Abstract
In the present study we have analyzed the role of SMAD family member 1 protein (SMAD1) gene products in relation to bone morphogenesis and osteoporosis. Out of 1045 single nucleotide polymorphisms (SNP) investigated, we find that one nonsynonymous SNP (nsSNP), rs1804647, to have significant damaging effects as predicted by all the tools used in the analysis. This nsSNP resulted in a change of amino acid from a positive charged residue, Lysine, to a strong negatively charged residue, Glutamate, and hence the change of residue with opposite charges might lead to structural defects and result in altered function. The results presented in this report will be a good starting point for genetic analysis of SMAD1 genes in patients with osteoporosis which might lead to more conclusive evidence of the association of this gene with osteoporosis.
Introduction
Osteoporosis is a disease associated with progressive deterioration of bone which results in fragility fractures that occur with very little trauma. 1, 2 It is a common disease that affects millions of people worldwide. It is estimated that over 200 million people have osteoporosis. 3 Genetic factors play a major role in the determination of bone mineral density (BMD) and osteoporosis risk. 4 Multiple chromosomal loci have been mapped by linkage approaches which potentially carry hundreds of genes involved in the determination of bone mass and quality. Association studies based on candidate gene polymorphisms and subsequent meta-analyses, and the more recent genome-wide association studies, have clearly identified a handful of genes associated with BMD and/or fragility fractures. 4 Molecular genetic studies have implicated the association of single nucleotide polymorphisms (SNP) with osteoporosis. 5 It is anticipated that both the diagnosis and the treatment of this disease will be revolutionized by the integration of genomics and informatics. 2 Also, it is predicted that a genetic algorithm will be developed to identify at-risk patients before they develop osteoporosis, so that preventive measures can be instituted.
The role of SMA-and MAD-related (SMAD) protein in bone morphogenesis is documented by researchers. 7, 9 Even though there is a lot of information on the clinical impact, diagnosis and treatment of osteoporosis, there are not many reports on the systematic analysis of SMAD1 genes in relation to osteoporosis. This report systematically analyzes the SMAD1 gene and its possible implications on osteoporosis using bioinformatics tools. Our results from the present study might be useful for further investigations on the human genetics of osteoporosis.
Materials and methods

Data set
The SNPs associated with SMAD1 gene were obtained from the single nucleotide polymorphism database (dbSNP) 10 which is a public-domain archive established by NCBI, having broad collection of SNPs of any gene of an organism serving as a public repository for genetic variation.
There are 1045 SNPs associated with SMAD1, these are commonly referred to by their reference sequence IDs (rsID), and they are listed in Table 1 .
Predicting damaging nssnPs using siFT
We used the program Sorting Intolerant from Tolerant (SIFT) designed by Ng and Henikoff to identify the damaging non-synonymous SNP's (nsSNP) in the data set. 11, 12 SIFT predicts whether an amino acid substitution affects protein function and it distinguishes between functionally neutral and deleterious amino acid changes in mutagenesis studies and on human polymorphisms. SIFT also searches for similar protein sequences from different species in the database, obtains the multiple alignments of these sequences, and then calculates from the alignment the tolerance index (from 0 to 1) for all possible substitutions at each position. The higher a tolerance index, the less functional impact a particular amino acid substitution is likely to have. [11] [12] [13] structural alteration by nssnPs using PolyPhen
The structural alteration as a result of nsSNP was determined using the program Polymorphism Phenotyping (PolyPhen). [14] [15] [16] PolyPhen assesses the possible damaging effect of amino acid substitution, based on whether the substitution was (i) in an active or binding site; (ii) affecting interactions with ligands present in the experimentally resolved structure;
(iii) leading to hydrophobicity or electrostatic charge change in a buried site; (iv) destroying a S-S bond; (v) affecting protein's solubility; (vi) inserting proline (P) in an α-helix or (vii) incompatible with the profile of amino acid substitutions observed at that site in the set of homologous proteins.
