Detection and analysis of disease-associated single nucleotide polymorphism influencing post-translational modification
© Kim et al.; licensee BioMed Central Ltd. 2015
Published: 29 May 2015
Post-translational modification (PTM) plays a crucial role in biological functions and corresponding disease developments. Discovering disease-associated non-synonymous SNPs (nsSNPs) altering PTM sites can help to estimate the various PTM candidates involved in diseases, therefore, an integrated analysis between SNPs, PTMs and diseases is necessary. However, only a few types of PTMs affected by nsSNPs have been studied without considering disease-association until now. In this study, we developed a new database called PTM-SNP which contains a comprehensive collection of human nsSNPs that affect PTM sites, together with disease information. Total 179,325 PTM-SNPs were collected by aligning missense SNPs and stop-gain SNPs on PTM sites (position 0) or their flanking region (position -7 to 7). Disease-associated SNPs from GWAS catalogs were also matched with detected PTM-SNP to find disease associated PTM-SNPs. Our result shows PTM-SNPs are highly associated with diseases, compared with other nsSNP sites and functional classes including near gene, intron and so on. PTM-SNP can provide an insight about discovering important PTMs involved in the diseases easily through the web site. PTM-SNP is freely available at http://gcode.kaist.ac.kr/ptmsnp.
Since the advance of next-generation sequencing (NGS) technologies, the number of single nucleotide polymorphisms (SNPs) keeps increasing precipitously and it becomes crucial issue to discovering functional implication of SNPs on the biological process and complex diseases. Especially, non-synonymous SNPs (nsSNPs), including missense SNPs that provoke amino acid mutation and stop-gain SNPs that terminate the peptide synthesis by generating stop codon, are thought to play key roles in causing diseases by changing the protein structure and functions. It is estimated that nsSNPs cover more than half of the disease-causing mutation in the Human Gene Mutation Database .
In this regard, several efforts have been made to elucidate the effect of nsSNPs on diseases recently. For example, CanPredict  and Diseasome  tried to predict the effect of nsSNPs on disease using SIFT  or PolyPhen . However, although previous studies predicted disease-associated nsSNPs, related biological process have not been provided and hence have limits to understand detailed disease mechanisms. Therefore, an additional in-depth study about correlation between the location of nsSNPs and functional site of proteins is necessary. David et al. reported that disease-associated nsSNPs in the protein interaction interface affect the protein stability and prohibit protein interactions . Alternatively, post-translational modification (PTMs) sites are another important functional parts of the protein and nsSNP influencing PTM sites should be considered.
Post-translational modification plays essential roles in the most biological pathways and disruption of PTM sites is known as the major cause of diseases . Radivojac, et al. predicted phosphorylation-related variants from disease-associated variants and found that phosphorylation binding site disrupting variants are correlated with somatic cancer mutations . Recently, several previous studies [9–11] detected PTM-related nsSNPs or protein sequence variations. Yang et al.  collected 15,738 experimental phosphorylation binding sites and found 1,515 coding-region SNPs in the flanking phosphorylation binding sites with position -7 to +7. Ryu et al.  collected 33,651 protein-sequence variations from the SwissVariant database and predicted the effects of variants on phosphorylation binding sites using a phosphorylation-related variant prediction tool. Ren et al.  collected 91,797 nsSNPs from dbSNP Build 130 and mapped nsSNPs onto mRNA/Protein sequences from RefSeq Build 31 . They collected 64,035 phosphorylation-related nsSNPs in 5 categories using GPS 2.0 , a phosphorylation binding site prediction tool. However, previous studies are tend to focus on phosphorylation even there exists other kinds of PTMs like ubiquitination that is related in protein degradation . They also didn't consider the statistical significance with disease-association of PTM-SNPs with genome-wide association studies.
Here, we built a comprehensive PTM-SNP database containing more than 50 kinds of experimentally validated PTMs and predicted PTMs. While previous studies considered phosphorylation, PTM-SNP can examine multiple PTMs simultaneously. We also matched PTM-SNPs with various disease-associated SNPs based on GWAS to find disease-associated PTM-SNPs. The statistical significance values of the disease-association from GWAS are provided for accurate consideration of disease-association. We found that PTM-SNPs are highly correlated with diseases, compared with other SNPs in functional units. In addition, the result of the case study about PTM-SNP on type 2 diabetes shows our PTM-SNP provides an insight into unveiling important PTMs involved in causing diseases and finding novel disease markers. The web interface of PTM-SNP make easier to access our data and be conjugated as useful evidences in biomedical studies like drug development or predicting diagnosis and drug response.
