the condition to develop. Mitochondrial disorders inherited in this pattern usually result from consanguineous descent.

Autosomal dominant mitochondrial disorders only require one copy of the mutated gene to bring about the conditions. Examples of this inheritance pattern are not uncommon, and usually caused by either sporadic de novo mutations or have late-presenting symptoms.

For mitochondrial conditions inherited in an X-linked manner, the affected genes are located on the X chromosome. If the mother is a carrier for an X-linked recessive disease, all female offspring would be carriers and all male offspring would be affected. Pyruvate dehydrogenase complex deficiency (E1 alpha), is an example of an X-linked recessive mitochondrial disorder.

Disease Mechanism

The general principle behind all forms of genetic mitochondrial disorder is inadequate oxygen phosphorylation (OXPHOS). OXPHOS subunits, also known as electron transport chain enzymes, are essential in generating adenosine triphosphate (ATP) by creating a proton motive force via protein complexes I through V (CI - CV). The proteins generate a proton gradient within the intermembrane space of mitochondria, and the efflux of protons creates an electrochemical gradient essential for ATP synthesis. Disruption of the OXPHOS process results in a decreased ability to produce ATP, which is the body’s main source of energy, with consequences ranging from cell injury to possibly apoptosis. The genetic coding for these complexes are expressed in complicated patterns involving both nDNA and mtDNA, with the exception of CII (which is coded by nDNA only). At the moment, a couple of disease-causing genetic mutations have been identified. The classical form of disease-causing mutation are mutations that directly cause impairments in an OXPHOS subunit. Other forms of disease-causing defects include altered mtDNA maintenance and defects in mitochondrial transcription and translation factors.  

Pathophysiology

There are many mitochondrial disease-causing genes that target either one or more of the OXPHOS subunits located within the mitochondria. Each of these genes interact with the structure and function of the OXPHOS units in varying manners. As such, it is challenging to identify the specific phenotypes caused by a mutated gene.

Genes associated with CI Deficiency

CI (NADH ubiquinone oxidoreductase) is in charge of translocating protons from the mitochondrial matrix into the intermembrane space. It does so by isolating protons from NADH and attaching it to water-soluble ubiquinone (UQ), which then diffuses into the intermembrane space.

Isolated CI deficiency appears in nearly one-third of all mitochondrial disorders. A large percentage of cases still lack proper diagnosis due to the complexity of the large enzyme and its dual genetic origin. The diagnosis of CI deficiency disorders include Leigh syndrome (LS), neonatal cardiomyopathy with lactic acidosis, fatal infantile lactic acidosis (FILA), macrocystic leukoencephalopathy, and isolated myopathy.

Diagram indicating different genes associated to mitochondrial disorders.

ACAD9

ACAD9 is a frequent mutation associated with the reduction in CI assembly and activity. Patients exhibit infantile hypertrophic cardiomyopathy, encephalopathy, and lactic acidosis. Other clinical features include severe neonatal symptoms including liver and kidney damage. Riboflavin is usually prescribed to temporarily alleviate symptoms for patients, although surviving patients often develop delayed-onset neurologic or muscular symptoms.

NUBPL/lnd1

Nucleotide-binding protein like (NUBPL) specifically inserts essential Fe-S centers into CI. Patients with compound heterozygous mutations in NUBPL are usually diagnosed with mitochondrial encephalopathy and CI deficiency.

Genes associated with CII Deficiency

CII acts to deliver electrons into the quinone pool, which is responsible for regulating electron transport. CII contains four protein subunits: succinate dehydrogenase (SDHA), succinate dehydrogenase iron-sulfur subunit (SDHB), succinate dehydrogenase complex subunit C (SDHC) and succinate dehydrogenase complex subunit D (SDHD). CII does not transport protons into the intermembrane space, hence has less involvement in the generation of the electrochemical gradient.

Isolated examples of defect-causing CII deficiency genes are observed in less than 10% of OXPHOS defective cases. CII deficiency is generally classified into two clinical presentations: mitochondrial encephalomyopathy and familial paragangliomas. The former is characterized by prevalence of LS, including other symptoms such as encephalopathy and isolated cardiomyopathy. The pathophysiology behind the latter, the induction of CII-associated paragangliomas, remains unknown.

SDHAF1

SDHAF1 is a protein associated with the incorporation of Fe-S centers into SDHB. Mutations in this area are shown to lead to reduced CII activity. To date, no mutations in SDHAF1 are present in people with paragangliomas.

SDHAF2

SDHAF2 is a chaperone responsible for regulating the refolding of SDHA for flavination. A missense mutation in SDHAF2 has been identified to show association to the expression of head and neck paragangliomas.

Genes associated with CIII Deficiency

The CIII protein extracts protons from the quinone pool generated by CII, translocating the protons into the intermembrane space.

CIII deficiency is rare in comparison to CI and CIV deficiencies. Disease-causing genes mainly compose of recessively inherited mutations in nDNA that affect structural subunits and assembly factors. Clinical presentations for these mutations are diverse.