15
Functional analysis and selection for nssnPs using FAsTsnP
We used the functional analysis and selection tool for single nucleotide polymorphisms (FASTSNP) web server that allows users to efficiently identify the SNPs that will most likely have functional effects. It prioritizes SNPs according to 13 phenotypic risks and putative functional effects, such as changes to the transcriptional level, pre-mRNA splicing, and protein structure.
17
The functional report on a SNP contains seven sections on the SNP's functional effects, namely:
(i) genomic information, presents the nearby sequence, the alleles and the allele frequency among different ethnic groups; (ii) functional effects summary, presents the risk assessment; (iii) transcription regulatory, shows the predicted transcription factor binding sites generated or disrupted by the different SNP alleles; (iv) alternative splicing regulatory, reports exonic splicing enhancer/silencer motifs changed by the SNP alleles leading to exon skipping or inclusion; (v) mRNA/protein domain effects, presents all spliced forms of mRNAs and protein variants extracted from GenBank. 18 The protein domains that the SNP locates in are highlighted; (vi) protein structure effects, reports whether the SNP may cause a significant structural change in a protein; and (vii) SwissProt 19 feature table, provides information regarding other known mutations or variations of the translated protein of mRNAs related to the SNP. 17 Some of these sections were specific to coding or noncoding SNPs and they will appear or not appear in the functional report accordingly. 17 gene information for nssnPs from ensembl
We found the gene related information for the nsSNPs from the Ensembl database. [20] [21] [22] The Ensembl database consists of comprehensive genome information system that features 
81
Influence of SMAD1 gene in osteoporosis rs43206128  rs41837184  rs41837183  rs41837182  rs41837181  rs41837180  rs41837179   rs41837178  rs41837177  rs41837176  rs41837175  rs41837174  rs41837173  rs41837172  rs41837171  rs41837170  rs41837169  rs41837168  rs41837167  rs41837166  rs41837165  rs41837164  rs41837163  rs41837162  rs41837161  rs41837160  rs41837159  rs41837158  rs41837157  rs41837156  rs41837155  rs41837154  rs41837153  rs41837152  rs41837151  rs41837150  rs41837149  rs41837148  rs41837147  rs41837146  rs41837145  rs41837144  rs41837143  rs41837142  rs41837141  rs41837140  rs41837139  rs41837138  rs41837137  rs16383403  rs16383402  rs16383401  rs16383400  rs16383399  rs16383398  rs16383397  rs15529039  rs15529035  rs15529034  rs15529032  rs15529030  rs15529027  rs15529025  rs15529023  rs15529021  rs15529018  rs15529016  rs15529014  rs15529011  rs15529009  rs15529006  rs15529004  rs15529002  rs15528997  rs15528993  rs15528990  rs15528988  rs15528985  rs15528983  rs15528980  rs15528978  rs15528976  rs15528973 rs38594114  rs38586608  rs38571957  rs38478504  rs38408710  rs38407259  rs38381145  rs38379589  rs38379316  rs38357614  rs38343211  rs38319122  rs38304454  rs38286771  rs38216312  rs38198301  rs38151706  rs38122743  rs38104923  rs38084479  rs38069777  rs38046037  rs38028566  rs38028025  rs38009371  rs37999190  rs37996620  rs37970866  rs37890809  rs37857319  rs37844585  rs37829955  rs37829805  rs37820241  rs37798728  rs37776122  rs37769262  rs37751971  rs37723161  rs37678138  rs37670970  rs37662143  rs37622281  rs37606105  