CC BY
11
Section 2. Life Sciences
https://doi.org/10.29013/ELBLS-21-4-15-26
Zixi Gao,
The experimental high school attached to Beijing Normal University, Beijing, China
E-mail: 1193004899@qq.com
SYSTEMATIC ANALYSIS OF TRPM8'S POLYMORPHISM AND THE PREDICTION OF ITS ASSOCIATION WITH DISEASES
Abstract
Introduction: TRPM8, Transient Receptor Potential Cation Channel Subfamily M Member 8, is located on the chromosome 2. TRPM8 is known for its ability to sense cold temperature or other cooling agents at the body level, including menthol, icilin, cold temperature. At the cellular level, TRPM8 is the non-selective cation channel that can control the movement of calcium ions. There has been enough research investigating TRPM8's function as a sensitive temperature receptor, but seldom research has examined its polymorphism and amino acids change.
Objective: Since TRPM8's SNPs (Single Nucleotide Polymorphisms) have not been fully analyzed, we hope to systematically identify and verify the SNPs that affect TRPM8's transmembrane structure and function, so that the polymorphism of the TRPM8 gene can be mapped to the changes in the structure and function of the protein it encodes. Also, we aim to use SNPs to investigate the relationship between TRPM8 and several diseases.
Methods: The dbSNP database is first used to download all SNPs of TRPM8, and wANNOVAR is used later to do the annotation. Then all SNPs are gathered, and Uniprot is used to find their corresponding exon and amino acids. The aforementioned steps are used to investigate TRPM8 systematically. In terms of pathological research, by setting the p-value at 0.01 as our filter condition, we use GWAS (Genome-Wide Association Study) Central to find the connection between TRPM8 and diseases. Ultimately, UCSC Genome Browser, UCSC Cell Browser, and Gene Expression Omnibus are used to verify the connection.
Results: There are, in general, 870 exonic nonsynonymous SNPs, which is 62.77% of all SNPs. At the amino acid level, 70 of the transmembrane amino acids can be affected by exonic SNPs. Among the 70 SNPs found related to amino acids, 34 of them are dangerous SNPs, making these dangerous SNPs potential, pathogenic sites. Interestingly, 61 intronic SNPs of TRPM8 are found related to diseases, such as monoclonal gammopathy, epilepsy, and so on. when the p-value is smaller than or equal to 0.0001, two intronic SNPs including rs11563199 and rs17869077 are related to psychiatric disorders. The upregulation of TRPM8 when psychiatric disorders occur was also investigated to try
to explain the possible mechanism. This connection is first discovered, suggesting an important role of TRPM8 as a possible biological marker of psychiatric disorders.
Keywords: TRPM8, SNP, non-selective cation channel, psychiatric disorders.
Introduction
TRPM8 is the last member of TRP's largest subfamily. TRPM8 mostly permeates calcium ions and is crucial in maintaining calcium homeostasis. All TRPM8, known for sensing cold, exists in a wide range of animals, including chicken, mouse, lizard, and human, showing its early existence.
As a transmembrane protein, TRPM8 serves to non-selectively transport cations, especially calcium ions. When activated by cooling agents, like menthol, or cold temperature, TRPM8 shows strong outward rectification and generates more negative electronic potential, so that depolarization occurs and increases the possibility of opening the TRPM8 cation channel (1-3). In addition to cold and menthol, TRPM8 is sensitive to voltage and phosphatidylinositol-4,5-biphosphate (PIP2) which hydrolysis can make TRPM8 desensitize [4].
Since TRPM8 can sense cold, it is widely distributed in the sensory neurons of the trigeminal ganglion and the dorsal root. Both the cold temperature and cooling agents, including menthol, can switch the activation curve to physiological membrane potentials while TRPM8 is activated [5]. N-Glyco-sylation is used to modulate TRPM8's sensitivity to cold temperature and cooling agents, and the loss of N-Glycosylation may reduce TRPM8 sensitivity [6].
