it is generally understood that every individual has a few mutations in their genome that did not exist in their parents ' genomes .
an estimate of the spontaneous mutation rate in humans is 1.8  10per nucleotide per generation . with 3.2 billion bases per genome
multiplying that by 6 billion people gives 690 billion mutations per human generation , or about 216 mutations for each nucleotide in the human genome .
our species is undergoing a massive worldwide mutational experiment , with every nucleotide in our genomes being tested by mutation .
what this means is that , if we look hard enough , we should find all non - lethal sequence variants in any given human gene .
the genetics research community has begun this search by identifying genes that , when defective , lead to a human disease .
the online mendelian inheritance in man ( omim ) database   is a repository for information on human genes .
omim suggests searching locuslink , limiting to human , the search with the term ' disease_known ' to find a human disease gene count ( 2,440 entries ) .
refining this search with ' disease_known and has_seq ' will limit the result to human diseases with a known gene sequence . as of 23rd september , 2004 , this number is 1,692 .
the current gene count in humans is 42,716 according to the genome alignment and annotation ( gala ) database .
therefore , about 4 per cent of human genes today have a disease associated with them . to show that a gene is responsible for a disease ,
the location of stop codons , splice - site mutations , in - frame deletions , frameshifts or missense mutations that affect function in patients but not in unaffected individuals proves that the gene is responsible for the phenotype of the disease ; however , this is only the beginning . if a disease is common or of longstanding interest to the research community , there is often a database started for that gene to compile the known mutations and phenotypes .
the human gene mutation database   has a link to 292 locus - specific mutation databases . because there is some duplication ( such as six different tp53 databases ) and some databases cover more than one gene ,
these 292 links cover a minimum of 257 different human genes . many are free to the public , but some require registration , a password or subscription -- as with the ' hypoxanthine guanine phosphoribosyltransferase- 1 ' ( hprt ) database these 257 genes currently represent 15 per cent of the 1,692 human disease genes with known sequences .
this list is not comprehensive , since it does not contain the hbvar database of haemoglobin variants .
what can be expected from intense scrutiny of a gene ? as mentioned above , if we look hard enough , mutations in every codon and even every nucleotide -- that do not cause a lethal phenotype -- should be discovered . in the real world , how close are we to achieving this hypothetical coverage ? herein , are presented six examples of well studied genes : brca1 and brca2 , the breast cancer susceptibility genes ; haemoglobinbeta ( hbb ) ; hprt1 ; the cystic fibrosis transmembrane conductance regulator ( cftr ) ; and the tp53 tumour suppressor .
the brca1 and brca2 genes are special cases of great human interest . more than 20,000 complete coding sequences of brca1 from patients with breast cancer or from their relatives have been determined .
the protein is 1,863 amino acids long , which is equivalent to 5,589 base pairs ( bp ) just for the coding sequence .
usually , the test that is ordered involves sequencing both brca1 and brca2 ( 27 exons , 3,418 amino acids , 10,254 bp for the coding region ) at a cost of $ 2,975 for the first member of a family and $ 350 for subsequent members .
the cost may be partially covered as part of a health insurance plan . because myriad genetic laboratories has a patent on the test , they perform all of these tests with uniform quality control , and all of the mutation data goes into one database , the breast cancer information core ( bic ) .
membership may be obtained by online application   and it is clearly intended for researchers , not patients .
results from the bic have been summarised [ 14 - 16 ] . as of 22nd september , 2004
since 20,000 individual sequence tests have been performed , one can infer that more than 10,000 of them are without a single mutation , else they would have been counted as an entry in the bic . for brca1 , 1,539 of the 9,556 entries are distinct mutations , polymorphisms or variants .
the remainder are duplicates , and some are very common founder mutations . of the 1,539 unique mutations , 878 were observed only once ( 57 per cent ) ; these are the rare variants mentioned above and are seen once in 20,000 sequences ( or even at a lower frequency , but discovered by accident in 20,000 sequences ) . by examining the bic database for mutations in the coding region
, it was shown that 850 of the 1,863 codons contain at least one mutation ( 46 per cent ) . by looking at the cdna mutation map ,
the largest non - mutated region is 43 bp , from nucleotides 4,530 to 4,572 .
the mutations on this histogram plot are so thick that the whole gene looks like a dense bar code .
it has 1,893 distinct mutations , polymorphisms or variants , with 1,146 reported only once ( 60 per cent ) ; this is very similar to brca1 . in brca2
, 1,323 of the 3,418 codons have at least one mutation ( 39 per cent ) . from the cdna mutation map ,
the largest non - mutated region is 48 bp , from nucleotides 1,282 to 1,329 .