rs37601352  rs37595569  rs37592912  rs37570279  rs37565989  rs37557932  rs37547978  rs37529033  rs37512483  rs37502918  rs37462850  rs37462363  rs37447335  rs37424711  rs37395093  rs37380944  rs37372985  rs37358262  rs37346703  rs37344855  rs37328013  rs37316393  rs37311870  rs37278533  rs37227073  rs37197406  rs37185197  rs37183904  rs37176820  rs37173505  rs37141214  rs37138939  rs37133086  rs37095121  rs37078372  rs37072603  rs37055222  rs37049288  rs37041092  rs37036754  rs37035385  rs37026804  rs37021065  rs37014646  rs36984116  rs36981578  rs36974734  rs36961475  rs36935369  rs36934698  rs36926658  rs36915208  rs36913122  rs36912217  rs36910109  rs36904384  rs36854139  rs36836173  rs36826819  rs36807995  rs36785399  rs36781909  rs36781325  rs36780156  rs36773895  rs36770138  rs36763326  rs36756065  rs36734144  rs36720417  rs36693953  rs36682785  rs36678280  rs36676848  rs36652809  rs36628123  rs36623863  rs36620169  rs36616718  rs36604460  rs36604173  rs36598919  rs36592125  rs36590740  rs36589398  rs36589170  rs36587668  rs36577742  rs36574562  rs36574154  rs36574562  rs36574154  rs36550928  rs36548387  rs36543793  rs36550928  rs36548387  rs36543793  rs36518106  rs36516399  rs36513012  rs36500074  rs36499024  rs36485545  rs36471433  rs36463229  rs36433155  rs36417697  rs36409573  rs36392860  rs36385065  rs36380669  rs36378657  rs36374178  rs36372803  rs36355585  rs36355433  rs36355396  rs36343803  rs36336450  rs36334006  rs36308116  rs36307375  rs36304409  rs36273831  rs36262476  rs36258235  rs13471524  rs6320822  rs6320755  rs6320308  rs6320203  rs6157027  rs41182187  rs41162462  rs41127100  rs41097188  rs41052054  rs41042179  rs41001288  rs40914529  rs40829302  rs40801804  rs40785650  rs40718880  rs40714934  rs40707256  rs40699558  rs40696624  rs40624834  rs40600779  rs22438341  rs22438340  rs22438338  rs22435243  rs22435242  rs22430308  rs22429717  rs22429143  rs22423498  rs22423496  rs22422179  rs22422178  rs22420143  rs22416419  rs22413764  rs22410926  rs22408159  rs22407129  rs22407128  rs22407127  rs22407126  rs22407125  rs22407124  rs22407110  rs22407109  rs22407108  rs22407107  rs22407106  rs22407105  rs22407104  rs22407103  rs22407101  rs22407094  rs22407093  rs22407092  rs22407091  rs22407090  rs22407089  rs22407088  rs22407087  rs22407086  rs22407079  rs22407078  rs22407077 (Continued ) )   rs22407076  rs22407075  rs22407074  rs22407073  rs22407072  rs22407071  rs22407070  rs22407064  rs22407062  rs22407060  rs22407059  rs22407057  rs22407056  rs22407055  rs22407054  rs22407052  rs22407049  rs22407047  rs22407045  rs22407044  rs22407043  rs22407042  rs22407041  rs22405061  rs22403115  rs22401316  rs22399442  rs22398238  rs22398211  rs22398210  rs22379962  rs22379948  rs22379160  rs22364266  rs22364212  rs22363681  rs22353531  rs22353529  rs22349453  rs22349452  rs22349451  rs22348997  rs22348996  rs9197660  rs9197659  rs9190264  rs9190263  rs9159692  rs9159691  rs9159690  rs9159689  rs9159688  rs9127901  rs9127900  rs9113580  rs9082059  rs9082058  rs9015277  rs9015276  rs9015275  rs8967641  rs8967640  rs8958253  rs8958252  rs8948231  rs8948230  rs8948229  rs8948228  rs8916864  rs8904715  rs8878619  rs8878618  rs8878617  rs8878616  rs8878615  rs8871149  rs8871148  rs8871147  rs8821428  rs8616390  rs8616389  rs8548136  rs8548135  rs8548134  rs8548133  rs8434954  rs8434953  rs8434952  rs8434951  rs8412032  rs26109380  rs25383970  