Materials and methods
Collection of nsSNP data and PTM site data
We collected total 517,466 human nsSNPs from the NCBI dbSNP database Build 135 . From 517,466 nsSNPs, 509,183 SNPs were missense SNPs and 14,878 SNPs were stop-gain (nonsense) SNPs. We linked the location of nsSNPs on protein sequences from Ref-Seq Build 37.3. The flanking peptide of nsSNP sites from -7 to +7 retrieved by using the SNP-MapLinkProtein table in dbSNP and defined as the SNP sequence.
Human PTM site data were retrieved from dbPTM3 , which includes 194,886 experimentally verified PTMs from MS/MS analysis based research articles and 10 external PTM-related resources. The dbPTM also provides 404,501 computationally predicted PTMs by the KinasePhos-like method based on hidden Markov models (HMMs). It includes 20 types of PTM sites predicted with the threshold of specificity 100% for minimizing false positive. Peptides of flanking PTM sites from -7 to +7, called the PTM site sequence, were retrieved by matching the PTM site with the protein sequence from UniProt .
Detection of PTM-SNP
We also extracted and integrated PTM-SNPs from the public resource, PhosSNP 1.0. PhosSNP includes phosphorylation-associated nsSNP from computationally translating mRNA sequences. However, about 18% of SNPs in PhosSNP have not yet been defined as nsSNP by dbSNP Build 135, therefor, were removed in the integrating process.
From GWASs using SNP arrays and NGS techniques, huge numbers of statistically significant disease-associated SNPs have been identified recently. National Human Genome Research Institute (NHGRI) GWAS catalog have been provided 8,771 trait-associated SNPs with p-values < 1.0 × 10-5 (Apr 02, 2013) . The genetic association database (GAD)  contains 29,578 disease-associated SNPs and 23,671 disease-associated SNPs are also available from Genotypes and Phenotypes (dbGaP) . To find a correlation between PTM-SNPs and diseases, we collected total 52,731 distinct disease-associated SNPs from NHGRI GWAS catalog, GAD and dbGaP after then matched these SNPs with our PTM-SNPs.
Furthermore, we analyzed type 2 diabetes (T2D) associated SNPs that were identified in our previous study  by using Wellcome Trust Case Control Consortium (WTCCC) datasets . T2D-associated SNPs with p-values < 1.0 × 10-5 were identified from quality controlled (QC) 409,656 SNPs, based on Cochran-Armitage trend test statistics using PLINK 1.07 . QC is applied as a sample missing genotype rate of > 3%, a SNP missing genotype rate of > 1%, Hardy-Weinberg Equilibrium (HWE) p-value ≤ 10-4, and minor allele frequency (MAF) < 1%. Linkage disequilibrium pruning was leaved out to preserve the PTM-SNP candidates. T2D PTM-SNPs were detected by mapping T2D-associated SNPs and PTM-SNPs. Significantly associated T2D PTM-SNPs were further studied to interpret disease mechanisms with various biological information resources. Known disease genes are mainly collected from OMIM , GAD, and KEGG . Drug targets are collected from DrugBank , PharmGKB Drug , and KEGG Drug.
Result and disscussion
PTM-SNP statitsics based on SNP locations
On the PTM site
On the flank of the PTM site
Stop-gain affected PTM site
Statistics of PTM Site based on PTM types with the Top 10 ratio
Number of PTM sites
Number of PTM Sites of PTM-SNPs
Correlation between PTM-SNP and Diseases
Statistics of disease-associated SNPs based on Functional Categories
Number of SNPs
Number of Disease-associated SNPs
Missense & Stop-gain (PTM-SNP)
Missense & Stop-gain (Non PTM-SNP)
Not only missense and stop-gain SNPs, we also compared different functional SNPs in dbSNP. Functional classes that contain more than 100 disease-associated SNPs were considered in comparison. In addition, 414,510 SNPs on miRNA-mRNA binding site were collected from MirSNP . The result shows that SNPs near the gene, such as NearGene-3, MirSNP, NearGene-5 and UTR-3 were higher disease-associated proportion than SNPs in intron because they might affect protein functions by indirect way like regulating protein expressions. On the other hand SNPs around PTM site can give a potent influence on the protein and, as a result, the ratio of disease-associated PTM-SNP was superior to other functional classes. Therefore, we can conclude that PTM-SNPs are highly associated with diseases compared with both other nsSNPs and other functional sites.
