pre - mrna splicing removes intervening sequences or introns from eukaryotic precursor messenger rnas ( pre - mrnas ) to ensure accurate gene expression ( 1 ) . apart from joining consecutive exons together , this process is capable of selective removal or inclusion of exonic and intronic segments in mrna , generating distinct transcripts from a single gene , often in a cell type - specific or developmental- or gender - dependent manner ( 24 ) . both constitutive and alternative splicing are controlled by sequence elements in the pre - mrna that are recognized by a large ribonucleoprotein complex termed the spliceosome ( 1 ) . these conserved but degenerate signals are located predominantly in introns and include 5-splice sites ( 5-ss ) and 3-splice sites ( 3-ss ) , with upstream polypyrimidine tracts and the branch point sequence . mutations in any of these cis - elements can dramatically alter splicing efficiency and result in genetic disease ( 58 ) , but their consequences for rna processing have been difficult to predict . the majority of mutations at the 5-ss or 3-ss consensus have been reported to cause skipping of one or more exons and activation of cryptic splice sites ( 9 ) . occasionally , splice - site mutations may also result in full intron retention or give rise to entirely novel pseudoexons or cryptic exons , often in repetitive sequences , particularly in short interspersed nuclear elements ( sines ) , including mammalian interspersed repeats and alus ( 1012 ) . finally , creation of de novo splice sites in large exons may remove internal exonic sequences and create pseudointrons . traditional splicing signals contain only a half of the information necessary for accurate splice site recognition ( 13 ) . the remaining information is provided by auxiliary signals in introns and exons , known as splicing enhancers and silencers ( 14,15 ) , that are thought to interact with trans - acting factors and/or contribute to critical rna structural motifs and a  splicing code ( 16,17 ) . in addition , as the splicing and transcription machineries are tightly linked , splicing outcomes can be influenced by pre - mrna processing kinetics and transcription ( 18 ) . as a result , a  splicing mutation may affect not only rna processing , but also transcription ( 19 ) and downstream expression pathways , including translation . for example creating or eliminating exons containing upstream open reading frames or altering splicing efficiency and general intron - mediated translation enhancement can dramatically influence the abundance of gene products ( 20,21 ) . the fraction of gene mutations or variants that influence splicing and gene expression is thus likely to be larger than previously thought . because copy - number or structural variants exceed single - nucleotides polymorphism by at least 2-fold ( 22 ) , the overall contribution of human variability to differential pre - mrna processing and downstream remodelling of ribonucleoprotein particles could be even higher . better understanding of the complicated interplay of factors that control splice site choice would clearly benefit from convenient access to a comprehensive resource that pools sequences from scattered mutation reports in a wide - range of biomedical journals . although rare , these reports provide valuable information about splice site selection in vivo . currently , the human gene mutation database ( hgmd ) ( 23 ) and other locus - specific mutation databases ( for example hprt at www.ibiblio.org/dnam/des_hprt.htm or cftr at www.genet.sickkids.on.ca/cftr/app and see www.hgvs.org/rec.html for other genes ) give a list of splicing mutations . however , none of these databases provide critical and comprehensive information needed to understand why aberrant splice sites are selected . apart from the initial overview of aberrant splicing ( 24,25 ) , a comprehensive , regularly updated and publicly available tool is missing . here , we describe dbass3 and dbass5 , the databases of aberrant 3-ss and 5-ss in human disease genes and discuss their utility and importance for studying splice site selection . both databases contain sequences of new exon  intron boundaries that were generated through naturally occurring and disease - causing variants or mutations , both germ - line and somatic . dbass3 and dbass5 contain mutation - induced and sequence - verified aberrant rnas published in peer - reviewed communications over the past 30 years ( from january 1981 to june 2010 ) . briefly , reports of cryptic and de novo splice sites were identified by searching pubmed ( http://www.ncbi.nlm.nih.gov/entrez/query.fcgi ; queries :  mutation , splicing , cryptic ;  mutation , splicing , new acceptor or mutation , splicing , new donor ) and home pages of peer - reviewed journals . a subset of case reports were identified by searching locus - specific mutation databases ( http://archive.uwcm.ac.uk/uwcm/mg/docs/oth_mut.html ) . reported sequences were manually checked against reference sequences available from human genome databases , including genbank ( http://www.ncbi.nlm.nih.gov/genbank ) ( 26 ) and ensembl ( http://www.ensembl.org ) ( 27 ) . the vast majority of aberrant transcripts were verified by amplifying reverse - transcribed total rna extracted from blood samples taken from affected individuals and/or their family members . neither database features exon skipping or full intron retention events in which no new exon  intron boundaries were generated , nor do they include polymorphisms that influence utilization of the nagnag type of 3-ss , which was reported elsewhere ( 28 ) . dbass3 and dbass5 were designed as retrieval and submission tools containing mutation - induced aberrant splice sites that resulted in a recognizable phenotype . the web application was created using the microsoft asp and asp.net server technology and sql server database software . a breakdown of updated dbass3 and dbass5 records by gene , phenotype and location of aberrant splice sites is shown in tables 1 and 2 , respectively . table 1.summary of aberrant 3-splice sites in dbass3location of cryptic or de novo 3-splice sitesexonintronbothmutationin the 3-ss consensus ( cryptic)elsewhere (  de novo)in the 3-ss consensus ( cryptic)elsewhere (  de novo)all mutationsnumber of genes72303970170number of phenotypes67323771165number of cryptic and de novo 3-ss ( % ) 107 ( 34.3)48 ( 15.4)49 ( 15.7)108 ( 34.6)312 ( 100)number of aberrant 3-ss affecting terminal exons1259428median distance ( nucleotides ) between authentic and aberrant 3-splice sites134941121the 3-ss consensus is yag / g ( y is a pyrimidine , slash is the intron  exon boundary ) . table 2.summary of aberrant 5-splice sites in dbass5location of cryptic or de novo 5-splice sitesexonintronbothmutationin the 5-ss consensus ( cryptic)elsewhere (  de novo)in the 5-ss consensus ( cryptic)elsewhere (  de novo)all mutationsnumber of genes1155711367255number of phenotypes1226112368281number of cryptic and de novo 5-ss ( % ) 203 ( 33.9)89 ( 15.2)221 ( 36.9)90 ( 14.0)603 ( 100)number of aberrant 5-ss affecting terminal introns ( % ) 826218median distance ( nucleotides ) between authentic and aberrant 5-splice sites435943189the 5-ss consensus is mag / guragu ( m is a or c , r is purine and slash is the exon summary of aberrant 3-splice sites in dbass3 the 3-ss consensus is yag / g ( y is a pyrimidine , slash is the intron  exon boundary ) . summary of aberrant 5-splice sites in dbass5 the 5-ss consensus is mag / guragu ( m is a or c , r is purine and slash is the exon the initial analysis of dbass3 and dbass5 records ( 29,30 ) confirmed that cryptic splice sites were , on average , intrinsically stronger than mutated authentic ( natural ) sites but generally weaker than their authentic , wild - type counterparts ( 31 ) . analysis of dbass3 and dbass5 sequences also showed that the maximum entropy ( me ) algorithm ( 32 ) gave the best overall discrimination between aberrant and authentic sites ( 29,30 ) , while algorithms based only on the weight matrix , such as the shapiro and senapathy score ( 33 ) performed less successfully . the density of silencers and enhancers in the segments between authentic and aberrant sites was intermediate between exons and introns , supporting a gradient concept of exon and intron definition ( 34 ) . analysis of dbass3 and dbass5 data also revealed the underlying mutation pattern of aberrant splice sites and their location . for example breakdown of dbass3 alterations showed that ag - creating mutations were much more common than previously estimated , totalling to 42% ( 29 ) , as opposed to the initial estimate of 13% ( 24 ) . de novo 5-ss in introns were stronger than their authentic counterparts ( 30 ) , but this did not apply to exonic de novo sites , suggesting that their activation in vivo is more reliant on exonic splicing enhancers or silencers , rather than on the intrinsic strength of the 5-ss consensus ( 30 ) . finally , dbass3 and dbass5 data has been employed to develop other prediction tools , such as cryp - skip ( http://www.dbass.org.uk/cryp-skip/ ) , which can distinguish between cryptic splice site activation and exon skipping upon mutation of 3-ss or 5-ss ( 35 ) or hot - skip ( http://www.dbass.org.uk/hot-skip/ ) , which computes the ess / ese profile for all possible point mutations at each exon position and identifies nucleotide substitutions that are most likely to skip the exon ( m. raponi et al . such free practical utilities are useful for molecular diagnostics to facilitate identification of exonic changes that interfere with rna processing and prediction of their phenotypic outcome . dbass3 and dbass5 provide a quick search page where the user needs to specify a gene symbol ( http://www.gene.ucl.ac.uk/nomenclature/ ) , phenotype or mendelian inheritance in man ( mim ) number ( www.ncbi.nlm.nih.gov/omim/ ) ( 36 ) or nucleotide sequences that match the input criteria . in an advanced search option , one can select a combination of several criteria , including phenotype , gene , mutation , location of aberrant splice sites in introns and exons , distance from authentic splice sites and relevant bibliographic references ( figure 1 ) . investigators studying coupled processes of rna splicing and end - processing can easily retrieve aberrant splice sites that were activated in terminal exons or introns . not in the least , the user can search for aberrant splice sites ( and their wild - type and mutated authentic counterparts ) by their intrinsic strength . this option permits retrieval of splice sites according to various splice - site scores and user - defined cut - off points or desired intervals . dbass3 and dbass5 provide a quick search page where the user needs to specify a gene symbol , phenotype or nucleotide sequence . in the advanced search option , one can select a combination of several criteria , including phenotype , gene , mutation , location of aberrant splice sites in introns and exons , distance from authentic ( natural ) splice sites , intrinsic strength of splice sites and bibliographic references . dbass3 and dbass5 provide a quick search page where the user needs to specify a gene symbol , phenotype or nucleotide sequence . in the advanced search option , one can select a combination of several criteria , including phenotype , gene , mutation , location of aberrant splice sites in introns and exons , distance from authentic ( natural ) splice sites , intrinsic strength of splice sites and bibliographic references . the search results can be expanded by clicking on the  view details button ( figure 1 ) , leading to full details in each record ( figure 2 ) , including gene , phenotype , mim numbers , mutation ( using traditional and official nomenclature where available ) , distance between authentic and aberrant splice sites , change of the reading frame ( 0 , + 1 and + 2  nt ) , literature references with pubmed hyperlinks and nucleotide sequences flanking the authentic and aberrant 5-ss . the ex vivo origin of aberrant rna is indicated in the comments fields of dbass3 and dbass5 records , which also contains additional information . greater than sign for nucleotide substitutions , by parentheses for deletions and by brackets for duplications or insertions . intronic sequences are shown in blue lower case , exons are shown in green upper case . greater than sign for nucleotide substitutions , by parentheses for deletions and by brackets for duplications or insertions . intronic sequences are shown in blue lower case , exons are shown in green upper case . finally , both dbass3 and dbass5 allow the users to submit newly published entries for their inclusion in the databases via a dedicated submission tool and also to register to obtain regular database updates by email . although mutations anywhere in intron or exon can impair pre - mrna splicing , most mutations that activate aberrant splice sites are found in or close to natural splice sites ( 2931 ) . if the mutation nomenclature incorporated the distance ( in nucleotides ) between mutation and natural exon intron junction , this information would help to identify mutations that activate aberrant splice sites . for exonic mutations , however , neither traditional ( 37 ) nor official ( www.hgvs.org/mutnomen/ ) mutation nomenclature assimilate these data . for intronic alterations , the distance can be derived from both the traditional and the official designation , although the latter does not provide this information when referring to a genomic sequence ( for example g.234g > t ) . as most human mutation reports currently adhere to the official nomenclature , exonic mutations that affect splicing are less likely to be recognized and are probably under - reported . to allow the investigators immediate access to this critical information , both dbass3 and dbass5 still show the traditional mutation nomenclature for all records . in exons , e ( for exon ) , which is followed by an exon number , a  plus sign and the distance ( in nucleotides ) between the last authentic intron  exon junction and the point mutation . in addition to sequence , dbass3 and dbass5 users