rs25350003  rs25327873  rs25326101  rs25321520  rs25306350  rs25297286  rs25294121  rs25292078  rs25285454  rs25281219  rs25279061  rs25263171  rs25241614  rs25239333  rs25238019  rs25231997  rs24891022  rs62446864  rs62446863  rs62446862  rs61613428  rs61128823  rs61098313  rs60956809  rs60584748  rs60532382  rs60323633  rs60103805  rs59784627  rs59525016  rs59034773  rs58891283  rs58572713  rs58441521  rs57305857  rs57232114  rs56022562  rs55811804  rs55731773  rs41275991  rs41275988  rs36125163  rs35821025  rs35711502  rs35482863  rs35425165  rs35302357  rs35241091  rs35151755  rs35045679  rs34957085  rs34885661  rs34725384  rs34668775  rs34495080  rs34457367  rs34230821  rs34149226  rs28937323  rs28937322  rs28936972  rs17159296  rs17159291  rs17159289  rs17159287  rs17159280  rs17159279  rs17159275  rs17159273  rs17159272  rs17159267  rs17159262  rs60939160  rs16875290  rs17159297  rs13228758  rs13228097  rs13228094  rs13228006  rs13226718  rs12532057  rs11982890  rs11981106  rs11980069  rs11979352  rs11976576  rs56650146  rs11761080  rs11553503  rs11553502  rs11553498  rs11553497  rs11465024  rs35809100  rs11454228  rs17778009  rs60345184  rs10951271  rs10487700  rs10487699  rs10280491  rs61212867  rs10265589  rs10264469  rs10249885  rs10239660  rs57200522  rs10239537  rs10233132  rs10229836  rs7808770  rs6970807  rs6950015  rs10347965  rs6947982  rs6945586  rs6945546  rs6945475  rs6945223  rs6945035  rs6462214  rs4000210  rs3886641  rs3840589  rs11403655  rs3840588  rs3807633  rs10386194  rs9792086  rs56638865  rs3807632  rs2970504  rs2952805  rs2952804  rs2952795  rs4461798  rs2893390  rs2893389  rs2893388  rs10378014  rs3735431  rs2709809  rs2709806  rs3807631  rs17700042  rs57637303  rs2709778  rs60761314  rs2709772  rs2709771  rs2529442  rs60428326  rs2529441  rs3779252  rs2529439  rs11553501  rs2529438  rs3764826  rs57476542  rs2527880  rs60677610  rs2527879  rs2527878  rs10378720  rs2270025  rs2240501  rs58255920  rs10378822  rs2240401  rs60101002  rs2240400  rs34886142  rs2230310  rs2214837  rs2190241  rs2074779  rs2072236  rs1986757  rs56754355  rs10338017  rs1986756  rs59471849  rs1558064  rs1468402  rs17413053  rs3189753  rs1130653  rs3189564  rs11553500  rs17856227  rs17856223  rs1049402  rs3189560  rs1049399  rs2709804  rs758000  rs61005429  rs728539  rs722423  rs17159306  rs1130593  rs3189633  rs2230311  rs2070205  rs1801345  rs1049584  rs3189770  rs4593  rs62343509  rs62343505  rs62343494  rs62343493  rs62343492  rs61303468  rs61102345  rs60788260  rs60537353  rs60369536  rs60139040  rs60073922  rs60072759  rs60014433  rs59984540  rs59971057  rs59933736  rs59909582  rs59846899  rs59268619  rs59193620  rs59063315  rs59057604  rs59054276  rs59029464  rs58968834  rs58913698  rs58688997  rs58688046  rs58468364  rs58339165  rs57963489  rs57797623  rs57612157  rs57581646  rs57545372  rs57257834  rs57044560  rs56986943  rs56985736  rs56837794  rs56756555  rs56695028  rs60478333  rs56250124  rs55948666  rs55835219  rs55700163  rs36007951  rs35999258  rs35986161  rs35956618  rs35923324  rs35882080  rs35842798  rs35813714  rs35812565  rs35774186  rs35756866  rs35747692  rs35710925  rs35668194  rs35612203  rs35584992  rs35528395  rs35483247  rs35476542  rs35386763  rs35281972  rs35241245  rs35184283  rs35058397  rs35046379  rs62343508  rs34978105  rs34968029  rs34959603  rs34880052  rs34832344  rs34791274  rs34791030  rs34629544  rs34610916  rs34504494  rs34443486  rs34405116  rs34373983  rs34333288  rs34318288  rs34303116  rs34169615  rs34142651  rs56664494  rs34107624  rs34082880  rs34079792  rs34077417  rs34013277  rs34011978 (Continued ) 
