DNA Polymerase gamma and mitochondrial replication disorders

DNA polymerase γ is the only known DNA polymerase in human mitochondria and is essential for mitochondrial DNA replication and repair. Over 160 coding variations in the gene encoding the catalytic subunit of DNA polymerase γ (POLG) have been identified. Human POLG was identified in 1996 (Ropp and Copeland, 1996) and the gene mutations causing human disease was first described in 2001 (Van Goethem, et al., 2001)

Background knowledge

Mitochondria have besides the nuclear DNA (nDNA) their own small 16.5 kb circular double-stranded DNA called mtDNA. The mtDNA contains 37 genes which encode 2 different molecules of ribosomal RNA (rRNA) , 22 different molecules of transfer RNA (tRNA) (at least one for each amino acid) and 13 polypeptideswhich are essential for electron transport and oxidative phosphorylation.

Mitochondrion

The rest of the 1000–1500 proteins that are imported into the mitochondria are encoded by the nuclear DNA (nDNA) . The nuclear gene mutations produce mtDNA alterations and cause mitochondrial depletion syndromes.

These nuclear genes are either

1. those whose products directly affect the mtDNA replication fork, such as POLG, POLG2, and TWINKLE,
2. or whose products supply the mitochondria with deoxynucleotide triphosphate pools needed for DNA replication, such as TK2, DGUOK, TP, SUCLA2, ANT1, and possibly MPV17.

Mitochondrial DNA (mtDNA) defects can be due to deletions, point mutations, or depletion, resulting in loss of oxidative phosphorylation. These mutations may be spontaneous, maternally inherited, or a result of inherited nuclear defects in genes that maintain mtDNA.

It should be noted that mitochondrial disorders are a very heterogenous group. Identical mtDNA mutations may not result in identical disorders (Genocopies) and identical disorders may not have the same nDNA or mtDNA mutation (Phenocopies). nDNA mutations often present in childhood while mtDNA mutations often present in late childhood or adult life

Mitochondrial replication

Mitochondrial replication disorders can affect a variety of organs, with variable ages of onset. The specific manifestations of mtDNA replication diseases are usually determined by the types of mutations within these sets of genes. There is generally a correlation with age of onset and tissue specificity for many symptoms. For example, the hepatopathy usually occurs early in life whereas skeletal muscle myopathy presents later in life. Onset of neuropathies do not appear to be age specific.

MtDNA is replicated by an assembly of proteins and enzymes including

• DNA polymerase γ (pol γ) and its accessory protein.( Of the 16 DNA polymerases in the eukaryotic cell, only pol γ is known to function in the mitochondria)
• single-stranded DNA binding protein (mtSSB)
• mtDNA helicase (Twinkle)
• and a number of accessory proteins and transcription factors

An increasing number of POLG mutations have been reported to be associated with a broad range of clinical phenotypes, some inherited in an autosomal recessive manner, others displaying autosomal dominant inheritance. The POLG mutation database can be found at http://tools.niehs.nih.gov/polg/

Mutations in these genes are studied using mouse models, yeast genetics and invitro biochemical analysis. Three well-established forms of MDS are known: myopathic, encephalomyopathic and hepatocerebral

POLG and PEO

Human pol γ is isolated from mitochondria as a complex containing two subunits, a catalytic subunit, pol γA,(139 kDa)encoded by POLG at chromosomal locus 15q25 and an accessory subunit, pol γB,(53 kDa)encoded by POLG2 at chromosomal locus 17q24.1 (2-5). The catalytic subunit is a family A DNA polymerase with separate polymerase and 3′-5′ exonuclease domains. Mutations in the catalytic subunit of human pol γ cause mitochondrial disorders.The accessory subunit is a 55 kDa protein (p55)and is required for tight DNA binding and processive DNA synthesis

The first POLG disease mutations identified in 2001 were associated with progressive external ophthalmoplegia (PEO). Subsequently, mutations in POLG were identified in patients with Alpers syndrome and other infantile hepatocerebral syndromes, ataxia–neuropathy syndromes, Charcot–Marie–Tooth disease, idiopathic parkinsonism, nucleoside reverse-transcriptase inhibitor (NRTI) toxicity, among others. These diseases are characterized by mtDNA deletions or depletion in symptomatic tissues.Approximately 150 disease mutations in POLG have been identified to date.