In addition to sensory neurons, TRPM8 also exists in the prostate, testis, heart, taste papillae, human lung epithelial cells, and other sites [7; 8; 9]. Since TRPM8 is widely distributed in the prostate cell, the relationship between TRPM8 and the cancer is widely investigated. Past research showed that, compared to normal prostate cell, in the prostate cancer cell, TRPM8, served as the calcium cation channel, will decide oncogenic status, dependent calcium ions signatures, that is important in prostate cell proliferation and apoptosis. TRPM8's expression will
increase in cancerous human prostate tissue, and decrease in cancerous human prostate [9]. There is also research showing TRPM8's existence in the bladder: TRPM8's agonists, including menthol, can activate TRPM8, and reduce the interval of micturition, and vice versa [10]. Except for prostate cancer, TRPM8 is also believed as the therapeutic target of migraine. Existing research showed that one of the TRPM8 nonsynonymous SNPs, rs10166942, can lower the risk of suffering from migraines. Having depression or anxiety disorder could lead to a higher risk of having a severe migraine. Although rs10166942's SNPs is related to migraine, the possibility of using TPRM8 agonists or antagonists as treating migraine still needs more investigation [11].
Past research analyzed how TRPM8 can sense cold from either the angle of SNP or amino acids, but none of them have compared and examined both in order to reach a more continuous and comprehensive level. Also, very little research has used TRPM8 SNPs to try to explain certain, relating diseases. Therefore, our goal is to 1) annotate all possible exonic, non-synonymous SNPs change and all pathogenic SNPs systematically, 2) identify the corresponding amino acids in relation to exonic nonsynonymous SNPs, 3) predict and analyze certain pathogenic SNPs or amino acids that can lead to certain diseases and try to explain the process and outcome.
Methods
SNP analysis SNPs extraction
The dbSNP database (https://www.ncbi.nlm. nih.gov/snp/), which is a system used to store and analyze biology, especially molecular biology, genetics, and others. It is also used to help scientists investigating the structure and function of molecules online, instead of doing experiments themselves [12]. This website has been used to
extract all TRPM8 SNPs. The data was saved as a VCF file.
SNP annotation and classification
We use wANNOVAR (https://wannovar.wglab. org) to annotate all TRPM8 SNPs, including the starting and ending point; the original and changeable nucleotide of SNPs; SNP's location: exonic, intronic, 5'-UTR region (UTR represents for untranslated region), 3'-UTR region, upstream, downstream, splicing site, intergenic region; SNPs type (synonymous, nonsynonymous, frameshift, etc.); and so on.
Amino acids analysis
Identify exons and amino acids affected by TRPM8 SNPs
By using data from Uniprot, a database containing a protein's size, function, changes in amino acids, protein subcellular localization in a cell, and other annotated data related to protein. We used the the reference mRNA transcript ENST00000324695 to show TRPM8 gene structure. Then we generate and analyzes figures of place these amino acids locate. Relating diseases analysis
Identify certain diseases connected with TRPM8 SNPs
By using GWAS Central database (https://www. gwascentral.org/), a database including the experiments related to a certain gene, always about a gene's location (mainly from SNP sites) related to certain diseases, we obtain TRPM8's relating diseases. First search the keyword, 'TRPM8', and then set the p-value equal to or smaller than 0.01, because we aim to identify diseases that show quite a strong relation with TRPM8. Then databases of certain diseases are compared to TRPM8 genome annotation in order to find out TRPM8 SNPs in those diseases.
The connection between TRPM8 and diseases
UCSC cell browser (https://cells.ucsc.edu) was used to examine TRPM8's expression at single cell level in body organs of patients compared with TRPM8 expression in organs of normal people. In addition, data sets related to differential TRPM8 in
normal human and patient tissues were downloaded from Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) in order to show TRPM8 expression upregulation or downreg-ulation is related to diseases found by GWAS.