Statistics of disease-associated SNPs based on PTMs
Number of Disease Associated PTM-SNP
Number pf Total PTM-SNP
Statistics of trait or disease-associated PTM-SNPs from GWAS catalogs
Number of SNP
Number of PTM-SNP
Coronary heart disease
Diabetes Mellitus, Type 1
Lupus Erythematosus, Systemic
Diabetes Mellitus, Type 2
Coronary Artery Disease
Case Study for Type 2 Diabetes associated PTM-SNP
Type 2 diabetes-associated PTM-SNPs
Known T2D gene
One of T2D-associated PTM-SNPs, rs1801282, is located on PPARG which is a popular T2D gene and a T2D drug target for a thiazolidinedione and changed the Pro12 to the alanine. The SNP is reported T2D from previous study , however, the detailed correlation between the SNP and T2D have not been clearly known yet. According to PTM-SNP, rs1801282 may enhance the phosphorylation on Ser8 by CSNK2A1, prohibit the phosphorylation on Ser14 by MTOR or MAPK10, and also may affect the phosphorylation on Ser16. Not only PPARG but CSNK2A1, MAPK10, and MTOR are known as T2D genes and participate on parts of T2D associated pathways such as type II diabetes mellitus, insulin signaling pathway, and Wnt signaling pathway in KEGG.
The associations with T2D, body mass index (BMI), and weight of a Val66Met PTM-SNP (rs6265) located on BDNF are discovered from NHGRI GWAS catalogs and WTCCC T2D dataset analysis. The association between obesity and diabetes is discovered with rs6265 due to GWASs on BMI, weight, and T2D identified G allele as a risk allele and A allele as a non-risk allele. Adjacent site Thr62 of BDNF is predicted as a phosphorylation site and kinases for Thr62 are predicted as ARAF, RAF1, MAP3K2, and MAP3K4 by GPS2.0 and filtrated by STRING. RAF1 is known as a T2D gene and a member of insulin signaling pathway.
PTMs play crucial roles in biological mechanisms, and PTM-SNPs may influence PTM network regulation and have effects on associated diseases. From disease-associated PTM-SNPs, we can find disease-associated PTM networks regulated by PTM-SNPs that may provide insight into the mechanisms of disease. Our case studies show the possibility of using PTM-SNPs, combined with various types of biological information including known disease genes, drug targets, protein-protein interaction networks, and pathway networks, to identify mechanisms underlying disease. Novel disease-candidate genes can be found from disease-associated PTM networks assembled from the relations of PTM enzymes and PTM substrate proteins.
In this study, we performed the investigation to associate PTMs, SNPs and disease using PTM-SNPs and disease-associated GWAS studies. PTM-SNP is a database based on dbSNP build 135, dbPTM3, and public GWAS databases of disease-associated SNPs that influence PTMs. In total, more than 50 kinds of PTMs were considered. PTM sites involved in signalling pathways such as phosphorylation, ubiquitination, and acetylation tended to have a greater proportion of PTM-SNPs than other PTM sites. Genes that contain disease-associated PTM-SNPs have been collected and matched against disease genes and drug targets to elucidate the effects of the SNPs and out analysis showed that PTM-SNP has higher disease-associations as compared with non-PTM-SNPs. We were also able to identify some of the mechanisms of disease-associated SNPs using PTM-SNP data. Researchers can match their own GWAS datasets with the PTM-SNP database to find PTM-related effects of the disease-associated SNPs easily by using the PTM-SNP website. We expect that provides an insight into unveiling important PTMs involved in causing diseases and finding novel disease markers.
Publication of this article has been funded by the Bio-Synergy Research Project (2012M3A9C4048759) of the Ministry of Science, ICT and Future Planning through the National Research Foundation (NRF), and by the KAIST Future Systems Healthcare Project from the Ministry of Science, ICT and Future Planning, and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2010-0028631).