BCS1L

All mutations of the BCS1L gene are highly associated with isolated CIII deficiency and reduced incorporation of Fe-S centers into CIII. BCS1L mutations can cause a diverse range of syndromes such as GRACILE syndrome, Björnstad syndrome, as well as other nonsense mutations and variants in splice sites.

Genes associated with CIV Deficiency

CIV (or COX) is the terminal oxidase of the ETC. Its functions include the translocation of protons into the intermembrane layer, and the reduction of oxygen to form water.

During infancy, the most severe clinical manifestation of CIV deficiency is LS. There are very few defect-causing mutations relating to the structural components of CIV, indicating that most mutations in this area are not suitable for extrauterine life. Instead, most disease-causing genes are associated with the incorporation of CIV prosthetic groups.

SCO1 and SCO2

Both SCO1 and SCO2 promote incorporation of copper ions in catalytic sites CuB and CuA of CIV subunit MTCO1 and MTCO2, thus inhibiting normal CIV function. Infants found with defective SCO1 and SCO2 are usually diagnosed with fatal encephalomyopathy.

Genes associated with CV Deficiency

CV, more commonly known as ATP synthase, uses energy generated by the proton gradient to convert inorganic phosphate and ADP into energy-storing ATP.

ATP-synthase deficiency due to nDNA mutations is often manifested through neonatal-onset hypotonia and hypertrophic cardiomyopathy. Two disease-causing mtDNA mutations have also been identified.

TMEM70

The prevalent TMEM70 mutation is a homozygous A-to-G transition in intron 2. The mutation results in unconventional splicing and the confusion of mRNA processing of the CV protein. Clinical severity varies, possibly due to variations between individuals in RNA decay systems. The most frequent symptoms include respiratory distress, cardiomyopathy, and delays in psychomotor functions.

MT-ATP6 and MT-ATP8

MT-ATP6 and MT-ATP8 are the only two mtDNA mutations found to be correlated to ATP-synthase deficiency. Heteroplasmic missense mutations in MT-ATP6 are associated with adult-onset NARP (neuropathy, ataxia, and retinitis pigmentosa) and LS. Conversely, the main clinical phenotype of MT-ATP8 mutations is hypertrophic cardiomyopathy.

Diagnosis

Biochemical Testing in Blood and Spinal Fluid

Initial mitochondrial disorder testing usually involves measurement of lactate and pyruvate levels in plasma and cerebrospinal fluid (CSF). Typical signs of mitochondrial disorder is the underutilization of pyruvate in the mitochondria, resulting in insufficient ATP synthesis. Lactate levels rise as a product of increased anaerobic respiration. However, lactate measurements are limited by sampling errors and handling issues. In a properly collected sample, an elevated plasma lactate level (>3 mmol/l) suggests potential mitochondrial dysfunction or organic acidemias and metabolic errors present in other diseases. For patients with associated neurological symptoms, elevated CSF lactate levels is a useful indicator of mitochondrial disease.

Pyruvate level measurements are more catered towards defects involving enzymes closely involved in pyruvate metabolism, such as pyruvate dehydrogenase and pyruvate carboxylase. The instability of pyruvate compounds acts as the main limitation of this method alongside other sampling and collection errors.

Amino acid analysis in blood or spinal fluid is commonly used to evaluate the possibility for mitochondrial disease. Defective mitochondria often induce altered redox reactions, leading to elevations in several amino acids including alanine, glycine, proline, and threonine. Elevations in urinary organic acids, creatine phosphokinase and uric acid are also good measures for diagnosing mitochondrial disorders.

DNA Testing

Developments in DNA sequencing allows for more DNA-testing options to be performed on mitochondrial disorders. Effective mtDNA coding and heteroplasmy analysis can now be performed in blood. Patients with low heteroplasmy of 5 ~ 10% are more prone to mitochondrial disorders. Advent technology such as massive-parallel and next-generation sequencing proved useful for detecting mtDNA deletion or duplication syndromes, while other options such as single gene testing on nDNA, and the recently more prevalent whole exome sequencing are also available.

Biochemical Testing of Tissue

Tissue testing for mitochondrial diseases are most often conducted on muscle tissue. Muscle biopsies allow for detection of mtDNA mutations in specific tissues as well as the presence of low heteroplasmy levels. Biopsies also often help validate the significance of unknown variants found in other tests.

Muscle histology analysis routinely includes light microscopy detecting structural changes, histochemical stains for the evaluation of specific enzymes, and examination of muscle ultrastructure via an electron microscope.

Treatment and Management

Currently there are no known cures for mitochondrial genetic disorders. However scientists are looking into methods that alleviate the symptoms of the disease.

Gene therapy treatment is an experimental method currently running on mice which tries to eliminate the damaged defect-causing mtDNA. This technique is also known as mitochondrially targeted zinc finger nuclease (mtZFN). It recognizes and eliminates mutated mitochondrial DNA by comparing differences in DNA sequences between healthy and mutated DNA.

Alternatively, in vitro fertilisation (IVF) transfer of mitochondria is a technique that removes affected mitochondria from an embryo and replaces them with healthy mitochondria. Therefore, mitochondrial disorder must be identified prenatally for IVF transfer to be successful.