this brings us back to the earlier question of how saturated is our mutation catalogue . to discuss this
, i will introduce the concept of mutation space . considering only single - base changes , and leaving out insertions and deletions ,
if each codon can theoretically be mutated by nine different one - base changes , then the mutation space for a protein - coding gene is nine times the number of codons . for illustrative purposes ,
thus , 1,539 distinct mutations is 9 per cent of the mutation space -- although that number does include frameshift mutants , so the 9 per cent value is slightly inflated . for brca2 ,
the percentage of mutation space is only 6 per cent . in other words , more than 90 per cent of
because the sampling of this space is so low , a high percentage of the mutants found in brca1 and brca2 screening will never have been seen before .
one of a kind missense mutations that do not truncate the protein will be the so - called ' variants of uncertain significance ' .
patients who have such a mutation are given a pamphlet entitled testing for hereditary cancer risk : what does a " variant of uncertain significance " mean ?
this is a looming question for geneticists faced with mutation data but no experimental data .
the paper of abkevich et al .   tries to address this by bioinformatics methods .
of 314 missense mutations in brca1 from the 20,000 sequence tests , they state that only 21 are classified as deleterious , or suspected deleterious , mutants . by comparing human brca1 with orthologues from other species , however , and by taking into account the properties of the side chains in the normal and mutated amino acids , they predicted that 50 more of these missense mutations would be suspected to be deleterious .
of the 243 other missense mutants , 14 had previously been deemed neutral or harmless .
their analysis added 92 more mutants into this category -- for a total of 177/314 mutants with predictions either of deleterious , suspected deleterious or of little clinical significance .
the authors caution that this method only gives predictions , and that care must be used in interpretation in a clinical setting .
it should be remembered that patients are making decisions about mastectomies , oophorectomies , chemotherapy options and radiation treatments based , in part , on this mutation information .
another study , by goldgar et al . , also uses a bioinformatics approach and integrates the results from several analyses into a weighted probability of significance .
their model was applied to three mutants each from brca1 and brca2 , allowing a risk to be assigned to five of the six mutants .
this was one of the earliest human sequences to be studied ; in fact , many of the mutations were found by protein sequencing methods , rather than dna sequencing .
hbb is among the most intensively studied human proteins ; it is responsible for sickle - cell anaemia and the beta - thalassaemias .
the omim entry for hbb has 1,157 references and entries for 522 allelic variants . in 1996 , huisman et al . stated that 138 of the 146 codons of the hbb gene have been mutated .
inspection of the hbvar database   reveals that this number is now 141/146 codons , with only thr-4 , thr-12 , thr-50 , pro-125 and val-137 not having a known mutation in humans .
note that the val after the start met has been assigned as amino acid 1 .
hbb has 461 distinct point mutants in 146 codons ; this equals 35 per cent of the mutation space ( 9  146 = 1,314 possible point mutants ) .
this value is much higher than that seen in the larger brca1 and brca2 genes .
one observation that affects this value is the near - complete lack of synonymous substitutions in this database , which reflects the protein rather than dna sequencing analyses .
certainly synonymous substitutions have been observed , but they do not seem to be reported . including synonymous mutations would make the 35 per cent figure significantly higher .
examination of the pfam database of 7,503 protein family sequence alignments   for the globin family reveals that thr-12 was seen as a ser in the mouse - eared bat ( myotis velifer ) and thr-50 was also often seen as a ser -- even in primates such as macaca mulatta .
pro-125 was almost always gln , except in a few primates such as the gorilla , human and chimpanzee .
val- 137 was an ile in the south american lungfish ( lepidosiren paradoxus ) and ile or leu in some frog hbb sequences .
thr-4 was outside the alignment edge in pfam , but rabbits and black lemur ( eulemur macaco ) both had a ser at this position .
these observations show that at least four of these five amino acids do vary in other mammals and probably could vary in humans without serious consequences .
val-137 seems to be the most invariant of the five , and it may have a significant effect if mutated to another residue .
mutations in the hprt1 gene lead to the lesch - nyhan syndrome , a defect in the purine salvage pathway .
the protein is fairly small , with only 218 amino acids ( 217 in the mature protein ) , yet there are 2,500 mutations reported in this gene .
the database for hprt1 is available only by subscription , so , without access , one is limited to published reports that give a detailed breakdown of the types of mutations and the number of unique mutations . in public sources ,
the human gene mutation database now lists 223 mutants , with 115 being missense or nonsense point mutants in 80 codons ( about 6 per cent of the mutation space ) . in people , very few mutants
somatic functional mutations in hprt1 can be selected in vitro in 6-thioguanine - resistant t lymphocytes from normal people .