PEO is a mitochondrial disorder associated with mtDNA depletion and/or accumulation of mtDNA mutations and deletions and is characterized by

• late onset (between 18 and 40 years of age)
• bilateral ptosis
• progressive weakening of the external eye muscles, resulting in blepharoptosis and ophthalmoparesis.
• Proximal muscle weakness and wasting, as well as exercise intolerance
• Skeletal muscles of PEO patients have decreased respiratory chain enzyme activity and show ragged red fibers pathologically.

Multiple large-scale deletions of mtDNA isolated from muscle biopsies were first demonstrated in Italian families with heritable autosomal dominant PEO (adPEO).To date, with one exception, all autosomal dominant POLG mutations responsible for PEO are in the polymerase domain of pol γ.

For reviews see Copeland 2008,Graziewicz 2006,Hudson 2006

POLG and Alpers Syndrome

Alpers syndrome is a rare but severe autosomal recessive Mitochondrial DNA depletion syndrome (MDS) disease of childhood. Spastic quadri-paresis and progressive cerebral degeneration within the first few years leading to mental deterioration and seizures, cortical blindness, deafness, liver failure, and eventual death.

MR in Alper's

To date, the number of reported Alpers-associated POLG mutations has risen to >35, from 46 different probands,,.POLG mutations in Alpers are recessive. Many of these same mutations can cause arPEO as well.The A467T mutation commonly found as a compound mutation in arPEO is the most frequent Alpers mutation and is found as either a homozygous or a heterozygous mutation combined with other mutations.

POLG and Ataxia-Neuropathy

POLG mutations can also cause ataxia-neuropathy syndrome with onset in the early teens to late thirties. This ataxia, also termed mitochondrial-associated ataxia syndrome (MIRAS), spino-cerebellar ataxia-epilepsy syndrome (SCAE), or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO), is caused by autosomal recessive mutations in POLG producing multiple mtDNA deletions in the affected individuals.

• Symptoms:
  • peripheral neuropathy
  • dysarthria
  • mild cognitive impairment
  • involuntary movements
  • psychiatric symptoms
  • myoclonus
  • seizures

POLG and Male Infertility

The human POLG gene contains a 10-unit CAG trinucleotide tract encoding a polyglutamine stretch near the N terminus of the mature protein.Some studies suggest that alteration of the CAG repeat is associated with loss of sperm quality and contributes to 5%–10% of the male infertility cases in the European population But this has not been confirmed in other independent studies.

POLG2 mutations

Recently, a single mutation in the gene encoding the accessory subunit, POLG2, was reported in a patient with adPEO. This mutation results in defective binding of the accessory subunit with the pol γ catalytic subunit and fails to stimulate DNA synthesis. This causes the complex to stall during DNA replication and results in accumulation of mtDNA deletions as detected in PEO.

The mitochondrial DNA Helicase,THE TWINKLE or PEO1 GENE

The mtDNA helicase encoded by the TWINKLE gene, also known as PEO1. This gene was first isolated as a locus for PEO on chromosome 10, C10orf2. Mutations in TWINKLE are mainly associated with adPEO, but one report has described a recessive TWINKLE mutation as a cause of SANDO.

Adenine nucleotide translocator ANT1

ANT1 is one of three adenine nucleotide translocator proteins found as inner-transmembrane mitochondrial proteins and is the most abundant protein in the mitochondria. ANT1 is highly expressed in heart, kidney, liver, and skeletal muscle. Its main function is to transport ATP out of the mitochondrial matrix in exchange for ADP. Mutations of ANT1 has been associated with adPEO and sporadic PEO.

Thymidine phosphorylase ECGF1

Thymidine phosphorylase (TP) is part of the pyrimidine salvage pathway required for the conversion of thymidine and phosphate to thymine and deoxyribose-1-phosphate. A defect in ECGF1, the gene encoding Thymidine phosphorylase (TP), causes the accumulation of thymidine and uracil in the blood.