Results
TRPM8 function and distribution
TRPM8 appears in many body organs, including the brain, prostate, the trigeminal ganglion dorsal root. The Figure down below, shows TRPM8 distribution in cell and body. TRPM8 is in the dorsal root and trigeminal ganglion to carry out its function: while depolarization, sensing cold to change its activation curve to control calcium influx.
Figure 1. TRPM8 distribution in the human body. In the figure, TRPM8 is widely distributed in the nervous system, reproductive system, and digestive system, which corresponds to the human brain, basal ganglion, prostate, and testis. The data is downloaded from Cell expression Atlas
The identification and categorization of TRPM8 SNPs
SNPs are nucleotide changing on one single locus, and the rate is at least one percent among all human populations. TRPM8 has 102,181 bases, 22.404 of which are SNPs. A part of this research focused on investigating analyzing TRPM8 SNPs and their relation with exons and amino acids.
Among all 22.404 SNPs, this part of the research focuses more on the 1.386 exonic SNPs and 18.225 intronic SNPs located on the coding region. Only402 are synonymous SNPs, SNPs that can change their nucleotide but do not affect amino acids' type, in these 1386 SNPs (Figure 2). Among all, nonsynonymous SNPs account for the highest portion, reaching 62.77%, meaning over half of the exonic SNPs in the coding region can affect TRPM8 gene coding the protein.
Figure 2. TRPM8 exonic SNPs classification. In the figure, take 'nonsynonymous, 870, 62.77%' as an example. 'nonsynonymous' represents the type of SNP, '870' represents the number of SNPs, and '62.77%' represents the ratio of the specific type of SNPs to all SNPs. As can be seen from the figure, only 29% of exonic SNPs are synonymous SNPs. Nonsynonymous SNPs are the most common SNPs among all exonic SNPs, with the ratio reaching 62.77%
TRPM8 exons and amino acids' relation with its functions
Another part is to find changeable exons and the corresponding region in TRPM8's transmembrane region. First, I divide the SNPs into 21% synonymous SNPs, 62.77% nonsynonymous SNPs, and 8.23% other SNPs. In 8.23% of all other SNPs, there is three nonframeshift insertion; five startloss SNPs that cause changes on exon 1, exon 2, exon 3; five stoploss SNPs which change on exon 9; seven nonframeshift deletion; seventeen frameshift insertion that change exon 1 to exon 10; 26 frameshift deletion that mianly focus on exon9, 17, 20; and 51 stopgain SNPs that mainly focus on exon11, 12, 16, 17, 22. All TRPM8's nonsynonymous SNPs in the coding region appear on exon 2 to exon 25. Exon 12 and
exon 20 are exons that can mutate the most, with 92 and 91 SNPs, respectively.
TRPM8 protein is a homotetramer including one pore helix, one ascending loop, and a transmembrane region. S1 to S6, representing helix 1 to helix 6, are interrelated to form a channel hole in the center; thus generating an ion channel (6). At the micro level, TRPM8 has 1,104 amino acids, and amino acid 692-712 (S1), 735-755 (S2), 760-780 (S3), 795-815 (S4), 830-850 (S5), and 959-979 (S6) are served as the six transmembrane helices. Each of the six helices has its unique function: S2 and S3 have binding sites for cooling chemicals such as menthol, the S4 fragment, and the intermediate part of S4 and S5 can sense voltage change, making TRPM8 the voltage-gated calcium channels, and
the channel hole is formed between S5 and S6. [13; 14], and investigating these transmembrane Therefore, the six transmembrane regions of S1- region's changes can be important to understand S6 are closely related to the function of TRPM8 how TRPM8's function is being affected.