This article has been published as part of BMC Medical Genomics Volume 8 Supplement 2, 2015: Selected articles from the 4th Translational Bioinformatics Conference and the 8th International Conference on Systems Biology (TBC/ISB 2014). The full contents of the supplement are available online at http://www.biomedcentral.com/bmcmedgenomics/supplements/8/S2.
- Stenson PD, Mort M, Ball EV, Howells K, Phillips AD, Thomas NS, Cooper DN: The Human Gene Mutation Database: 2008 update. Genome medicine. 2009, 1 (1): 13-10.1186/gm13.View ArticlePubMedPubMed CentralGoogle Scholar
- Kaminker JS, Zhang Y, Watanabe C, Zhang Z: CanPredict: a computational tool for predicting cancer-associated missense mutations. Nucleic acids research. 2007, 35 (Web Server): W595-598. 10.1093/nar/gkm405.View ArticlePubMedPubMed CentralGoogle Scholar
- J O, and Sohn, T K: An integrated database-pipeline system for studying single nucleotide polymorphisms and diseases. BMC bioinformatics. 2008, 9 (Suppl 12): S19-10.1186/1471-2105-9-S12-S19.View ArticleGoogle Scholar
- and Ng, P C: Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nature protocols. 2009, 4: 1073-1081. 10.1038/nprot.2009.86.View ArticleGoogle Scholar
- I A, V E, A S, Sunyaev SR: A method and server for predicting damaging missense mutations. Nature methods. 2010, 7: 248-249. 10.1038/nmeth0410-248.View ArticleGoogle Scholar
- David A, Razali R, Wass MN, Sternberg MJ: Protein-protein interaction sites are hot spots for disease-associated nonsynonymous SNPs. Hum Mutat. 2012, 33 (2): 359-63. 10.1002/humu.21656.View ArticlePubMedGoogle Scholar
- Li S, Iakoucheva LM, Mooney SD, Radivojac P: Loss of post-translational modification sites in disease. Pacific Symposium on Biocomputing. 2010, 337-347.Google Scholar
- Radivojac P, Baenziger PH, Kann MG, Mort ME, Hahn MW, Mooney SD: Gain and loss of phosphorylation sites in human cancer. Bioinformatics,. 2008, 24 (16): i241-247. 10.1093/bioinformatics/btn267.View ArticleGoogle Scholar
- Yang CY, Chang CH, Yu YL, Lin TC, Lee SA, Yen CC, Yang JM, Lai JM, Hong YR, Tseng TL, et al: PhosphoPOINT: a comprehensive human kinase interactome and phospho-protein database. Bioinformatics. 2008, 24 (16): i14-20. 10.1093/bioinformatics/btn297.View ArticlePubMedGoogle Scholar
- Ryu GM, Song P, Kim KW, Oh KS, Park KJ, Kim JH: Genome-wide analysis to predict protein sequence variations that change phosphorylation sites or their corresponding kinases. Nucleic acids research. 2009, 37 (4): 1297-1307.View ArticlePubMedPubMed CentralGoogle Scholar
- Ren J, Jiang C, Gao X, Liu Z, Yuan Z, Jin C, Wen L, Zhang Z, Xue Y, Yao X: PhosSNP for systematic analysis of genetic polymorphisms that influence protein phosphorylation. Molecular & cellular proteomics. 2010, 9 (4): 623-634. 10.1074/mcp.M900273-MCP200.View ArticleGoogle Scholar
- Pruitt KD, Tatusova T, Maglott DR: NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic acids research. 2007, 35 (Database): D61-65. 10.1093/nar/gkl842.View ArticlePubMedGoogle Scholar
- Xue Y, Ren J, Gao X, Jin C, Wen L, Yao X: GPS 2.0, a tool to predict kinase-specific phosphorylation sites in hierarchy. Molecular & cellular proteomics. 2008, 7 (9): 1598-1608. 10.1074/mcp.M700574-MCP200.View ArticleGoogle Scholar
- Han Y, Lee H, Park JC, Yi GS: E3Net: a system for exploring E3-mediated regulatory networks of cellular functions. Molecular & cellular proteomics. 2012, 11 (4): O111 014076-10.1074/mcp.O111.014076.View ArticleGoogle Scholar
- Sherry ST, Ward MH, Kholodov M, Baker J, Phan L, Smigielski EM, Sirotkin K: dbSNP: the NCBI database of genetic variation. Nucleic acids research. 2001, 29 (1): 308-311. 10.1093/nar/29.1.308.View ArticlePubMedPubMed CentralGoogle Scholar
