Friday, October 2, 2009

Dynamic Genomics

During the Persian Gulf War, some 700 000 individuals were exposed to a whole range of environmental hazards, including low-level chemical warfare agents, investigational drugs (inclucing pyridostigmine bromide, used as a prophylactic against nerve agents), organophosphate, carbamate, and other pesticides and insect repellents, low levels of nuclear and electromagnetic radiation, toxic combustion products from oilwell fires, diesel exhaust products and airborne particles, all collectively known to be genotoxic, or capable of causing harm through effects on the genetic material. The veterans were also exposed to multiple vaccinations, also of questionable safety. A significant proportion of the veterans developed a pattern of symptoms that have been referred to as Persian Gulf War-Related Illnesses, or Gulf War Syndrome (GWS): rash, fatigue, muscle and joint pain, headache, irritability, depression, unrefreshing sleep, gastrointestinal and respiratory disorders and cognitive defects. These were eventually defined as a clinical entity in 1998.

Most Gulf War I veterans (GWIVs) received oral poliovirus vaccine before deployment. Persistent enterovirus infection has been implicated in the chronic fatigue syndrome, one of the major disorders of GWIVs. There has already been a report that enterovirus-specific RNA was found in the sera of patients with chronic fatigue syndrome. For these reasons, Howard Urnovitz, Scientific Director of the Chronic Illness Research Foundation, and his colleagues decided to search for virus-specific nucleic acids in the sera of the GWIVs by using virus-specific primers to amplify RNA sequence. The sera from 24 GWIV with GWS deployed approximately 5 years previously were compared with serum samples from 50 controls, for the most part matched by age, sex and race.

When the amplified RNAs were separated according to size by running the mixtures through an agarose gel in an electric field, a striking difference between the GWIVs and controls was seen. Controls typically gave no more than three faint RNA bands, all less than 350 nucleotides (nt) in length. The sera from GWIVs, in contrast, contained numerous bright bands of very large RNAs, most of them longer than 750nt and especially longer than 2 000 nt. Most of the bands, moreover, did not belong to either the poliovirus or enterovirus. Both viral RNAs tended to be found more frequently in the sera of GWIVs, but the differences from controls were not significant.

The team sequenced two of the many bands that were found only in GWIVs, one 414nt and the other, 759nt, from three different samples. They were 99% identical between samples, but unrelated to each other, and were not homologous (similar) to any sequence found the public DNA database GenBank. However, short stretches of 14 to 15nt were homologous to segments in a region on the short arm of chromosome 22, 22q11.2. It is as though something had chopped up that region into pieces, shuffled them, and joined them up together again.

Thus, 3 sequences of 15nt and 8 of 14nt in the 759nt RNA had 100% homology to short segments of chromosome 22q11.2. Five of these segments occur only on chromosome 22q11.2. For the 414nt RNA, there were 2 sequences of 15nt and 4 of 14nt with 100% homology to the 22q11.2 region, but these segments also occur on other chromosomes, so it cannot be excluded that other chromosome regions were also involved in this gene shuffling exercise. Another important feature is that 6 of the segments in the 759nt RNA and 2 of those in the 414nt RNA occur near, between, or in Alu elements ("Molecular genetic engineers in junk DNA?", this series) that are capable of multiplying and jumping around the genome, and are hence thought to be involved in genetic recombination or gene shuffling.

This is a surprising finding. After all, GWS is generally considered to be a ‘multifactorial’ disease, ie, a disease due to multiple causes, possibly one for each of the symptoms. And yet, for the first time, Urnovitz and his colleagues have demonstrated that there could be a common molecular marker for the disease.

Not only that, using the same techniques, Urnovitz and colleagues were able to identify another unique RNA molecular marker in patients with multiple myeloma (malignant transformation of blood plasma precursor cells) and related disorders. They analysed 65 patients with multiple myeoloma (MM) 3 with Waldenstrom’s macroglobulinemia (WM), 2 with monoclonal gammopathy of undetermined significance (MGUS), and 50 healthy controls.

A 713nt plasma RNA occurred in 16/18 of MM patients in relapse, 5/8 MM patients who were untreated, 2/3 WM patients and ½ MGUS patients. None of the MM patients in remission, nor the 50 healthy controls was positive. The homology of the 713nt RNA between four samples was > 99.7% and matched (99.6%) a 704ng sequence of the flanking region of the peroxisome proliferator activator receptor gene, located in the same genome region, chromosome 22q11.2. A 255nt sequence within the 713nt RNA had a 90.2% homology with an Alu consensus sequence.

There is reasonable evidence that multiple myeloma is associated with exposure to industrial chemicals, pesticides or other environmental insults, as in the case of GWS.

This raises key questions: what is the origin of these RNAs? What is the possible role of these RNAs and of chromosome 22q11.2 in these diseases? Have environmental genotoxins played a role in causing disease? And finally, could the RNA molecular markers offer diagnostic tools for the diseases?

