New Hazards in GMOs from Synonymous Mutations

Prof Joe Cummins

Institute of Science in Society

Change in base sequence that does not alter amino acid sequence of proteins encoded nevertheless may result in alterations of the protein that make it unsafe.

Single nucleotide mutations form the majority of genetic polymorphisms (single nucleotide polymorphisms, SNPs) in populations. When found in gene protein coding regions, SNPs can be synonymous, i.e., causing no change in the amino acid encoded, or non-synonymous, when the amino acid is altered.  Until the current decade, synonymous mutations were assumed to be neutral, with no effect on the protein or any other functions of the organism.  Sequencing a vast array of genomes has revealed surprisingly, that many synonymous mutations were causing dysfunctions and illnesses in plants and animals. Synonymous mutations may lead to changes in protein folding related to translation pausing, RNA splicing, and alterations in enzyme specificity [1].

All current GM crops use protein coding genes from bacteria that have been  altered by introducing synonymous  codons, replacing plant-preferred codons for the  bacterial codons in order to enhance the production of protein from the transgenes. However, the toxicity of the transgenic proteins in animals and humans was not studied. Instead the original proteins produced in bacteria were used as surrogates in feeding trials.  The synonymous codons were assumed to be neutral and to have no effect on the transgenic proteins, and presumed to be safe [2] (Bt Toxins in Genetically Modified Crops: Regulation by Deceit, SiS 22).

The genetic code is made up of 64 three letter codons (code words) for twenty amino acids (61 codons) plus words for translation start and stop. The number of code words for amino acid varies from one for methionine and tryptophan to six for arginine, leucine, and serine. The first two positions of the codon are fixed for a particular amino acid while the third position is said to ‘wobble’, allowing for alternate code letters, hence two or more codons for most amino acids. The frequency with which different codons are used varies between groups of organisms; so for example, genes from bacteria are poorly read in higher plants and vice versa.  For optimum expression, the code for a transgene frequently needs to be rewritten. The codon bias characteristic of each group of organisms is believed to be caused by the presence of distinct transfer RNA families in the different groups of organisms. In synthesizing transgenes for GM crops, say, a Bacillus thuringiensis (Bt) cry toxin gene, a table of plant preferred codons is used to substitute for the bacterial codons [3]. Sometimes, it is necessary to substitute one or more of the amino acids (non-synonomously) so that the final cry toxin can function in the plant cell environment [4]. As plant genetic engineering has “advanced” the crucial active domains of toxins, and enzyme are “improved” to such an extent that the gene for the original source protein is hardly recognizable.

There are many examples of synonymous mutations that are not neutral.  Synonymous mutations may lead to ribosome stalling, thereby changing protein folding pathways affecting enzyme activity or antigenicity [5].  Synonymous mutations  in  the HIV gene Rev enhance HIV-1 replication,  providing resistance to the drug enfuvirtide [6].  Synonymous codons in the oncogenes of the rabbit papillomavirus increased oncogenicity and immunogenicity [7]. A single synonymous mutation was sufficient to alter the substrate specificity of a multidrug resistance phenotype in mammalian cells [8].Synonymous mutations affect the stability of mRNA secondary structure in mammals [9].

To Conclude

It is now clear that synonymous mutations are not neutral but instead frequently adversely affect the proteins encoded.  Essentially all commercial GM crops contain bacterial genes with codons altered to enhance the expression of transgenes in plants. The products of such transgenes carrying synonymous mutations were risk assessed using surrogates produced originally in bacteria and free from synonymous mutation.  Thus the regulatory approval of GM crops is based on the false assumption that synonymous mutations are neutral, and hence illegitimate. It cannot be assumed that the crops are free of defect just because millions have consumed GM food. GM food has not been labelled in the market, and no effort has been made to trace the impact of consuming such food. Nevertheless, there has been an apparent “marked deterioration of health” reported with the introduction of GM crops based on inspection of official data (see [10] Scientific American Disinformation on GMOs, SiS 60).   It is reasonable to assume that the synonymous mutations may have created proteins with adverse phenotypes and contributed to the deterioration in public health, and this must now be thoroughly investigated together with mandatory labelling of GM products.

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References

1. Parmley JL, Hurst LD. How do synonymous mutations affect fitness? Bioessays 2007, 29(6), 515-9.
2. Cummins J.  Bt toxins in genetically modified crops: regulation by deceit.  Science in Society 22, 32-3, 2004.
3. Fischoff D. and Perlak F. Synthetic plant genes 1996 United States Patent 5,500,365 pp1-59
4. Payne J, Cummings D, Cannon R, Narva K. and Stelman S. Bacillus thuringiensis genes encoding lepidopteran-active toxins 1998 United States Patent 5,723,758 pp 1-32
5. Tsai CJ, Sauna ZE, Kimchi-Sarfaty C, Ambudkar SV, Gottesman MM, Nussinov R. Synonymous mutations and ribosome stalling can lead to altered folding pathways and distinct minima J Mol Biol. 2008 Nov 7;383(2):281-91. doi: 10.1016/j.jmb.2008.08.012.
6. Ueno M, Kodama EN, Shimura K, Sakurai Y, Kajiwara K, Sakagami Y, Oishi S, Fujii N, Matsuoka M. Synonymous mutations in stem-loop III of Rev responsive elements enhance HIV-1 replication impaired by primary mutations for resistance to enfuvirtide. Antiviral Res 2009, 82(1), 67-72. doi: 10.1016/j.antiviral.2009.02.002
7. Cladel NM, Budgeon LR, Hu J, Balogh KK, Christensen ND. Synonymous codon changes in the oncogenes of the cottontail rabbit papillomavirus lead to increased oncogenicity and immunogenicity of the virus. Virology 2013, 438(2), 70-83. doi: 10.1016/j.virol.2013.01.005.
8. Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, Ambudkar SV, Gottesman MM. A “silent” polymorphism in the MDR1 gene changes substrate specificity. Science 2007 315(5811), 525-8.
9. Chamary JV, Hurst LD. Evidence for selection on synonymous mutations affecting stability of mRNA secondary structure in mammals. Genome Biol 2005, 6(9), R75.
10. Ho MW, Sirinathsinghji E and Saunders PT. Scientific American disinformation on GMOs. Science in Society 60, 2-4, 2013.

Tags: Farm Wars, farmwars, genetic engineering, GMO, Health, isis, joe cummins, mutations, toxicity, transgenic proteins