Figure. 2. Homo sapiens TLR4 mRNA for toll-like receptor 4, complete cds: GenBank: AB445638.1. When the primer is designed, the highlighted c (red) is altered to g (red) in the F primer to fit with the restriction site Hinf1 (GANTC)= gaatc. N= any nucleotide. This SNP is known as rs4986791 or 1196 C/T which means the nucleotide cytosine 1196 (C1196) is mutated to threonine 1996 (T1196). The wild type codon (aCc) is Threonine (Thre) and its product size= 406 bp. The SNP codon (aTc) is Isoleucine (Ile) and its product size= 406-29= 377 bp. Thus, the amino acid Thr399 is changed to Ile399. Therefore, the SNP is also called TLR4 Thre399Ieu
PCR reactions were prepared by mixing 10 µl Add start mastermix 2 x (Add Bio Inc, Korea) with 1 µl of each primer (5 pM) and 10 µl genomic DNA. The reactions were run in a thermal cycler (Master cycler nexus, Eppendorf AG, Hamburg, Germany) by initial denaturation at 95°C for 5 minutes; followed by 40 cycles of 95°C for 30 seconds, annealing temperature at 62 for 45 seconds and 72°C for 30 seconds; and finally extended at 72°C for 5 minutes. Ten (10) µl PCR products were digested by 1 µl of Hinf1 restriction endonucleases (New England Biolabs, Ipswich, MA, USA) in 4 µl Hinf1 buffer 4x and 10 µl nuclease free water (Promega) incubated at 37°C for 2 hours. The digested products were mixed with gel loading dye, Purple (6X) (New England Biolabs, Ipswich, MA, USA) and explored on 2% agarose gel, stained with 10 µl Prime safe dye (GENET BIO CO., Daejeon, KR), run with 1x TBE buffer at 100 v, 80 mA for 2 hours. The wild type product size (406 bp) is cleaved with Hinf1 restriction enzyme to form 377 bp and 29 bp in case of the mutant variant.
To test the Hardy-Weinberg equilibrium, Gene Calculators online program (Chi-square, P>0.05) was used (https://www.genecalculators.net/pq-chwe-check.html).
3. Results and discussion
Current study investigated TLR4 Thr399Ile variant in Kurdish populations in Sulaymaniyah province using PCR-RFLP. The results of this study showed that 6 out of 85 (7.1 %) of the samples have carried the heterozygous mutant alleles of the TLR4 Thr399Ile variant. An example was shown in Supplementary Figure 1. No homozygous variant was identified and the genotype frequency was not deviated from Hardy-Weinberg equilibrium (Chi-square P=0.735). This variant is infrequent among other populations in middle eastern countries. For example, 5.8 % of TLR4 Thr399Ile heterozygous mutant was determined in healthy Iranian populations and homozygous mutants were only found in chronic cutaneous leishmaniasis cases . Likewise, no homozygous mutants of TLR4 Thr399Ile were found in Turkish populations ,but the heterozygous variants were 9% . A study also suggested weak selective pressure of the TLR4 Thr399Ile variant in fighting infections since the alleles are heterogeneous among Iranian ethnic populations including Iranian Kurds . It has been shown that TLR polymorphisms are associated with both reduced responses to bacterial infections and susceptibility to Gram negative bacterial infections due to LPS signaling impairment . Several studies have focused on laboratory mice, or different ethnic populations including Caucasians and Koreans , Iranians [12,14], sub-Saharan Africa, Europe and East Asia . For instance, the TLR4 Thr399Ile SNPs (rs4986791) is only 0.79% in Africans, 8.51% in Europeans, and 0% in East Asians and this SNP is considered as ‘possibly damaging’ to the function and structure of the protein . Interestingly, in Caucasian populations, TLR4 Thr399Ile is 17.7% heterozygous variant with no any homozygous mutants were found . Up to my best knowledge by the time of writing this manuscript (08/11/2019), no articles were found in Pubmed- NCBI data base when searching for key words as ‘TLR4 SNP Iraq’. Although, when searched for ‘TLR4 polymorphism Iraq’ only one article was published that was about TLR4 Asp299Gly SNP (rs4986790) and the samples were taken from Jordanian populations but one of its authors is from Iraq . Similarly, no data were found for TLR4 SNP in Syria as a country of having Kurdish populations.
In spite of its limitations, this is the first time, TLR4 gene polymorphisms are investigated in Kurdistan Region of Iraq. However, when searching in google for ‘TLR SNPs Iraq, in other provinces of Iraq, where the majority of populations are almost Arabs, several articles have been published in several journals which were not cited in this manuscript due to their poor quality. The limitation of the current study is that the other SNP, TLR4 Asp299Gly variant was not investigated. It is worth to mention that this SNP is often co-segregated with the SNP investigated in this study. The co-segregated TLR4 Asp299Gly/Thr399Ile mutants play an important role in inflammatory response to bacterial LPS challenge similar to the wildtype variants known as ‘functional neutrality’ . Both segregated mutations may have been originated from Africa approximately 60,000 years ago, spreading out through Arabian Peninsula and middle eastern route to Europe as a result of genetic drift . Further researches are required to investigate both SNPs in Kurdish ethnic populations in all Kurdish inhabiting provinces and countries including Iraq, Iran, Turkey and Syria. Particularly, comparing the mutants among the main Kurdish tribes will give an idea about the selective pressure and homogeneity of the TLR4 SNP alleles in Kurdish ethnic populations.
Genetic variations, together with historical, demographic, linguistic, anthropological and archaeological findings, can be exploited as a population marker to study human evolution . Discovery of both Neanderthal and Denisovan like haplotypes of TLR1, 6, and 10 in modern human indicates gene flows among archaic human species . Investigations of gene polymorphisms in Kurdish people might be of archaeological and anthropological interests since the area was a homeland of ancient civilizations and archaic human species. For instance, in Shanidar, (an ancient cave in Zagros mountain belonging to Iraqi Kurdistan), Neanderthal bones were found and dated back to about 40,000-70, 000 years ago . This is apparently the period when both homo sapiens and Homo Neanderthals encountered and interbred [19,20] Research about ancient DNA has not been recorded yet in human species in the Kurdistan caves. It might be fascinating for both archaeologists and evolutionary geneticists to seek polymorphisms in innate immune genes between the two human species.
In conclusions, Iraqi Kurdish populations, lived in Sulaymaniyah province, bear only the heterozygous variant of TLR4 Thr399Ile (7.1%). This suggested the rarity of the homozygous mutant in the province. Further study with large size samples is recommended to confirm this, taking samples from other provinces in Kurdistan region. This also suggests that TLR4 Thr399Ile SNP in Kurdish people is neither very common as in Caucasian nor rare as in East Asian nor uncommon as in African people. This result also suggests that the Kurds has TLR4 Thr399Ile SNP frequency similar to that of Europeans or Middle Eastern nations as in Iran; it supports both Indo-Iranian and Endo-European language families. It seems to be exciting to compare the TLR4 SNPs between modern and ancient human species found in the caves of Kurdistan. Finally, this investigation unlocks a main gate for further research on genetic associations of this innate immune gene with infectious, immunological or non-infectious diseases, and it is also anthropologically and genetically important for studying Kurdish population.
Dr. Sirwan M. Amin, Mr. Mariwan AbdulRahman, Ms. Sakar Nariman, Ms. Soma Mahmood, Mr, Chya Mustafa Othman, and Ms. Dekan Kamaran helped me during sample collections. I acknowledge all staffs and students who donated the blood samples.
Supplementary information related to this article can be found at: (Supplementary ) Niranji, Sh. S. Passer 1 (2020) 32- 36