can see both the distance between mutation and authentic splice site and the distance between authentic and aberrant splice sites . rather than relying merely on the nomenclature , this design ultimately facilitates rapid verification of splicing mutations and auxiliary splicing signals , giving the user immediate access to the sequence of newly intronized or exonized segments and the underlying dna change . new releases of dbass3 and dbass5 data will reflect the growing number of studies reporting new aberrant splice sites . the updates will be facilitated by user - assisted submissions and proper reporting of disease - associated aberrant splices in biomedical journals , which should be endorsed by journal editors . with recent realization that single nucleotide variability is less extensive than insertion / deletion or copy - number variability ( 22 ) , it will be interesting to catalogue structural alterations that influence pre - mrna processing and alter the relative expression of pre - existing , alternatively spliced mrnas , particularly in conserved gene families . dbass5 and dbass3 entries are also planned to be linked to external resources other than omim and ensembl , such as the snp databases ( http://www.ncbi.nlm.nih.gov/snp/ ) . both databases contain sequences of new exon  intron boundaries that were generated through naturally occurring and disease - causing variants or mutations , both germ - line and somatic . dbass3 and dbass5 contain mutation - induced and sequence - verified aberrant rnas published in peer - reviewed communications over the past 30 years ( from january 1981 to june 2010 ) . briefly , reports of cryptic and de novo splice sites were identified by searching pubmed ( http://www.ncbi.nlm.nih.gov/entrez/query.fcgi ; queries :  mutation , splicing , cryptic ;  mutation , splicing , new acceptor or mutation , splicing , new donor ) and home pages of peer - reviewed journals . a subset of case reports were identified by searching locus - specific mutation databases ( http://archive.uwcm.ac.uk/uwcm/mg/docs/oth_mut.html ) . reported sequences were manually checked against reference sequences available from human genome databases , including genbank ( http://www.ncbi.nlm.nih.gov/genbank ) ( 26 ) and ensembl ( http://www.ensembl.org ) ( 27 ) . the vast majority of aberrant transcripts were verified by amplifying reverse - transcribed total rna extracted from blood samples taken from affected individuals and/or their family members . neither database features exon skipping or full intron retention events in which no new exon  intron boundaries were generated , nor do they include polymorphisms that influence utilization of the nagnag type of 3-ss , which was reported elsewhere ( 28 ) . dbass3 and dbass5 were designed as retrieval and submission tools containing mutation - induced aberrant splice sites that resulted in a recognizable phenotype . the web application was created using the microsoft asp and asp.net server technology and sql server database software . a breakdown of updated dbass3 and dbass5 records by gene , phenotype and location of aberrant splice sites is shown in tables 1 and 2 , respectively . table 1.summary of aberrant 3-splice sites in dbass3location of cryptic or de novo 3-splice sitesexonintronbothmutationin the 3-ss consensus ( cryptic)elsewhere (  de novo)in the 3-ss consensus ( cryptic)elsewhere (  de novo)all mutationsnumber of genes72303970170number of phenotypes67323771165number of cryptic and de novo 3-ss ( % ) 107 ( 34.3)48 ( 15.4)49 ( 15.7)108 ( 34.6)312 ( 100)number of aberrant 3-ss affecting terminal exons1259428median distance ( nucleotides ) between authentic and aberrant 3-splice sites134941121the 3-ss consensus is yag / g ( y is a pyrimidine , slash is the intron  exon boundary ) . table 2.summary of aberrant 5-splice sites in dbass5location of cryptic or de novo 5-splice sitesexonintronbothmutationin the 5-ss consensus ( cryptic)elsewhere (  de novo)in the 5-ss consensus ( cryptic)elsewhere (  de novo)all mutationsnumber of genes1155711367255number of phenotypes1226112368281number of cryptic and de novo 5-ss ( % ) 203 ( 33.9)89 ( 15.2)221 ( 36.9)90 ( 14.0)603 ( 100)number of aberrant 5-ss affecting terminal introns ( % ) 826218median distance ( nucleotides ) between authentic and aberrant 5-splice sites435943189the 5-ss consensus is mag / guragu ( m is a or c , r is purine and slash is the exon summary of aberrant 3-splice sites in dbass3 the 3-ss consensus is yag / g ( y is a pyrimidine , slash is the intron  exon boundary ) . summary of aberrant 5-splice sites in dbass5 the 5-ss consensus is mag / guragu ( m is a or c , r is purine and slash is the exon the initial analysis of dbass3 and dbass5 