these are not naturally occurring mutants , they arise by inactivation of the hprt1 gene .
these in vitro mutants make up the majority of the hprt1 database that is not publicly available .
since they are inactivating mutations , they have to be at critical sites in the gene .
examined the known mutations ( including patient mutations ) and related them to the crystal structures of the hprt protein .
report that 155 of the 217 amino acids ( 72 per cent ) have known missense mutations that cause an amino acid change : these arise from a subset of 963 single - base - substitution mutants that cause missense mutations . the number of synonymous mutations in this set is not given .
in addition to the 963 missense mutations , there are 51 ( of a possible 66 ) nonsense mutations in the database ; this gives a total of 1,014 single base mutations in 217 codons for 52 per cent of the sequence space ( 9  217 = 1,953 ) .
after accounting for missense , nonsense and synonymous mutations , there remain 46/217 codons with no known mutations .
there are currently 1,338 distinct mutations listed in the cftr mutation database . by examining the list of mutations and
not including the mutations in non - coding regions or insertions and deletions , there were 702 unique point mutations in 501 of the 1,481 codons ( 34 per cent ) .
this covers about 5 per cent of the cftr gene 's mutation space , which is very similar to that of the brca2 gene .
the international agency for research on cancer ( iarc ) tp53 mutation database   version r9 of june 2004 , contains 19,809 somatic mutations ( 74 per cent missense , 7 per cent nonsense ) and 264 germline mutations .
more than 1,700 different point mutations at more than 310 distinct codons ( out of a total of 393 codons ) have been found as described in the slideshow on the database site .
1,700 mutations represent 48 per cent of the mutation space for 393 codons ( 9  393 = 3,537 ) ; this is an even higher percentage than that seen in the hbb gene .
the vast majority of the mutations are in the middle third of the gene , coding for the dna - binding portion of the protein .
this was one of the earliest human sequences to be studied ; in fact , many of the mutations were found by protein sequencing methods , rather than dna sequencing .
hbb is among the most intensively studied human proteins ; it is responsible for sickle - cell anaemia and the beta - thalassaemias .
the omim entry for hbb has 1,157 references and entries for 522 allelic variants . in 1996 , huisman et al . stated that 138 of the 146 codons of the hbb gene have been mutated .
inspection of the hbvar database   reveals that this number is now 141/146 codons , with only thr-4 , thr-12 , thr-50 , pro-125 and val-137 not having a known mutation in humans .
note that the val after the start met has been assigned as amino acid 1 .
hbb has 461 distinct point mutants in 146 codons ; this equals 35 per cent of the mutation space ( 9  146 = 1,314 possible point mutants ) .
this value is much higher than that seen in the larger brca1 and brca2 genes .
one observation that affects this value is the near - complete lack of synonymous substitutions in this database , which reflects the protein rather than dna sequencing analyses .
certainly synonymous substitutions have been observed , but they do not seem to be reported . including synonymous mutations would make the 35 per cent figure significantly higher .
examination of the pfam database of 7,503 protein family sequence alignments   for the globin family reveals that thr-12 was seen as a ser in the mouse - eared bat ( myotis velifer ) and thr-50 was also often seen as a ser -- even in primates such as macaca mulatta .
pro-125 was almost always gln , except in a few primates such as the gorilla , human and chimpanzee .
val- 137 was an ile in the south american lungfish ( lepidosiren paradoxus ) and ile or leu in some frog hbb sequences .
thr-4 was outside the alignment edge in pfam , but rabbits and black lemur ( eulemur macaco ) both had a ser at this position .
these observations show that at least four of these five amino acids do vary in other mammals and probably could vary in humans without serious consequences .
val-137 seems to be the most invariant of the five , and it may have a significant effect if mutated to another residue .
mutations in the hprt1 gene lead to the lesch - nyhan syndrome , a defect in the purine salvage pathway .
the protein is fairly small , with only 218 amino acids ( 217 in the mature protein ) , yet there are 2,500 mutations reported in this gene .
the database for hprt1 is available only by subscription , so , without access , one is limited to published reports that give a detailed breakdown of the types of mutations and the number of unique mutations . in public sources ,
the human gene mutation database now lists 223 mutants , with 115 being missense or nonsense point mutants in 80 codons ( about 6 per cent of the mutation space ) . in people , very few mutants
somatic functional mutations in hprt1 can be selected in vitro in 6-thioguanine - resistant t lymphocytes from normal people .
these are not naturally occurring mutants , they arise by inactivation of the hprt1 gene .