Figure 3. We sort out all TRPM8 exon's nonsynonymous SNPs on the translated region. Exon 12 and exon 20 have the most SNPs, and exon 24 has the least SNPs. Most of the exons have SNPs in the range 20-60, and the average is 41.083. The peak-like occurrence was frequent
At the TRPM8 SNPs level, some researches have shown that, nonsynonymous SNP rs11562975 turning from GG to GC will lead to changes in sensitivity of TRPM8 to cold and cooling agents [15]. TRPM8 has 22.404 SNPs that can mutate, which is 21.94% of the total TRPM8 bases. Among the 22.404 SNPs, 19611, that is 87.53% of SNPs located on the coding area, which can directly affect the coding of TRPM8 protein. Among these SNPs, rs7577262 is considered the most closely related to cold adaption [16].
At the protein level, existing research shows that TRPM8 amino acids change can make a severe influence on TRPM8 function as the cation channel. For example, the absence of Y908, a side chain of Y908, results in almost complete loss of TRPM8 stimulation of cold and menthol [17]. The change from as-paragine to glutamine in 934 amino acids will lead to N-Glycosylation loss, which causes TRPM8 to lose its sensitivity to cold and menthol, and reduces expression [18]. The leucine to proline change of amino acid 1089 at the site was also found to reduce TRPM8 expression and channel activity [16]. Mutations Y1005A and L1009R, as scientists researched, can block the activation of menthol, and changes in
LYS-995, ARG-998, and ARG-1008 will reduce the sensitivity of TRPM8 to PIP 2 [19].
We hope to summarize all amino acids that can change the sensitivity of TRPM8 as a cation channel. Therefore, we use TRPM8's annotation and selected all transmembrane amino acids and SNPs and exons.
70 SNPs were concluded to be linked to amino acids in the transmembrane region, corresponding to exon 16, 17, 18, 19, and exon21. Excluding the repeated 15 amino acids corresponding to several SNPs, there are a total of 55 amino acids that could mutate. The average possible changeable amino acid is 9.17, indicating that nearly half of the amino acids are likely to change their types and mutate in the transmembrane region. S1, S3, and S6 have 10-12 amino acids, accounting for about 50%-57% of all amino acids, while S2, S4, and S5 account for about 33%-38% of the amino acids in the transmembrane region. Such a high proportion indicates that the amino acids of TRPM8 have a very high possibility of changing the structure of the protein. Moreover, in combination with previous experiments, changes in amino acid types may seriously affect the channel function of TRPM8, and even be associated with related diseases [10, 18].
nortsynonymous SM Pa
2 nonSynanymaus SNPh (rameshrtt
nonsynonymous SNPs & irameshil)
3 or more Ihan three nonsynonymous SNPs
Figure 4. The distribution of SNPs locates on TRPM8 amino acids of the transmembrane region. S6 has the most SNPs among all six transmembrane regions. S3 and S6 are helices with the most SNPs; S2 and S4 are helices with the least SNPs. Existing researches have shown that the change of amino acids 934 will lower the cold sensitivity function of TRPM8, and the change of amino acids 1089 will lower TRPM8's expression [6, 18]
Table 1.- Brief information of TRPM8 SNPs *
SNP Reference Alteration Function Exon Amino acids change
1 2 3 4 5 6
rs762170786 A T nonsynonymous exon 16 N692Y
rs767999589 T A nonsynonymous exon 16 I696N
rs1422384639 T A stopgain exon 16 C698X
rs200853140 T C nonsynonymous exon 16 I701T
rs1407748410 T C nonsynonymous exon 16 I702T
rs766440724 A G nonsynonymous exon 16 I702M
rs1350887877 C A/G nonsynonymous exon 16 P703T/P703A
rs1411533847 T - frameshift deletion exon 16 V705Gfs*
