as a part of a large sequencing project in iceland ( methods summary ) , we sequenced 78 trios , a total of 219 distinct individuals , to over 30x average coverage ( fig . forty - four of the probands ( offspring ) have autism spectrum disorder ( asd ) , and 21 are schizophrenic ( sz ) . the other thirteen probands were included for various reasons , including the construction of multi - generation families . in addition , 1859 other icelanders , treated as population samples , were whole genome sequenced ( all > 10x , 469 > 30x ) . ( i ) all variants that have likelihood ratio lik(ar)/lik(rr ) or lik(aa)/lik(rr ) > 10 , where r denotes the reference allele and a the alternative allele , in any of the 1859 population samples , were excluded . some recurrent mutations could have been filtered out , but the number should be small . the de novo mutation calls further satisfy the conditions that ( ii ) there are at least 16 quality reads for the proband at the mutated site , ( iii ) the likelihood ratio lik(ar)/lik(rr ) is above 10 , and ( iv ) for both parents the ratio lik(rr)/lik(ar ) is above 100 . further examination led us to apply additional filtering ( v ) by including only snps where the number of a allele calls is above 30% among the proband s quality sequence reads . this was considered necessary because there was an abnormally high number of putative mutation calls where , despite extremely high lik(ar)/lik(rr ) ratios for the proband , the fraction of a calls were low ( supplementary fig . 1 ) . applying ( v ) eliminated 1,285 candidate mutations ( supplementary information ) . with high coverage , the false negatives resulting from ( v ) is estimated to be a modest 2% ( supplementary information ) . after 3 more candidates were identified as false positives by sanger sequencing ( see section on validation below ) , a total of 4,933 de novo mutations , or an average of 63.2 per trio , were called . for the five trios where a child of the proband was also sequenced , parent of origin of each de novo mutation called was determined as follows . if the paternal haplotype of the proband was transmitted to his / her child , and the child also carries the mutation , then the mutation was considered to be paternal in origin . if the child carrying the paternal haplotype of the parent does not have the mutation , then it is inferred that the mutation is on the maternal chromosome of the proband . similar logic was applied when the child inherited the maternal haplotype of the proband . in the five trios , the average number of paternal and maternal mutations is 55.4 and 14.2 respectively ( table 1 ) . if mutations were purely random with no systematic difference between trios , their number should be poisson distributed with the variance equal to the mean . this is much more striking for the paternal mutations ( variance = 428.8 , p = 1.2 10 ) than the maternal mutations ( variance = 48.7 , p = 0.016 ) . moreover , the number of paternal mutations has a monotonic relationship with the father s age at conception of the child . here , the mean number of paternal mutations is substantially higher than the mean number of maternal mutations ( ratio = 3.9 ) , but the difference is even greater for the variance ( ratio = 8.8 ) . hence , variation of de novo mutation counts in these individuals is mostly driven by the paternal mutations . relationships between parents age and the number of mutations ( paternal and maternal combined as they could not be reliably separated without data from a grandchild ) were examined using all 78 trios ( fig . the number of mutations increases with father s age ( p = 3.610 ) with an estimated effect of 2.01 mutations per year ( standard error ( se ) = 0.17 ) . mother s age is substantially correlated with father s age ( r = 0.83 ) and , not surprisingly , is also associated with the number of de novo mutations ( p = 1.910 ) . however , when father s age and mother s age were entered jointly in a multiple regression , father s age remained highly significant ( p = 3.310 ) , but not mother s age ( p = 0.49 ) . based on existing knowledge about the mutational mechanisms in sperm and eggs , the results support the notion that the increase in mutations with parental age manifests itself mostly , maybe entirely , on the paternally inherited chromosome . given a particular mutation rate , due to random variation , the number of actual mutations is expected to have a poisson distribution . after taking poisson variation into account , with a linear fit ( effect = 2.01 mutations per year ) , father s age explains 94.0% ( 90% confidence interval ( ci ) : [ 80.1% , 100% ] ) ( supplementary information ) of the remaining variation in the observed mutation counts . when an exponential model is fitted ( red curve in fig . 