man 's yesterday may ne'er be like his morrow ; nought may endure but mutabililty .
percy bysshe shelley ( 1816 ) mutability the first hint that the cytosine - guanine ( cpg ) dinucleotide might constitute a hotspot for pathological mutations in the human genome came nearly 25 years ago with the finding that two different cga  tga ( arg  term ) nonsense mutations had recurred quite independently at different locations in the factor viii ( f8 ) gene causing haemophilia a .
the potential generality of this phenomenon was supported by the finding that 12 of the 34 ( 35 per cent ) single base - pair ( bp ) substitutions then known to cause human inherited disease were c  t and g  a transitions within cpg dinucleotides .
further studies soon confirmed that the cpg dinucleotide was a mutation hotspot in a variety of different human disease genes , including pah , f9 , ldlr , rb1 , hprt1 and dmd .
as mutation data accumulated , cga  tga transitions were encountered particularly frequently as a cause of human genetic disease ; such nonsense mutations are inherently more likely to come to clinical attention than missense mutations . from the outset , it was realised that the hyper - mutability of the cpg dinucleotide was related to its role as the major site of cytosine methylation in the human genome .
the reason traditionally put forward to explain this association has been that , while cytosine spontaneously deaminates to uracil ( which is efficiently recognised as a non - dna base and removed by uracil - dna glycosylase ) , the spontaneous deamination of 5-methylcytosine ( 5mc ) yields thymine ,   thereby creating gt mismatches whose removal by methyl - cpg binding domain protein 4 ( mbd4 ) and/or thymine dna glycosylase followed by base excision repair is error prone [ 12 - 16 ] .
it remains possible , however , that mcpg transitions are not exclusively caused by the spontaneous deamination of 5-methylcytosine and may also arise through the action of other mechanisms and processes [ 17 - 19 ] .
( 1998 )   estimated that the rate of cg  tg ( and cg  ca on the other strand ) transitions was five times the base mutation rate .
subsequent estimates of 5mc hypermutability -- derived from various studies of polymorphism , disease mutations or evolutionary divergence -- have ranged between four - fold and 15-fold [ 20 - 26 ] .
it has been known for some time that cytosine methylation also occurs in the context of cpnpg sites in mammalian genomes , where n represents any nucleotide . since the intrinsic symmetry of the cpnpg trinucleotide would support a semi - conservative model of replication of the methylation pattern ( as with the cpg dinucleotide ) , it comes as no surprise that both maintenance and de novo methylation occurs at cpnpg sites in mammalian cells . in their recent paper on the human methylome , lister et al
.   reported abundant dna methylation in cphpg trinucleotides ( where h = a , c or t ) .
specifically , some 17.3 per cent of 5mc in embryonic stem cells was found to occur within cpapg , cpcpg and cptpg , with a further 7.2 per cent of 5mc occurring in cphph .
although lister et al .   suggested that non - cpg methylation is almost entirely lost upon differentiation ( a conclusion based solely upon the analysis of foetal lung fibroblasts ) , others have noted cpnpg methylation within human genes in a variety of different somatic tissues . although the extent of non - cpg methylation in the germ - line remains unclear , if we were to assume not only that cphpg methylation occurs in the germline , but
also that 5mc deamination can occur within a cphpg context , then it is very likely that methylated cphpg sites would constitute mutation hot - spots .
indirect evidence that this might indeed be the case has come from a disproportionately high number of c  t and g  a transitions at cpnpg sites in studies of the human nf1 and brca1 genes . in the light of the above , we have revisited the question of cpg dinucleotide hyper - mutability and explored the potential contribution that cphpg transitions might make to human inherited disease .
according to the april 2010 release of the human gene mutation database ( hgmd ; http://www.hgmd.org ) ,   56,457 pathological mutations have been reported in a total of 2,242 human genes .
a subset of 54,625 pathological missense and nonsense mutations in 2,113 genes , with  2 bp genomic dna sequence flanking the site of mutation , was retrieved from the hgmd .
the numbers of c  t and g  a mutations in this mutation dataset that were located within either cpg dinucleotides or cphpg trinucleotides were counted and termed ' mutations in di / trinucleotide ' ( table 1 ) . only these c  t and g  a transitions , found in the context of a cpg dinucleotide or cphpg trinucleotide , would be compatible with a model of methylation - mediated deamination of 5mc .
the remaining mutations in this hgmd dataset that were located in non - cpg or non - cphpg di / trinucleotides within the genes in question were also counted and termed ' mutations not in di / trinucleotide ' ( table 1 ) .
thus , 18.2 per cent of the studied missense / nonsense mutations causing human inherited disease are located in the cpg dinucleotide , while the corresponding proportion for the cphpg trinucleotide is 9.9 per cent . to assess the significance of these figures , the number of all possible c  t and g  a mutations within either cpg dinucleotides or cphpg trinucleotides within the coding regions of the mutated genes , termed ' possible mutations in di / trinucleotides ' ,
all possible single bp substitutions that occurred in a non - cpg dinucleotide or non - cphpg trinucleotide context ( as well as mutations other than c
 t and g  a in cpg and cphpg ) within the coding regions of the mutated genes were counted as ' possible mutations not in di / trinucleotide ' ( table 1 ) . a weak positive correlation was noted between the number of cpg mutations in the 2,113 genes analysed and the number of possible cpg mutations in these genes ( pearson 's correlation 0.129 , p = 2.45  10 ) , implying that the cpg mutation frequency is influenced to some extent by the frequency of occurrence of the underlying cpg dinucleotide .