records ( 29,30 ) confirmed that cryptic splice sites were , on average , intrinsically stronger than mutated authentic ( natural ) sites but generally weaker than their authentic , wild - type counterparts ( 31 ) . analysis of dbass3 and dbass5 sequences also showed that the maximum entropy ( me ) algorithm ( 32 ) gave the best overall discrimination between aberrant and authentic sites ( 29,30 ) , while algorithms based only on the weight matrix , such as the shapiro and senapathy score ( 33 ) performed less successfully . the density of silencers and enhancers in the segments between authentic and aberrant sites was intermediate between exons and introns , supporting a gradient concept of exon and intron definition ( 34 ) . analysis of dbass3 and dbass5 data also revealed the underlying mutation pattern of aberrant splice sites and their location . for example breakdown of dbass3 alterations showed that ag - creating mutations were much more common than previously estimated , totalling to 42% ( 29 ) , as opposed to the initial estimate of 13% ( 24 ) . de novo 5-ss in introns were stronger than their authentic counterparts ( 30 ) , but this did not apply to exonic de novo sites , suggesting that their activation in vivo is more reliant on exonic splicing enhancers or silencers , rather than on the intrinsic strength of the 5-ss consensus ( 30 ) . finally , dbass3 and dbass5 data has been employed to develop other prediction tools , such as cryp - skip ( http://www.dbass.org.uk/cryp-skip/ ) , which can distinguish between cryptic splice site activation and exon skipping upon mutation of 3-ss or 5-ss ( 35 ) or hot - skip ( http://www.dbass.org.uk/hot-skip/ ) , which computes the ess / ese profile for all possible point mutations at each exon position and identifies nucleotide substitutions that are most likely to skip the exon ( m. raponi et al . , submitted for publication ) . such free practical utilities are useful for molecular diagnostics to facilitate identification of exonic changes that interfere with rna processing and prediction of their phenotypic outcome . dbass3 and dbass5 provide a quick search page where the user needs to specify a gene symbol ( http://www.gene.ucl.ac.uk/nomenclature/ ) , phenotype or mendelian inheritance in man ( mim ) number ( www.ncbi.nlm.nih.gov/omim/ ) ( 36 ) or nucleotide sequences that match the input criteria . in an advanced search option , one can select a combination of several criteria , including phenotype , gene , mutation , location of aberrant splice sites in introns and exons , distance from authentic splice sites and relevant bibliographic references ( figure 1 ) . investigators studying coupled processes of rna splicing and end - processing can easily retrieve aberrant splice sites that were activated in terminal exons or introns . not in the least , the user can search for aberrant splice sites ( and their wild - type and mutated authentic counterparts ) by their intrinsic strength . this option permits retrieval of splice sites according to various splice - site scores and user - defined cut - off points or desired intervals . dbass3 and dbass5 provide a quick search page where the user needs to specify a gene symbol , phenotype or nucleotide sequence . in the advanced search option , one can select a combination of several criteria , including phenotype , gene , mutation , location of aberrant splice sites in introns and exons , distance from authentic ( natural ) splice sites , intrinsic strength of splice sites and bibliographic references . dbass3 and dbass5 provide a quick search page where the user needs to specify a gene symbol , phenotype or nucleotide sequence . in the advanced search option , one can select a combination of several criteria , including phenotype , gene , mutation , location of aberrant splice sites in introns and exons , distance from authentic ( natural ) splice sites , intrinsic strength of splice sites and bibliographic references . the search results can be expanded by clicking on the  view details button ( figure 1 ) , leading to full details in each record ( figure 2 ) , including gene , phenotype , mim numbers , mutation ( using traditional and official nomenclature where available ) , distance between authentic and aberrant splice sites , change of the reading frame ( 0 , + 1 and + 2  nt ) , literature references with pubmed hyperlinks and nucleotide sequences flanking the authentic and aberrant 5-ss . the ex vivo origin of aberrant rna is indicated in the comments fields of dbass3 and dbass5 records , which also contains additional information . greater than sign for nucleotide substitutions , by parentheses for deletions and by brackets for duplications or insertions . intronic sequences are shown in blue lower case , exons are shown in green upper case . greater than sign for nucleotide substitutions , by parentheses for deletions and by brackets for duplications or insertions . intronic sequences are shown in blue lower case , exons are shown in green upper case . finally , both dbass3 and dbass5 allow the users to submit newly published entries for their inclusion in the databases via a dedicated submission tool and also to register to obtain regular database updates by email . although mutations anywhere in intron or exon can impair pre - mrna splicing , most mutations that activate aberrant splice sites are found in or close to natural splice sites ( 2931 ) . if the mutation nomenclature incorporated the distance ( in nucleotides ) between mutation and natural exon intron junction , this information would help to identify mutations that activate aberrant splice sites . for exonic mutations , however , neither traditional ( 37 ) nor official ( www.hgvs.org/mutnomen/ ) mutation nomenclature assimilate these data . for intronic alterations , the distance can be derived from both the traditional and the official designation , although the latter does not provide this information when referring to a genomic sequence ( for example g.234g > t ) . as most human mutation reports currently adhere to the official nomenclature , exonic mutations that affect splicing are less likely to be recognized and are probably under - reported . to allow the investigators immediate access to this critical information , both dbass3 and dbass5 still show the traditional mutation nomenclature for all records . in exons , single - nucleotide substitutions are simply preceded by  e ( for exon ) , which is followed by an exon number , a plus sign and the distance ( in nucleotides ) between the last authentic intron  exon junction and the point mutation . in addition to sequence , dbass3 and dbass5 users can see both the distance between mutation and authentic splice site and the distance between authentic and aberrant splice sites . rather than relying merely on the nomenclature , this design ultimately facilitates rapid verification of splicing mutations and auxiliary splicing signals , giving the user immediate access to the sequence of newly intronized or exonized segments and the underlying dna change . new releases of dbass3 and dbass5 data will reflect the growing number of studies reporting new aberrant splice sites . the updates will be facilitated by user - assisted submissions and proper reporting of disease - associated aberrant splices in biomedical journals , which should be endorsed by journal editors . with recent realization that single nucleotide variability is less extensive than insertion / deletion or copy - number variability ( 22 ) , it will be interesting to catalogue structural alterations that influence pre - mrna processing and alter the relative expression of pre - existing , alternatively spliced mrnas , particularly in conserved gene families . dbass5 and dbass3 entries are also planned to be linked to external resources other than omim and ensembl , such as the snp databases ( http://www.ncbi.nlm.nih.gov/snp/ ) . juvenile diabetes research foundation international ( 1 - 2008 - 47 ) ; ec grant eurasnet - lshg - ct-2005 - 518238 .
dbass3 and dbass5 provide comprehensive repositories of new exon boundaries that were induced by pathogenic mutations in human disease genes . 
 aberrant 5- and 3-splice sites were activated either by mutations in the consensus sequences of natural exon 
 intron junctions ( cryptic sites ) or elsewhere (  de novo sites ) . 
 dbass3 and dbass5 currently contain approximately 900 records of cryptic and de novo 3- and 5-splice sites that were produced by over a thousand different mutations in approximately 360 genes . 
 dbass3 and dbass5 data can be searched by disease phenotype , gene , mutation , location of aberrant splice sites in introns and exons and their distance from authentic counterparts , by bibliographic references and by the splice - site strength estimated with several prediction algorithms . 
 the user can also retrieve reference sequences of both aberrant and authentic splice sites with the underlying mutation . 
 these data will facilitate identification of introns or exons frequently involved in aberrant splicing , mutation analysis of human disease genes and study of germline or somatic mutations that impair rna processing . 
 finally , this resource will be useful for fine - tuning splice - site prediction algorithms , better definition of auxiliary splicing signals and design of new reporter assays . 
 dbass3 and dbass5 are freely available at http://www.dbass.org.uk/.