rs1273059733 G C/T nonsynonymous exon 16 G706R/G706C
rs1219957101 G A nonsynonymous exon 16 C707C
rs1280983305 G A nonsynonymous exon 16 V710I
rs200217624 G A nonsynonymous exon 17 V736M
rs779488832 G A nonsynonymous exon 17 V737I
rs377385182 T C nonsynonymous exon 17 S739P
rs778493522 C A stopgain exon 17 Y745X
1 2 3 4 5 6
rs200649506 G A nonsynonymous exon 17 A747T
rs1286988090 T C nonsynonymous exon 17 Y754H
rs199991435 G A nonsynonymous exon 17 V755M
rs763131949 T C/G nonsynonymous exon 17 V755A/V755G
rs1350788437 C T nonsynonymous exon 17 H761Y
rs149382347 A G nonsynonymous exon 17 H761R
rs201939575 C T nonsynonymous exon 17 S762L
rs1464646092 G A nonsynonymous exon 17 V763M
rs1172210298 C T nonsynonymous exon 17 P764S
rs770879940 - C frameshift insertion exon 17 E768Rfs*1
rs991220444 A G nonsynonymous exon 17 H765R
rs774214043 C - frameshift deletion exon 17 E768Sfs*1
rs201204922 C A nonsynonymous exon 17 H765Q
rs201080817 C A/G nonsynonymous exon 17 P766T/P766A
rs778608904 C A/T nonsynonymous exon 17 P766H/P766L
rs202105112 C A/G/T nonsynonymous exon 17 P767T/P767A/ P767S
rs148846269 C A/G nonsynonymous exon 17 P767H/P767R
rs749366311 G A nonsynonymous exon 17 E768K
rs200296802 C A stopgain exon 17 S773X
rs200296802 C T nonsynonymous exon 17 S773L
rs199516053 G A/C nonsynonymous exon 17 V777I/V777L
rs766286039 T C nonsynonymous exon 17 V777A
rs753818944 C T nonsynonymous exon 17 L778F
rs1156234321 G A stopgain exon 18 W798X
rs1430954187 A G nonsynonymous exon 18 D802G
rs777936976 A G nonsynonymous exon 18 T803A
rs200017552 C T nonsynonymous exon 18 T803M
rs1293814216 G A nonsynonymous exon 18 G805R
rs771167003 G A nonsynonymous exon 18 G805E
rs761976363 - T frameshift insertion exon 18 Y808Lfs*13
rs560346678 T C nonsynonymous exon 18 Y808H
rs201621223 G A nonsynonymous exon 18 A811T
rs769614714 G A/T nonsynonymous exon 18 V814I/V814L
rs1235983967 - A frameshift insertion exon 18 F815Ifs*6
rs200252660 T A nonsynonymous exon 18 F815I
rs201483334 T C nonsynonymous exon 19 V830A
rs867129706 C A nonsynonymous exon 19 L834M
rs1286708460 T C nonsynonymous exon 19 I838T
rs1235272940 A C/T nonsynonymous exon 19 T840P/T840S
1 2 3 4 5 6
rs1305952489 A G nonsynonymous exon 19 R842G
rs1278968434 G A nonsynonymous exon 19 V849I
rs765594459 T C/G nonsynonymous exon 19 V849A/V849G
rs148696315 G T nonsynonymous exon 19 S850I
rs752284591 T — frameshift deletion exon 21 L959Rfs*66
rs781648773 G A/C nonsynonymous exon 21 V960M/V960L
rs1250600746 G A nonsynonymous exon 21 C961Y
rs556646442 A G/T nonsynonymous exon 21 Y963C/Y963F
rs1042643351 G T nonsynonymous exon 21 M964I
rs1335105286 A C nonsynonymous exon 21 T967P
rs923789976 CCAA-CAT - frameshift deletion exon 21 N968Cfs*55
rs756513083 A C nonsynonymous exon 21 N968H
rs751406672 C A nonsynonymous exon 21 L971M
rs780680561 T C/G nonsynonymous exon 21 V972A/V972G
rs1490992389 - T frameshift insertion exon 21 L975Afs*16
rs1450467773 A G nonsynonymous exon 21 M978V
* Not synonymous, exonic SNPs and their relating amino acids are selected. There are, in general, 70 SNPs that can change amino acids. SNPs mainly locate on exon16,17,18,19, and exon 21, and exon 17 has the most SNPs. Nonsynonymous SNPs are 82.86% of all 70 SNPs, meaning amino acid changing type is a common way of TRPM8s structural change
Among all the changing forms, the proportion of nonsynonymous SNVs is 82.86%; while in TRPM8 exonic SNPs annotation, the proportion of nonsynonymous SNVs is 88.41%. This indicates that the types and number of SNPs changes in the transmembrane region of TRPM8 are not significantly different from that of TRPM8 as a whole, but the number of other missense changeable SNPs except nonsynonymous SNPs is slightly reduced.