unsurprisingly , a significantly greater proportion ( approximately ten - fold ) of observed pathological missense / nonsense mutations within these genes were c  t and g  a transitions within cpg dinucleotides than would have been expected ( by chance alone ) for all possible mutations ( table 1 ; p < 10 ) . a weak positive correlation ( pearson 's correlation 0.251 , p = 1.01  10 )
was also noted between the number of cphpg - located mutations and the number of cphpg trinucleotides in these genes , implying that the cphpg mutation frequency is influenced to some extent by the frequency of occurrence of the underlying cphpg trinucleotide .
once again , a greater proportion ( approximately two - fold ) of observed pathological missense / nonsense mutations within these genes were c  t and g  a transitions within cphpg trinucleotides than would have been expected by chance alone for all possible mutations ( table 1 ; p < 10 ) . numbers of c  t and g  a mutations found in cpg dinucleotides and cphpg trinucleotides in a dataset of 54,625 missense and nonsense mutations in 2,113 different human genes ( hgmd ) and the numbers of possible c  t and g  a mutations in cpg dinucleotides and cphpg trinucleotides within the coding regions of the mutated genes . from the data presented in table 1 , we estimate that ~11.8 per cent of the 9,947 cpg mutations ( ie 1,176 ) occurred within this dinucleotide by chance alone and hence would not necessarily have originated via the methylation - mediated deamination of 5mc . in a similar vein
, we estimate that ~46 per cent ( 2,460 ) of the cphpg mutations ( 5,402 ) occurred within these trinucleotides by chance alone and hence may not have originated via methylation - mediated deamination of 5mc .
the other side of this particular coin , however , is that the remaining 54 per cent of the 5,402 observed cphpg mutations in hgmd ( ie the excess 2,842 over expectation , or ~5 per cent of all the missense / nonsense mutations analysed ) may well be attributable to methylation - mediated deamination of 5mc within a cphpg context . as far as we are aware , this is the first ( albeit crude ) estimate of the potential impact of cphpg mutations on human inherited disease .
a similar analysis was performed for 1,766 regulatory mutations ( identified in the promoters of 191 human genes ) retrieved from the hgmd .
the numbers of actual and possible cpg and cphpg mutations were counted as before , using the promoter sequences of each gene . in order to determine the total numbers of possible cpg / cphpg and non - cpg / cphpg mutations ,
the wild - type promoter sequences for each gene ( total length , 22,051 bp ) were used ( table 2 ) .
as with the missense / nonsense mutations , an approximately twofold higher proportion of observed pathological regulatory mutations within these genes were c
 t and g  a transitions within cpg dinucleotides than would have been expected by chance alone for all possible mutations ( table 2 ; p = 6.03  10 ) .
we estimate that ~55 per cent of the 94 cpg mutations ( ie ~52 ) probably occurred within these dinucleotides by chance alone rather than via methylation - mediated deamination of 5mc .
by contrast , a lower than expected proportion of c  t and g  a regulatory mutations located in cphpg trinucleotides was observed ( p = 0.011 ) .
the absence of any excess of c  t and g  a mutations located in cphpg trinucleotides indicates that most , if not all , the promoter cphpg mutations probably occurred by chance alone , making it unnecessary to invoke methylation - mediated deamination of 5mc to account for them .
since neither cpg nor cphpg were found to be under - represented in the examined promoter regions as compared to the coding regions , we surmise that the reduced ( cpg ) or absent ( cphpg ) preponderance of c  t and g  a promoter mutations in the methylatable di / trinu - cleotides may be due to the relative paucity of cytosine methylation within the promoter regions   that would render unmethylated cpg and cphpg di / trinucleotides no more mutable than any other di / trinucleotide .
numbers of c  t and g  a mutations found in cpg dinucleotides and cphpg trinucleotides in a dataset of 1,766 regulatory mutations of 191 gene promoters ( hgmd ) and the numbers of possible c  t and g  a mutations in cpg dinucleotides and cphpg trinucleotides .
although two specific examples of non - cpg methylation altering the binding of transcription factors to promoter elements within human genes have so far been reported ,   the functional role of most non - cpg methylation in the human genome is still unclear .
irrespective of the functionality or otherwise of this specific type of post - synthetic dna modification in the human genome , it would appear that methylation of the cphpg trinucleotide may leave a significant imprint on the spectrum of missense / nonsense mutations causing human genetic disease .
 the cytosine - guanine ( cpg ) dinucleotide has long been known to be a hotspot for pathological mutation in the human genome . 
 this hypermutability is related to its role as the major site of cytosine methylation with the attendant risk of spontaneous deamination of 5-methylcytosine ( 5mc ) to yield thymine . 
 cytosine methylation , however , also occurs in the context of cpnpg sites in the human genome , an unsurprising finding since the intrinsic symmetry of cpnpg renders it capable of supporting a semi - conservative model of replication of the methylation pattern . 
 recently , it has become clear that significant dna methylation occurs in a cphpg context ( where h = a , c or t ) in a variety of human somatic tissues . 
 if we assume that cphpg methylation also occurs in the germline , and that 5mc deamination can occur within a cphpg context , then we might surmise that methylated cphpg sites could also constitute mutation hotspots causing human genetic disease . to test this postulate , 
 54,625 missense and nonsense mutations from 2,113 genes causing inherited disease were retrieved from the human gene mutation database http://www.hgmd.org . 
 some 18.2 per cent of these pathological lesions were found to be c  t and g  a transitions located in cpg dinucleotides ( compatible with a model of methylation - mediated deamination of 5mc ) , an approximately ten - fold higher proportion than would have been expected by chance alone . 
 the corresponding proportion for the cphpg trinucleotide was 9.9 per cent , an approximately two - fold higher proportion than would have been expected by chance . 
 we therefore estimate that ~5 per cent of missense / nonsense mutations causing human inherited disease may be attributable to methylation - mediated deamination of 5mc within a cphpg context . 