Chromosome 22q11.2 has been identified as a region full of hotspots for genetic deletions and translocations correlated with multiple myelomas and related disorders, as well as with rearrangements of the immunoglobulin lambda light chains in the normal immune response. Chromosome 22 appears to be involved in the so-called Goldenhar complex, a birth defect possibly associated with GWS.

That region is full of Alu sequences, previously thought to be nothing but junk DNA. But it is becoming increasingl clear that they have important regulatory functions. Alu expression is induced when cells are stressed by heat shock, or genotoxic agents, and may be part of the detoxification response. Alu sequences are known to be involved in genetic recombination or gene shuffling. Alu-Alu rcombinants are generated by both extrachromosomal and chromosomal genetic mechanisms.

Thus, it seems reasonable to conclude that exposure to toxic substances had activated retrotransposable Alu elements, possibly in specific parts of the genome, which results in gene shuffling to produce the unique sequences of RNAs circulating in the serum.

These circulating RNAs appear to be derived from white blood cells that have died, and are enclosed in proteolipid vesicles that protect them from being broken down. There is evidence that such plasma RNAs account for at least some of the illnesses. They are capable of transforming the blood cells of healthy animals in a mouse model, and are associated with immune suppression, making them more susceptible to infections.

At a conference celebrating the Centennial of the University of Michigan Department of Microbiology and Immunology in May 2003, Urnovitz, presented the new concept of "the dynamic genome", the idea that the genome contains "an operating system that instructs the organism how to both use and adapt genomic elements to the constant challenges of a dynamic environment."

This concept led to a practical breakthough, surrogate marker blood tests for yet another condition, mad cow disease, which can be performed on live animals. And, he also mentioned potential public health application for understanding the role of the genome in epidemics ranging from influenza-like pandemics (SARS) to "Gulf War syndrome, chronic fatigue syndrome, and AIDS".

What led him to the idea of the dynamic genome is the discovery that blood borne particles, or "microvesicles" contain "non-blueprint" RNA. In the past, they were assumed to be foreign, and hence mistaken as viruses.

He rejects the theory that a coronavirus is the cause of SARS. The virus was isolated from lab cultures that showed sick and dying cells. "Transmissible factors don’t have to kill a cell to be part of the disease," Urnovitz says, "they could just dysregulate cell function without killing the host cell."

He has carried out his own analysis on the so-called SARS-related coronavirus gene sequence. "Frankly, I do not see a virus. I see a unique and complete rearrangement of genomic elements. For example, when I look at what is believed to be the gene sequence coding for the spike protein of this coronavirus, I see a complicated gene rearrangement of a region of human chromosome 7." As with the Gulf War Syndrome, gene rearrangements like this immediately says to him, "search for an associated catastrophic environmental event that could have caused such genomic rearrangement."

He sees a correlation between nuclear and chemical weapons deployment over the last 100 years and the associated occurrence of flu-like pandemics. He postulates that when animals are exposed to nuclear or chemical weapons, entirely new regulatory gene set are expressed and packaged into non-viral RNA regulatory microvesicles. The risk of turning an epidemic into a pandemic is increased when the exposed animals are migratory birds that frequent gene-swapping hot spots like southeast China. He says, "The recent sightings in eastern China and Hong Kong of rare migratory birds – white cranes, grey cranes, and swans – that spend significant time feeding in the radioactive-contaminated regions of Siberia suggest that international efforts should be focussed on not only hunting for weapons of mass destruction but also on cleaning up the ones that have already been released into the environment."

He rejects the common belief that vaccines are the key to stopping epidemics: "While the current dogma states that vaccines stop viral epidemics, the historical data do not support that claim. From smallpox to polio to HIV, all vaccine attempts have been ineffective or hazardous to the vaccinee."

His company, Chronix Biomedical, develop screening and diagnostic tests based on the detection of non-viral RNA regulatory microvesicles for both veterinary and human diseases. Is it making a profit? "Not yet," he answered.

The blood test for mad cow disease, or bovine spongiform encephalitis (BSE) —the first that can be performed on live animals—is under development in the laboratory of Professor Bertram Brenig, Director of the Institute of Veterinary Medicine, Georg-August University, Göttingen, Germany. Urnovitz’s collaboration with Brenig’s laboratory has resulted in the detection of a specific RNA unique to cows at risk for developing, or that have confirmed cases of BSE.

Urnovitz claims that the BSE blood test is 100% sensitive on all 6 BSE cows confirmed with a licensed prion test, and 100% specific on all 46 animals from known healthy herds. They found that 3.5% of cohort animals (two animals out of 57) showed a positive response in the surrogate blood marker for BSE. Cohorts are animals born and/or raised in the same herd as a confirmed BSE case within approximately 12 months before and after the date of birth of the BSE case. Positive cohort cases may represent animals at risk for developing BSE.
If Urnovitz is right, we have to seriously rethink environmental health.


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