83
Influence of SMAD1 gene in osteoporosis Table 1 (Continued )   rs60015014  rs33928689  rs57951197  rs28607764  rs28579035  rs28541642  rs56683612  rs28534855  rs28522520  rs28495533  rs28464746  rs28408896  rs28397904  rs17797966  rs17797805  rs17741593  rs17426247  rs59063334  rs17020321  rs17020317  rs17020315  rs17020313  rs17020310  rs17020304  rs17020302  rs17020297  rs17020284  rs17020281  rs17020269  rs17020255  rs17020253  rs17020251  rs59444418  rs17020248  rs17020237  rs17020236  rs58221759  rs17020235  rs17020202  rs59633267  rs17020201  rs56640118  rs58653480  rs17020200  rs16998662  rs16998659  rs13435697  rs13149786  rs13149771  rs13149754  rs13145825  rs13144151  rs13138805  rs58040113  rs13137598  rs13120843  rs13118865  rs13113936  rs13110369  rs13104775  rs17426240  rs12646702  rs12504239  rs58910572  rs11946962  rs11946830  rs11945076  rs58166623  rs11944685  rs11944363  rs11939520  rs11939179  rs11938489  rs11937875  rs11937328  rs11936636  rs11935522  rs57735613  rs11935237  rs13129371  rs11934722  rs11931456  rs13107924  rs11930852  rs13104519  rs11930324  rs11736932  rs11725101  rs56452565  rs58073224  rs11724813  rs11547180  rs11547178  rs11537832  rs58395199  rs11392205  rs11355954  rs11302430  rs33926573  rs11293293  rs33995597  rs11285972  rs11100885  rs11100884  rs17798152  rs60525943  rs56482779  rs11100883  rs56920353  rs11100882  rs10715854  rs35752574  rs10713518  rs10694111  rs10677232  rs36210455  rs10599198  rs10557196  rs33996344  rs10548153  rs10519733  rs10033759  rs9992345  rs9968492  rs9685596  rs9683725  rs7699931  rs7699352  rs59616816  rs7698944  rs7695203  rs61314879  rs7694604  rs7694086  rs56553319  rs7689005  rs7686994  rs60923728  rs7685592  rs7685387  rs7684336  rs7683590  rs58089363  rs7682401  rs7681713  rs7677879  rs7677863  rs7677160  rs7675811  rs59990034  rs7673821  rs7672606  rs59927196  rs7672306  rs60192542  rs7670486  rs7670403  rs17798495  rs7662543  rs56661211  rs7662541  rs56554331  rs7661162  rs7658436  rs7658004  rs57170185  rs7655568  rs7435311  rs17435952  rs6854034  rs61107117  rs6852200  rs61409198  rs6848098  rs6845829  rs57611059  rs6833260  rs6829932  rs6829091  rs6823513  rs6822700  rs6816306  rs6813833  rs6537359  rs13121278  rs17797649  rs6537356  rs17426233  rs57727279  rs6537355  rs60611965  rs35037580  rs5862739  rs5862738  rs4395527  rs3948257  rs3943707  rs3816967  rs3775325  rs3775323  rs3775322  rs60112569  rs3756021  rs57309032  rs17741322  rs2289737  rs17227189  rs2118438  rs57644485  rs2068991  rs61103877  rs2043779  rs57261547  rs2013367  rs59728727  rs17741821  rs1992165  rs1804647  rs1497126  rs1437827  rs1437826  rs1370571  rs57628021  rs1055440  rs3816966  rs58152281  rs1016792  rs60988923  rs1016791  rs60581465  rs959641  rs3775324  rs920261  rs58547099  rs768139  rs763560  rs17798104  rs714195  rs15392 rs11416. rs14270 rs62343506
an integrated set of genome annotation, databases and other information for chordate and selected model organism and disease vector genomes. [20] [21] [22] The Ensembl genome browser provides visualization for genome annotations, alignments, variation and functional genomics data and supporting additional data integration.