Because mitochondria rely heavily on salvage pathways for generating intramitochondrial dNTP pools, the mitochondria take up the excess thymidine. This in turn stimulates the synthesis of excess deoxythymidine triphosphate (dTTP) by thymidine kinase 2 in the mitochondria. The resulting unbalanced mitochondrial deoxynucleotide pools cause mtDNA depletion, multiple deletions, and point mutations.

ECGF1 mutations results in the autosomal recessive disorder Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) due to multiple deletions and mutations of mtDNA. To date, more than 30 mutations in ECGF1 are known to be associated with MNGIE.

• Characteristics:
  • Onset between the second and fifth decades of life
  • ptosis, PEO
  • gastrointestinal dysmotility
  • cachexia, peripheral neuropathy
  • myopathy
  • leukoencephalopathy.
• Potential treatment
  • Hemodialysis has been shown to transiently reduce thymidine levels in blood.
  • Allogeneic stem cell transplantation has had some success in restoring TP activity and lowering plasma thymidine levels.
  • Repeated platelet infusions can reduce thymidine levels in blood in MNGIE patients.

Mitochondrial thymidine kinase TK2

TK2 mutations primarily affect muscle tissue with no effect on liver, brain, heart, or skin. Quantitation of TK2 activity in various tissues relative to mtDNA or cytochrome c oxidase activity helps to explain the tissue specificity of TK2 deficiency.

Deoxyguanosine kinase DGUOK

Deoxyguanosine kinase is the other mitochondrial deoxyribonucleoside kinase that phosphorylates the purine nucleosides into nucleotide monophosphates. To date, 13 mutations have been described in the DGUOK gene, most presented as homozygous mutations from 14 probands Phenotypes are highly variable, including one patient who developed liver failure but responded well to liver transplant muscular weakness, and exercise intolerance due to a severe mitochondrial myopathy. and infantile hepatoencephalopathies

Deoxynucleotide carrier SLC25A19

Amish lethal microcephaly (MCPHA), an autosomal recessive disorder, with a unusually high incidence of at least 1 in 500 births in this population characterized by severe congenital microcephaly, severe 2-ketoglutaric aciduria, and death usually within the first year was described in 2002 by Kelley et al.among the Old Order Amish of Lancaster County, Pennsylvania.

The gene was later localized to chromosome 17q25 and it was later confirmed that the condition was associted with a missense mutation of themitochondrial deoxynucleotide carrier gene (DNC or SLC25A19) contained in this region.

Mouse knockout studies,yeast studies and in vitro transport assays have shown that the G177A DNC did not cause a defect in ribonucleotide or deoxynucleotide transport into the mitochondria, but caused reduced levels of Thiamine pyrophosphate (ThPP)within the mitochondria.. Thus, reduction of ThPP levels causes the inability of the α-ketoglutarate dyhydrogenase complex to function properly, which leads to the high levels of α-ketoglutaric acid in these patients.

Succinyl-CoA synthetase SUCLA2

An autosomal recessive encephalomyopathy associated with mtDNA depletion and linked to a homozygous deletion of the SUCLA2 gene on chromosome 13 was first reported in 2005 in a small Muslim pedigree.

MR in SUCLA2 mutation

Later SUCL2 mutations were shown associated with an autosomal recessive mitochondrial encephalomyopathy and elevated methylmalonic acid identified in the Faroe Islands population. Elevated methylmalonic acid is characteristic of methylmalonic acidemia (MMA), a heterogeneous group of disorders with symptoms that include vomiting, dehydration, lethargy, seizures, failure to thrive, progressive encephalopathy, and developmental delays.

Succinyl-CoA synthetase deficiency

Succinly CoA synthetase is a mitochondrial matrix enzyme that catalyzes the reversible synthesis of succinate and ATP (or GTP) from succinyl-CoA and ADP in the tricarboxylic acid (TCA) cycle. SUCLA2 encodes the β-subunit of succinyl-CoA synthetase. The reverse reaction occurs in the Krebs cycle. The synthesis of succinyl-CoA is needed for activation of ketone bodies and heme synthesis.