2 ) , the number of paternal and maternal mutations combined is estimated to increase by 3.23% per year . this model explains 96.6% ( 90% ci : [ 83.2% , 100% ] ) of the remaining variation . a third model fitted ( blue curve in fig . 2 ) assumes that the maternal mutation rate is a constant at 14.2 and paternal mutations increase exponentially . this explains 97.1% ( 90% ci : [ 84.3% , 100% ] ) of the remaining variation and the rate of paternal mutations is estimated to increase by 4.28% per year , which corresponds to doubling every 16.5 years and increasing by 8 fold in 50 years . seventy six of the 78 trios have father s age between 18 and 40.5 , a range where the differences between the 3 models are modest . hence , while it appears that the number of paternal de novo mutations increases at a rate that accelerates with father s age , more data at the upper age range are needed to better evaluate the nature of the acceleration . among the de novo mutations originally called , two were observed twice , both in siblings , one on chromosome 6 and one on chromosome 10 . the one on chromosome 6 is not actually de novo as it was seen in the mother also . the one on chromosome 10 was confirmed , i.e. it was observed in both siblings , who share the paternal haplotype in this region , but not the parents . this supports the theory that de novo mutations in different sperms of a man are not entirely independent . our trios include seven sibpairs with 921 de novo mutations called . a false de novo mutation call for one sib resulting from a missed call in the parent would show up in the other sib also about 50% of the time . only one such false positive was detected indicates that this type of error accounts for a small percentage ( 2/(920/2 ) = 0.43% ) of the called mutations . to evaluate the overall number of false positives , 111 called de novo mutations were randomly selected for sanger sequencing . six did not produce results of good quality in at least one member of the corresponding trio ( supplementary information ) . for the remaining 94 cases , 93 were confirmed as de novo mutations , i.e. the mutated allele was observed in the proband but not in the parents . one false positive , where the putative mutation was not observed in the proband , was identified . the 17 cases that could not be verified are more likely to be located in genomic regions that are more difficult to analyse and hence probably have higher false - positive rates than average . even so , the overall false - positive rate for the de novo mutation calls can not be high . false positives that are poisson distributed , while adding noise , would not create bias for the effect estimates in either the linear or exponential models for father s age , nor would they bias the estimate of the fraction of variance explained after accounting for poisson variation . in general , they do not create substantial bias for analyses of differences and ratios . however , if the variance of the false positives is higher than the mean , resulting from systematic effects that affect trios differently , such as dna quality and library construction , it would increase the unexplained variance and reduce the fraction of variance explained by father s age . the candidates filtered out by criterion ( v ) , if kept , would have introduced false positives of this kind ( supplementary information ) . since father s age explains such a high fraction of the systematic variance of the currently called de novo mutations , false positives with this property can not be common . consistent with other epidemiological studies , in iceland , the risk of schizophrenia increases significantly with father s age at conception ( n = 569 , p = 210 ) . the observed effect is limited to non - familial cases ( n = 631 , p = 5.410 ) , defined as those whose closest asd relative is farther than cousins . the epidemiological results , the effect of father s age on de novo mutation rate shown here , together with other studies that have linked de novo mutations to autism and schizophrenia , including three recent studies of autism through exome sequencing , all point to the possibility that , as a man ages , the number of de novo mutations in his sperm increases , and the chance that a child would carry a deleterious mutation ( not necessarily limited to snp mutations ) that could lead to autism or schizophrenia increases proportionally . however , this scenario does not imply that the relationship observed here between mutation rate and father s age would have been much different if the probands studied were chosen to be all non - asd / sz cases instead . for example , assume that autism / schizophrenia is in each case caused by only one