Ensembl gene sets are created using an automated analysis pipeline that has been significantly optimized based on the completeness of the genome sequence.
20-22

Functional significance of nsSNPs by UTr scan
The untranslated regions (UTR) functional significance of each SNP was determined using UTResource, an internet based UTR analyzing tool, consisting of a resource of sequence analysis of 5' and 3' UTR of eukaryotic mRNAs (http://www.ba.itb.cnr.it/BIG/UTRScan). Briefly, two or three sequences of each UTR SNP which have different nucleotide at SNP position were analyzed by UTRscan which looks for UTR functional elements by searching through user submitted sequence data for the patterns defined in the UTRsite collection.
The collection of functional sequence patterns located in 5' or 3' UTR sequences were stored in UTRsite. If different sequences of each UTR SNP were found to have different functional pattern(s), that UTR SNP is predicted to have functional significance. 13 
Modeling wild and mutant protein structures
We utilized the SWISS-MODEL [23] [24] [25] workspace, a web-based integrated service dedicated to protein structure homology 
3-D viewing and rMsD calculations
The Swiss PDB viewer (Deep View) version 4.01 was used for viewing the modeled structures and for calculation of the root mean square deviation (RMSD) between the native and mutant structures. 26 DeepView is an application that provides a user friendly interface to analyze several proteins at the same time. The proteins can be superimposed in order to deduce structural alignments and compare their active sites or any other relevant parts. Amino acid mutations, hydrogen bonds, angles and distances between atoms can be obtained using Deep View.
26

Results and Discussion
We used the SIFT tool to predict whether an amino acid substitution at a particular position in a protein molecule will have a phenotypic effect. We find that of 1045 rs IDs associated with SMAD1 gene, only two, rs1804647 and rs17159287, are non-synonymous.
SIFT gives the results of these nsSNPs with their amino acid change and the tolerance index score which predicts the functional impact of the protein of these SNPs. The lesser the score, the more functional impact, ie, the corresponding SNP is predicted to be damaging. Of these two SNPs, rs1804647 is predicted to be damaging with a tolerance index score of 0.01. (Score of #0.05 is the cut-off to identify damaging SNPs) [11] [12] [13] as shown in Table 2 . The nucleotide change is in the 510 position where AAA is replaced with GAA ie, Adenine to Guanine resulting in the substitution of amino acid Lysine to Glutamate. (The base represented in bold caption is the nsSNP). We employed the PolyPhen based methodology [14] [15] [16] to compute the position-specific independent counts (PSIC) score difference of the two identified nsSNPs (rs1804647 and rs17159287) to assess the functional impact of the amino acid substitution predicted by SIFT. To evaluate the ability of the PolyPhen method to distinguish between damaging and neutral amino acid types; we applied it to the data on known deleterious mutations and to the data on species divergence. 15 The deleterious data set consists of natural replacements known to cause disease phenotypes, variants observed in individuals affected by genetic disorders and artificial replacements known to damage structure, function and the stability of a protein. 15 Score of $1.5 is the cut-off to identify damaging SNPs by Polyphen. SNP with PSIC score difference of higher value is predicted to be damaging. We found that of the two nsSNPs, rs1804647 is predicted to be damaging with a PSIC score difference of 1.852, as depicted in Table 3 .
FASTSNP was employed to efficiently identify and prioritize high risk nsSNPs according to their phenotypic risks and putative functional effects. 17 FASTSNP rates the phenotypic deleterious risks from 0 to 5 point scale; 0 representing no known effect and 5 indicating very high damaging effect, while a score of 3-4 indicates moderate to high damaging effect. 17 We found rs17159287 to have low to medium level of risk, whereas rs1804647 was identified to have significantly damaging effect with a score in the range of 3-4; as shown in Table 4 . This may be due to splicing regulation functional effect by possibly breaking the exonic splicing enhancer/silencer binding site in the coding sequence leading to abolished protein domain. 17 Gene related information the non-synonymous SNPs associated with SMAD1, with their corresponding change in alleles and amino acids were obtained from Ensembl database and we found three of the 1045 associated with SAMD1 listed as nsSNPs. These are: rs1804647, rs61748163; and rs62343507, depicted in Table 5 . 
