Laayouni et al. were interested in changes of immune genes of two populations that lived in the same geographical area, in this case Europeans (Romanians) and Rroma (Gypsies), and were subjected to the same diseases. They used the geographical origin of the Rroma, northern India, to locate a reference population that was assumed to have remained untouched by the plagues that swept through Europe during the recent one thousand years. The Rroma settled in Romania with the Europeans around the 11th century after migrating from India and thus, would be subjected to any infectious diseases that passed through Europe in the last millennium.
This study recruited 100 individuals of European/Romanian descent and 100 individuals of a Rroma/Gypsy ethnic background and took a blood sample from each. In addition, 500 individuals descended from a North Indian population consented to the study and donated a blood sample. The isolated DNA was used to analyze 196,524 single-nucleotide polymorphisms (SNPs) with the Illumina immunochip array. Software analysis demonstrated differences among all three populations, confirming the Rroma and Northern Indians were genetically related whereas the Europeans were genetically distinct from both Rroma and Indians.
To discover which genes or gene regions may have undergone selection in the European and Rroma populations but not in the North Indian, the SNP results were subjected to two tests. These tests identified genetic differences and changes in allele frequencies and examined the most divergent loci in both tests to detect positive selection. The tests sought similar outliers in Romanian and Rroma populations that were not found in the Indian population as well as genetic differentiation between Rroma and Indians and between Romanians and Indians. One gene cluster that was overrepresented (highly selected) in the Rroma and Romanian populations was the Toll-like receptor (TLR)/cytokine–mediated signaling pathway group comprised of the genes TLR1, TLR6 and TLR10 on chromosome 4. Further analysis using derived allele frequency (DAF) confirmed that this overrepresentation was a real phenomenon shared by Romanians and Rroma and not the Indian population. The TLR genes were not the only ones that seemed to be selected in the European and Rroma populations, but TLRs were of interest based on their role in recognizing invading microbes and activating innate immunity.
TLRs are known to recognize bacteria like Yersinia pestis, which is the causative agent of the Black Death, the devastating plague that swept through Europe in the mid 14th century. The plague exacted a heavy toll, killing an estimated 30–50% of the global population and acting as a potential selective force. TLR2, also part of the same gene cluster found on chromosome 4, recognizes Y. pestis when it invades the human body. TLR1 and TLR6 are known coreceptors for Y. pestis recognition, but the role of TLR10 is less well known for this organism. To investigate the role of TLRs in the immune response to Y. pestis, the authors transfected human embryonic kidney (HEK) cells with TLR2 only, TLR10 only and TLR2+TLR10, then exposed the HEK cells to Y. pestis and Y. pseudotuberculosis (the parent to Y. pestis). TLR2 alone induced a large cytokine response to both Yersinia species while TLR10 alone did not. However, when the two TLRs were cotransfected, TLR10 modulated the cytokine response of TLR2. A complementary experiment using an anti-TLR10 antibody to deplete TLR10 in monocytes exposed to Y. pestis and Y. pseudotuberculosis demonstrated that without TLR10, TLR2 responded by producing increased levels of cytokines. Thus, TLRs do play a role in immune reponse to infection by Y. pestis, suggesting the Black Death may have played a role in selecting for inherited TLR gene variants.
To correlate the SNPs found in the TLR1/TLR6/TLR10 gene cluster to a response to Y. pestis, peripheral blood mononuclear cells (PBMCs) were isolated from individuals who were wildtype, heterozygous or homozygous for SNPs in each of the TLR genes and were exposed to Y. pestis. Those homozygous for the variant modulated the cytokine response compared to wildtype; a lesser effect was seen with heterozygous PBMCs. A known polymorphism in TLR4, a gene that did not show selection in the initial analysis, did not change the PBMCs response to Y. pestis.
This study investigated the possibility of convergent positive selection of immune genes in two genetically distinct populations in a single location that were subjected to the same infection pressure and found a gene cluster of Toll-like receptors that have a role in microbial recognition. The authors looked at Yersinia pestis specifically due to its high mortality in the 14th century and thus, likely selective pressure on the human population. Their preliminary evidence does suggest that TLR1/TLR6/TLR10 polymorphisms found in the Rroma and Romanian populations are more common compared to the Indian population that showed no selection of these SNPs. However, the authors acknowledge that there were other diseases that could have influenced the inheritance of these SNPs. The Black Death was just the most likely candidate. Laayouni et al. took advantage of a unique situation with three populations (two of which are related genetically and two of which are associated geographically) and managed to find some preliminary evidence of immune system selection from disease pressure.
Laayouni, H., Oosting, M., Luisi, P., Ioana, M., Alonso, S., Ricaño-Ponce, I., Trynka, G., Zhernakova, A., Plantinga, T.S. and Cheng, S.C. (2014) Convergent evolution in European and Rroma populations reveals pressure exerted by plague on Toll-like receptors. Proceedings of the National Academy of Sciences of the United States of America, PMID: 24550294
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