these in vitro mutants make up the majority of the hprt1 database that is not publicly available . since they are inactivating mutations
examined the known mutations ( including patient mutations ) and related them to the crystal structures of the hprt protein .
report that 155 of the 217 amino acids ( 72 per cent ) have known missense mutations that cause an amino acid change : these arise from a subset of 963 single - base - substitution mutants that cause missense mutations .
in addition to the 963 missense mutations , there are 51 ( of a possible 66 ) nonsense mutations in the database ; this gives a total of 1,014 single base mutations in 217 codons for 52 per cent of the sequence space ( 9  217 = 1,953 ) .
after accounting for missense , nonsense and synonymous mutations , there remain 46/217 codons with no known mutations .
there are currently 1,338 distinct mutations listed in the cftr mutation database . by examining the list of mutations and not including the mutations in non - coding regions or insertions and deletions ,
there were 702 unique point mutations in 501 of the 1,481 codons ( 34 per cent ) .
this covers about 5 per cent of the cftr gene 's mutation space , which is very similar to that of the brca2 gene .
the international agency for research on cancer ( iarc ) tp53 mutation database   version r9 of june 2004 , contains 19,809 somatic mutations ( 74 per cent missense , 7 per cent nonsense ) and 264 germline mutations .
more than 1,700 different point mutations at more than 310 distinct codons ( out of a total of 393 codons ) have been found as described in the slideshow on the database site .
1,700 mutations represent 48 per cent of the mutation space for 393 codons ( 9  393 = 3,537 ) ; this is an even higher percentage than that seen in the hbb gene .
the vast majority of the mutations are in the middle third of the gene , coding for the dna - binding portion of the protein .
the data on these six genes present a trend that will be followed with other disease genes .
more and more sequence data will be collected until a high percentage of all the possible mutations are found .
we could simply perform a ' thought experiment ' and ask : ' what does one do when all possible ( non - lethal ) point mutants in a gene are documented in real people and the missing mutants are made and tested for function ? '
we are heading in this direction -- as indicated by the 12th february , 2004 request for application ( rfa ) from the national institutes of health , entitled ' revolutionary genome sequencing technologies : the $ 1,000 genome ' .
once the low - cost genome is available , perhaps in 10 years , millions of human genomes will have been sequenced and every gene will have sequence data exceeding what is now available for the tp53 gene .
first , genotype must be linked to phenotype by examining family histories ; this will already have been done for the best - studied disease genes .
of course , this is not possible on a mutation that is seen only once .
secondly , the bioinformatics approach   will predict a mutation 's probable significance by sequence comparison to orthologues and by rating the severity of an amino acid change .
an enhancement of this method would be to scan for any sequence motifs for structure or targeting that may be affected by the mutation .
thirdly , examine the crystal structure of the protein , or of a close relative of that protein if known , to see if the mutation would cause a significant structural effect .
fourthly , assay the mutant protein in a variety of function tests or apply spectroscopic methods to detect changes in chromophores .
the assay method may not be possible if the function of the protein is unknown .
in many cases , the primary biology of the protein will need to be discovered -- that is , the pathway in which it participates , any interacting proteins , expression levels , subcellular localisation , post - translational modifications , rates of turnover , etc .
this is really an outline for the biochemistry , cell biology and molecular biology of human beings at the level of every gene .
considering the cost of analysing each mutation , there must be some point that crosses a practical return on investment .
do we know everything we need to know about mutants of haemoglobin - beta , or should we make the remaining 843 point mutants just to be sure we have not missed anything ?
nicholas murray butler ( 1862 - 1947 ) , president of columbia university , wrote : ' an expert is one who knows more and more about less and less ' .
i would like to thank the owners of the databases mentioned in this paper , without which there could be no paper : the breast cancer information core ( bic ) , online mendelian inheritance in man ( omim database ) , locuslink , the gala genome alignment and annotation database , the human gene mutation database , hprt database , hbvar database , pfam database , cftr mutation database and iarc tp53 mutation database .
 the rapid accumulation of mutation data has led to the creation of nearly 300 locus - specific mutation databases . 
 these sites may contain a few dozen to almost 20,000 mutations for a given gene . 
 many of the mutations are uncharacterised and have no known effects on the gene product , the ' variant of uncertain significance ' . here , the statistics of mutation distribution are examined for six different gene databases : brca1 and brca2 , haemoglobin - beta ( hbb ) , hprt1 , cftr and tp53 . 
 the percentage of all possible point mutations for a protein ( the mutation space ) is calculated for each gene and the question ' how much mutation data is enough ? ' is raised . 
Mutations and human diseases
None
Haemoglobin-beta
HPRT1
CFTR
TP53
Interpreting the data
Acknowledgements