85
Influence of SMAD1 gene in osteoporosis
There is a significant correlation between all the SNP tools with respect to rs1804647 and hence this SNP might be expected to have high deleterious impact on the function of the bone morphogenesis process. The other two SNPs (rs61748163 and rs62343507) did not show any result with other tools used and hence, we did not consider them for our study.
To determine the functional significance of the nsSNP rs1804647, we used UTRScan and found a change in a pattern in 3' UTR region. Polymorphism in the 3' UTR region may affect gene expression by affecting RNA half-life or influencing ribosomal translation of mRNA.
27
The functional element change related to this nsSNP was found to be 15-lipoxygenase differentiation control element (15-LOX-DICE). 15-LOX-DICE controls 15-LOX synthesis which catalyses the degradation of lipids and is an important factor responsible for the degradation of mitochondria during reticulocyte maturation. 13 The mutation Lysine→Glutamate is observed in the protein due to nucleotide change in the coding sequence of the SNP in 510 position where AAA is replaced with GAA. From this we infer that the nsSNP, rs1804647 is significantly damaging which might result in osteoporosis.
The nsSNP, rs17159287 has tolerance index score of 0.59 with SIFT, a PSIC score difference of 0.298 with PolyPhen, a relatively low risk score of 2-3 with FASTSNP and was not listed in Ensembl database. Hence we did not consider this nsSNP to be significantly damaging.
SNP rs61748163 has amino acid change from Serine to Asparagine, both are of same properties and showed no change in structure when native and mutant proteins were modeled. SNP, rs62343507 has amino acid change from Tryptophan to Arginine, also showed no change in structure when native and mutant proteins were modeled. Hence, we did not consider these nsSNPs (rs61748163 and rs62343507) listed in Ensembl to be significantly damaging and they did not show any results with other bioinformatics tools.
To assess the 3-D structural change brought about by the nsSNP rs1804647, we performed structural analysis comparing the native and mutant protein structures. The native structure for protein Q15797 and mutant structure for nsSNP rs1804647 were modeled using SWISS-MODEL. To analyze their structural differences, superimposition of both the structures was done using the SWISS PDB Viewer tool and we found an RMSD of 2.95 Å between the native and mutant protein structures as shown in Figure1. This nsSNP also showed a high PSIC score difference of 1.852. Hence, there might be a considerable change in the structure because of this nsSNP. Even though, there is only one amino acid change, the change in amino acid is of the different type. Here the amino acid Lysine is a cationic residue in the native protein and the substituted residue in the mutant protein molecule is Glutamate which is an anionic residue. Due to the complete change in charges brought about by this mutation, there seems to be a considerable change in structure.
The function of a protein is dependent on its 3-D structure and shape which is determined by its primary structure or sequence of amino acids. Since there is a change in amino acid sequence due to the single nucleotide polymorphism, non functional protein may be produced which will lead to altered overall function and hence this particular nsSNP may be responsible for a defective bone morphogenic protein and result in osteoporosis.
Results obtained from this in-silico study open new prospects for understanding osteoporosis and its consequences. These results, if correlated with clinical data, will be very useful in understanding the genetics of osteoporosis.
The methodology used to examine the effects of SNPs using a succession of tools could be structured and proposed as a standard screening process for SNPs' effects assessment on genes.
Conclusion
The nsSNP rs1804647 is predicted to be damaging by SIFT, PolyPhen, FASTSNP and listed as deleterious in Ensembl database. The change of nucleotide in 510th position from A to G (AAA→GAA) results in the amino acid change from Lysine to Glutamate. Since the change of amino acid residues is of opposite charge it may lead to conformational change of the three dimensional structure of the protein and hence result in altered function.
This observation can be considered as a good beginning for further investigations to study the genetic mechanisms behind osteoporosis, and when considered along with different clinical patterns of osteoporosis will result in more definitive evidence for the genetic origin of osteoporosis.
Disclosure
The authors report no conflicts of interest in this work.
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