TRPM8 changeable SNPs and amino acids cause diseases
Some of TRPM8 exonic, nonsynonymous SNPs can be pathogenic. We first use TRPM8 annotation to select 34 dangerous SNPs. Dangerous SNPs of S6 accounted for 71.43% of all not synonymous SNPs showing its danger of changing SNPs. Based on the fact that S6 and S5 constitute the channel pore, it can be inferred that the variation of S6 may cause the channel to flood with too many or too little calcium ions, making it pathogenic. In contrast, there are only
20% dangerous SNPs in the S3 region, and changes in SNPs and amino acids do not significantly affect the function of the amino acid, i.e. their ability to bind to specific agonists or antagonists. Dangerous SNPs of the six helices accounted for 40.96% of the total, indicating that the variation of SNPs was likely to have a significant impact on TRPM8 functionality.
Since TRPM8's change its amino acids can lead to its functional change, and it is widely distributed, TRPM8 has been considered several diseases' potential targets. According to data from GWAS, when setting a p-value smaller than or equal to 0.01, eight diseases are related with 43 TRPM8 SNPs. The diseases are psychiatric disorders, rheumatoid arthritis, and type II diabetes, astigmatism, monoclonal gammopathy, coronary artery disease, epilepsy, and migraine, and the body indicators are blood glucose, blood pressure, hearing, and so on. Although there are eight diseases relate to 43 SNPs, there is no diseases is related with exonic SNPs, meaning currently
there are no SNPs that can directly affect the type of amino acids to lead to diseases.
There are eight diseases that have been found related to psychiatric disorders, while few research has researched the relationship between psychiatric disorders and TRPM8. Actually, like mental disorders, psychiatric disorders' biological causes have not been yet discovered, let alone finding pathogenic sites. Eventually, instead of the systematic study on the relationship between the SNPs of TRPM8 and these 8 diseases, we will focus on the relationship between TRPM8 and psychiatric disorders, to provide new opinion on the cause of psychiatric disorders.
According to incomplete statistics, thirty hundred million people all over the world are affected by psychiatric disorders. Although the trend is getting stronger, scientists still have not found the cause of psychiatric disorders and their biological marker in the body.
Currently, very few research has been found on investigating the relationship between the TRPM8
gene and psychiatric disorders. Therefore, we examine the relationship between TRPM8 and psychiatric disorders thoroughly. we show that two TRPM8 intronic SNPs, rs11563199 and rs17869077, are related to psychiatric disorders.
We also try to testify how TRPM8 is related to psychiatric disorders. First, we use UCSC cell browser to investigate TRPM8 expression in autism patients and normal human. In the control group, which is the normal human's nervous system, there are 732 TRPM8-expressed cells were found. In the nervous system of people with ASD, 608 TRPM8 proteins were found, which is only 83.06% of the normal amount. However, ASD patients has more cells expressing TRPM8 2.19-5.02 (the highest expression amount). Therefore, in most ASD patients' cells, TRPM8 expression is diminished or disappeared. The downregulation of TRPM8 in most ASD patients' cells is likely to lead to unsatisfied cell demand for calcium ions.
