Gene machines at fault in eye paralysis

A molecular machine known as POLG, responsible for copying DNA in cells, has been shown to be the entity at fault in the debilitating ocular disorder known as progressive external ophthalmoplegia (PEO).
The disorder, characterised by deterioration in the muscles that move the eyes, advances to the point at which a patient must turn his or her head to follow an object. The disease has no current medical treatment.

The disorder has been linked to mitochondrial DNA (mtDNA), an extra-chromosomal compendium of genes. In humans, mtDNA is transmitted exclusively down the maternal line. As a result, affected males with mtDNA disease do not transmit the genetic defect.
PEO now joins an expanding list of human diseases traceable to this ancient stretch of DNA. Pathogenic mtDNA defects affect an estimated one in 15,000 adults.

Onset of PEO typically occurs in adulthood. Characteristic features include drooping of the upper eyelid (ptosis), external ophthalmoplegia, and slowly progressive skeletal muscle weakness. An accumulation of abnormal mitochondria at the periphery of muscle fibres gives them a 'ragged-red' appearance when viewed under a microscope.
To appreciate the reasons behind the severity of the pathology associated with mtDNA, you have to look at where mtDNA comes from and how it functions.

To understand mtDNA, you have to look at the history of the human cell. That history is founded on the so-called endosymbiont hypothesis. According to the theory, modern animal cells, such as those in your own eyes and elsewhere throughout your body, derive from a mutually beneficial merger which occurred about one billion years ago between a bacterium and a primitive animal cell.

At that point in time, the bacterium was highly efficient at generating energy via photosynthesis whereas the primitive animal cell was less efficient. Joining forces allowed a trade off: the animal cell could provide the bacterium with nourishment and protection; in return, the bacterium provided a ready-made energy supply.

Ultimately these bacteria evolved to become regular features of human cells. Today, we refer to these highly adapted 'bacteria' as mitochondria. And each mitochondrion possesses its own genome, the mtDNA.

In the human cell, mitochondria are responsible for oxidative phosphorylation or more simply, power supply. As any engineer will tell you, power is everything. Without power, all machines, including biological ones, grind to a halt. For this very reason any malfunctioning in the mtDNA can have very serious consequences.

Generally, mtDNA faults can be broadly divided into two groups: deletions and point mutations. Deletions, from a few hundred base pairs to over 10,000, can cause disorders such as PEO or Kearns-Sayre syndrome while point mutations may cause disorders such as Leber's hereditary optic neuropathy (LHON).

The mitochondrial genome consists of 16,569 bases and comprises 37 genes (13 polypeptides, 22 transfer RNAs (tRNA) and two ribosomal RNAs (rRNA). Partly due to poor repair mechanisms, mtDNA accumulates mutations more rapidly than nuclear DNA.
This feature, in combination with exclusive maternal inheritance, has made mtDNA analysis useful in forensics. One prime example of the application of mtDNA analysis was the identification of bones unearthed in Russia in 1991 as those of Czar Nicholas II.

Until recently, a puzzling aspect of PEO was that patients with the disorder were found to have accumulated various mutations in different genes of their mtDNA. This accumulation of mutations and deletions within mtDNA led researchers to look closely at the genes responsible for the maintenance of mtDNA.

That changed last year when a European research group identified a mutation on human chromosome 15 in the polymerase y gene (POLG). POLG is a molecular machine known as a polymerase and is the only DNA polymerase known to be responsible for mtDNA replication.

Led by Christine Van Broeckhoven PhD, of the Flanders Interuniversity Institute for Biotechnology at the University of Antwerp in Belgium, the group worked with the DNA of a Belgian family. The family was chosen because its members transmitted the disease from generation to generation in a predictable way.

Dr Broeckhoven's team sequenced the suspect gene of approximately 3,700 bases and found, at base number 2864, a change from A (adenine) to G (guanine). The change of codon altered the corresponding amino acid from tyrosine to cysteine, disrupting a critical part of the polymerase.

A quick computer search showed that the mutation identified was highly conserved in a number of species including yeast and flies. If a certain DNA sequence is shown to be the same across a number of different species, it is highly likely that it performs some essential function.

This vital finding immediately focused the efforts of other research teams to try to figure out why this mutation was causing so many problems.
Enter the research group led by William Copeland PhD at the Laboratory of Molecular Genetics at Research Triangle Park in the American State of North Carolina. Dr Copeland's team got to work studying the enzymology of POLG. Soon, Dr Copeland's group discovered that the mutation identified in the Belgian family caused the error rate to double when POLG attempted to replicate the mtDNA. This immediately explained the clinical pathology observed in patients with PEO.

The disease usually manifests in patients between 30 and 40 years of age. Because there are generally large numbers of mitochondria in each cell, it can take a long time for the effects of the error rate to accumulate. Eventually, the power supply is affected and clinical symptoms begin to appear.

Dr Copeland's team also suspects that interruption in energy supply may not be the only problem caused by the mutation in the POLG gene. Disruption of oxidative phosphorylation can result in a leakage of electrons from the electron transport chain where they may combine with oxygen to form superoxide anions. These anions cause cellular damage; researchers believe these anions may also contribute to the natural process of ageing.

These important research reports from the laboratories of Dr van Broeckhoven and Dr Copeland add to a growing body of evidence that genes involved in DNA replication can cause disease by introducing mutations into mtDNA

It is no coincidence then that disorders in mtDNA manifest more quickly in tissues that have a high energy demand such as the eyes, brain and kidneys. In eye muscles such as the lateral rectus, mitochondria comprise approximately 60% of the cell volume, indicating high energy requirements and consequently an increased sensitivity to faults in mtDNA replication.

In terms of devising a solution to this devastating disorder, there is some positive news: a therapeutic solution can now be focused on one gene rather than trying to cover all the mistakes brought about through the errors of that gene. POLG will now surely come under the spotlight of intense research to investigate how to manipulate, neutralise or replace this defective gene.

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