- Lu CT, Huang KY, Su MG, Lee TY, Bretana NA, Chang WC, Chen YJ, Huang HD: dbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications. Nucleic acids research. 2013, 41 (D1): D295-305. 10.1093/nar/gks1229.View ArticlePubMedGoogle Scholar
- Apweiler R, Bairoch A, Wu CH, Barker WC, Boeckmann B, Ferro S, Gasteiger E, Huang H, Lopez R, Magrane M, et al: UniProt: the Universal Protein knowledgebase. Nucleic acids research. 2004, 32 (Database): D115-119.View ArticlePubMedPubMed CentralGoogle Scholar
- Rice P, Longden I, Bleasby A: EMBOSS: the European Molecular Biology Open Software Suite. Trends in genetics. 2000, 16 (6): 276-277. 10.1016/S0168-9525(00)02024-2.View ArticlePubMedGoogle Scholar
- Hindorff LA, Sethupathy P, Junkins HA, Ramos EM, Mehta JP, Collins FS, Manolio TA: Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proceedings of the National Academy of Sciences of the United States of America. 2009, 106 (23): 9362-9367. 10.1073/pnas.0903103106.View ArticlePubMedPubMed CentralGoogle Scholar
- Becker KG, Barnes KC, Bright TJ, Wang SA: The genetic association database. Nature genetics. 2004, 36 (5): 431-432. 10.1038/ng0504-431.View ArticlePubMedGoogle Scholar
- Mailman MD, Feolo M, Jin Y, Kimura M, Tryka K, Bagoutdinov R, Hao L, Kiang A, Paschall J, Phan L, et al: The NCBI dbGaP database of genotypes and phenotypes. Nature genetics. 2007, 39 (10): 1181-1186.View ArticlePubMedPubMed CentralGoogle Scholar
- Kang C, Yu H, Yi GS: Finding type 2 diabetes causal single nucleotide polymorphism combinations and functional modules from genome-wide association data. BMC medical informatics and decision making. 2013, 13 (Suppl 1): S3-10.1186/1472-6947-13-S1-S3.View ArticlePubMedPubMed CentralGoogle Scholar
- WTCCC: Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature. 2007, 447 (7145): 661-678. 10.1038/nature05911.View ArticleGoogle Scholar
- Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, et al: PLINK: a tool set for whole-genome association and population-based linkage analyses. American journal of human genetics. 2007, 81 (3): 559-575. 10.1086/519795.View ArticlePubMedPubMed CentralGoogle Scholar
- Oyston J: Online Mendelian Inheritance in Man. Anesthesiology. 1998, 89 (3): 811-812. 10.1097/00000542-199809000-00060.View ArticlePubMedGoogle Scholar
- Kanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic acids research. 2000, 28 (1): 27-30. 10.1093/nar/28.1.27.View ArticlePubMedPubMed CentralGoogle Scholar
- Knox C, Law V, Jewison T, Liu P, Ly S, Frolkis A, Pon A, Banco K, Mak C, Neveu V, et al: DrugBank 3.0: a comprehensive resource for 'omics' research on drugs. Nucleic acids research. 2011, 39 (Database): D1035-1041. 10.1093/nar/gkq1126.View ArticlePubMedGoogle Scholar
- Hewett M, Oliver DE, Rubin DL, Easton KL, Stuart JM, Altman RB, Klein TE: PharmGKB: the Pharmacogenetics Knowledge Base. Nucleic acids research. 2002, 30 (1): 163-165. 10.1093/nar/30.1.163.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu C, Zhang F, Li T, Lu M, Wang L, Yue W, Zhang D: MirSNP, a database of polymorphisms altering miRNA target sites, identifies miRNA-related SNPs in GWAS SNPs and eQTLs. BMC Genomics. 2012, 13: 661-10.1186/1471-2164-13-661.View ArticlePubMedPubMed CentralGoogle Scholar
- Saxena R, Voight BF, Lyssenko V, Burtt NP, de Bakker PI, Chen H, Roix JJ, Kathiresan S, Hirschhorn JN, Daly MJ, et al: Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science. 2007, 316 (5829): 1331-1336.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.