Figure 5. Differential expression of TRPM8 in different cell types of human autistic-spectrum disorder brain tissue. L2/3, L4 represents layer2/3, layer4, and served as excitatory neurons, and many cells express TRPM8 here, showing its function in basal ganglion root. L5/6-CC are deep layer cortico-cortical excitatory projection neurons. Neu-NRGN-II and Neu-NRGN-I represent Neurogranin expressing neurons. OPC represents Oligodendrocytes precursor cells and has cells showing high TRPM8 expression. Neu-mat is immature cells that do not contain many cells expressing TRPM8, showing TRPM8 does not appear significantly while the early stages. IN-SST is somatostatin interneurons. IN-PV represents Parvalbumin interneurons that have many cells expressing TRPM8. IN-SV2C and IN-VIP are interneurons. AST-FB and AST-PP are astrocytes
The data of GEO also showed that compared schizophrenia was generally down-regulated, leading with the control group, the expression of TRPM8 in to the decreased expression of TRPM8 in neurons.
Figure 6. GEO data shows TRPM8 expression in neuron. The red lines represent the relative expression level of TRPM8, and blue dots show TRPM8's rank among all genes in the version. Compared to the control group, TRPM8 expression in neurons with schizophrenia is attenuated [20]
Discussion
Based on the result, we try to find out the relation between TRPM8 downregulation expression in neurons and psychiatric disorders. Since TRPM8 served as cation channels, the reduced TRPM8 expression in neurons can lead to less calcium ions influx into neurons. Existing study showed that although psychiatric disorders' relating channel protein-coding genes (TRPM8 in this research) may show relative low expression, these genes and relating calcium pathways can still play important though transient role in the developing process [21].
We also try to explain how rs11562199 and rs17869077's nucleotide change can lead to changes at the cell level. By using data from genome UCSC, we find out that rs11563199 can transform from cy-tosine to thymine. Since DNA methylation is common on cytosine, changing from cytosine to thymine may lead to the lost of DNA methylation. The possibility of switching from cytosine to thymine is 14.584%. The same change occur on rs17869077. The nucleotide of rs17869077 changes from C to T, which may lead to not only DNA methylation lost
on cytosine but also histone modification. Existing researches also show that DNA methylation is necessary to mediate memory consolidation in hippocampus [22]. And connections between memory and psychiatric disorders have also been investigated. Among many stress-associated diseases, including major depressive disorder, memory capacity seemed to be disrupted [23].
Since patients with major depression disorder may have trouble in recalling, and DNA methylation plays important role in long-term memory solidification, the lost of DNA methylation, which occurs when rs11563199 or rs17869077 change their nucleotides from cytosine to thymine, shows strong link with long-term memory loss, even to depression. We try to explain the relations between TRPM8 two intronic SNPs, rs11563199 and rs17869077, from two angles: the reduction of voltage-controlled calcium channel and the lost of DNA methylation.
Conclusion
Through the above studies, we summarize 986 TRPM8 exonic, not synonymous SNPs, accounting for 79% among all TRPM8 exonic SNPs. To be
broader, exon 12 and exon 20 become exons that have the most exonic, nonsynonymous SNPs, and TRPM8's sixth helix become the helix that have the most exonic SNPs affecting amino acids, having 11 changeable amino acids. By examining TRPM8 SNPs' relation with certain diseases, a total of 43 SNPs are found to be associated with asthma, astigmatism, diabetes, psychiatric disorders, coronary artery disease, epilepsy, migraine, monoclonal gam-mopathy, and rheumatoid arthritis. Specifically, the change of rs11563199 and rs17869077 nucleotide from cytosine to thymine may lead to changes in TRPM8 expression, which may affect the absorption
of calcium ions or influence on long-term memory solidification, and be associated with schizophrenia, depression, or other types of psychiatric disorders.
For the next step, we will: 1) identify one amino acids SNPs from the dangerous SNPs and use experiments to testify how the SNPs change can affect TRPM8's function, making it a confirmed pathogenic site, 2) create the wild-type and mutant of TRPM8 to verify whether the changes of rs11563199 and rs17869077 nucleotide will lead to the upregulation or downregulation of TRPM8 expression, thereby reducing/ increasing the inflow of calcium ions into the cell, and become pathogenic sites.
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