Deficiency of succinyl-coA synthetase results in accumulation of the substrate of the enzyme, succinyl-CoA, which in turn leads to elevated methylmalonic acid, because the conversion of methylmalonyl-CoA to succinyl-CoA is inhibited.

Immunoprecipitation experiments have shown that succinyl-CoA synthetase is in complex with mitochondrial nucleotide diphosphate kinase. The defect in SUCLA2 might disrupt association with the nucleotide diphosphate kinase, causing a defect in the last step of mitochondrial dNTP salvage by nucleotide diphosphate kinase, which leads to decreased dNTPs and subsequent mtDNA depletion. This might explain how how a defect in the citric acid cycle and accumulation of methylmalonic acid is related to mtDNA depletion.

Mutations in the succinyl-CoA gene should be considered in patients with early/neonatal-onset encephalomyopathy, dystonia, deafness, and Leigh-like MRI abnormalities mainly affecting the putamen and the caudate nuclei.

MPV17

MPV17 was recently identified as candidate loci for mitochondrial disorders. Mutations were shown to be linked to hepatic failure early in life. Mutation of the MPV17 gene has also been associated with the Navajo neurohepatopathy, an autosomal recessive multisystem disorder found in the Navajo of the southwestern United States. It is characterized by liver failure, severe sensory neuropathy, corneal anesthesis and scarring, cerebral leukoencephalopathy, failure to thrive, and acidosis.

Inducible ribonucleotide reductase RMM2B

The nucleotide precursors required for DNA replication can be directly obtained by reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates by ribonucleotide reductase.

Ribonucleotide reductase is made up of two subunits: a large catalytic subunit, R1, and the smaller R2 subunit. Cells have two forms of the R2 subunit: a cell cycle regulated form that is maximally expressed in S-phase, and a p53-inducible form known as p53R2. The p53R2 form is required for a basal level of DNA repair and mtDNA synthesis in nonproliferating cells.

The RRM2B gene encoding p532B has been identified as a candidate gene in a condition with severe muscle mtDNA depletion, resulting from nonsense, missense,splice-site mutations and inframe deletions.

The RRM2B gene might thus have a role in mtDNA nucleotide metabolism and mitochondrial disease.

Summary

	POLG1	DGUOK	MPV17	TWINKLE	TK2	RRM2B	SUCLG1	SUCLA2
Age of onset	Variable	Neonatal	Neonatal	Neonatal	Infancy, childhood	Neonatal, infancy	Neonatal	Infancy
Liver disease	Hepatic failure	Hypoglycaemia, hepatic failure,	Hypoglycaemia, hepatic failure,	Hepatic failure	None	None	Hepatomegaly	None
Muscular features	Myopathy, external ophthalmoplegia	Hypotonia	Hypotonia	Myopathy, hypotonia, ophthalmoplegia	Myopathy, elevated CK	Trunk hypotonia	Hypotonia, lactic acidosis	Hypotonia, lactic acidosis
CNS features	Poliodystrophy, ataxia, epilepsia partialis continua	Nystagmus, dystonic movements	Ataxia, neuropathy, dystonic movements	Athetosis, sensory neuropathy, ataxia, epilepsy	Occasionally PEO, seizures	Microcephaly and global developmental delay hearing loss	Connatal encephalopathy	Leigh-like syndrome, dystonia
mtDNA depletion	Liver, muscle	Liver	Liver	Liver	Muscle	Muscle, kidney	Muscle, liver	Muscle
Inheritance	AR	AR	AR	AR	AR	AR	AR	AR
Other features	Valproate hepatotoxicity	–	Corneal scarring mental retardation scoliosis	Infantile-onset spinocerebellar ataxia (IOSCA)	SMA1-like, SMA3-like, muscular dystrophy	Tubulopathy nephrocalcinosis	Dysmorphisms, methylmalonic aciduria	Deafness methylmalonic aciduria

source:Spinazzola et al 2008

Future research

• pathogenesis and tissue specificity of mitochondrial depletion disorders
• animal models of mtDNA replication
• development of therapies

References

Contributors to this page

Biju Hameed