de novo mutation . then autism / schizophrenia cases would on average have more de novo mutations than population samples . the magnitude could be substantial if the distribution of father s age has a large spread in the population , but then most of the difference would be due to the cases having older fathers . if we control for the age of the father at the conception of the individual , this difference in the average number of de novo mutations between autistics / schizophrenics and controls would be reduced to approximately one ( supplementary information ) . examination of the 4933 de novo mutations revealed that 73 are exonic , including two stopgain snp and 60 nonsynonymous snp ( supplementary table 2 ) . one non - familial schizophrenic proband carries a de novo stopgain mutation ( p.arg113x ) in the gene nrxn1 , previously associated with schizophrenia . one non - familial autistic proband has a stopgain de novo mutation ( p.r546x ) in the cullin 3 gene ( cul3 ) . de novo loss of function mutations in cul3 have been reported to cause hypertension and electrolyte abnormalities . recently , a separate stopgain de novo mutation ( p.e246x ) in cul3 was reported in an autistic case . another one of our mutations is a nonsynonymous variant ( p.g900s ) two bases from a splice site in the eph receptor b2 ( ephb2 ) , a gene implicated in the development of the nervous system . a de novo stopgain mutation ( p.q858x ) in this gene has recently been described in another autistic case . given the small number of loss of function de novo mutations we and others have reported ( approximately 70 genes in the three autism exome scans ) , the overlap is unlikely to be a coincidence . hence , cul3 and ephb2 can be added to the list of genes that are relevant for asd . effective genome coverage , computed by discounting regions that have either very low ( <  genome average ) or very high ( > 3 times genome average ) local coverage , the latter often a symptom of misaligning reads , was estimated to be 2.63 billion basepairs ( supplementary information ) . from that , 4933 mutations correspond to a germline mutation rate of 1.2010 per nucleotide per generation , falling within the range between 1.110 and 3.810 previously reported . tables 2 and 3 summarize the nature of the de novo mutations with respect to sequence context . moreover , there is clear difference between mutation rates at cpg and non - cpg sites . cpg dinucleotides are known to be mutational hotspots in mammals , ostensibly because spontaneous oxidative deamination of methylated cytosines leads to an increase in transition mutations . the observed rate of transitions here is 18.2 times that at non - cpg sites , somewhat higher but not inconsistent with previous estimates of 13.3 ( ref . ) and 15.4 ( ref . ) . transversion rate is also higher at cpg sites , 2.55 fold that at non - cpg sites . most of this increased transversion rate at cpg sites is presumably due to general mutation bias favouring mutations that decrease g+c content . the rate of mutations that change a strong ( g : c ) basepair to a weak one ( a : t ) is 2.15 times higher than mutations in the opposite direction . this mutational pressure in the direction of a+t is observed for both transitions ( ratio = 2.24 ) and transversions ( ratio = 1.82 ) and can not be solely explained by cpg mutations . father s age does not appear to affect the ratios between the rates of these different classes of mutations , i.e. as a man ages rates of all mutation types increase by a similar factor . the average number of mutations for each chromosome separately and the effect of father s age are displayed in fig . 3 . the effect of father s age is significant ( p < 0.05 ) for 14 of the 22 chromosomes when evaluated individually . the solid line in the figure corresponds to a model where the linear effect of father s age is proportional to the mean number of mutations on the chromosome , or that father s age has a uniform multiplicative effect across the chromosomes . all 22 95% cis overlap the line , indicating that the results are consistent with the model . recombination rate is higher for women than men , and children of older mothers have more maternal recombinations that those of young mothers . however , men transmit a much higher number of mutations to their children than women . furthermore , even though our data also show some over dispersion in the number of maternal de novo mutations , it is the age of the father that is the dominant factor in determining the number of de novo mutations in the child . seeing an association between father s age and mutation rate is not surprising , but the large linear effect of over two additional mutations per year , or the estimated exponential effect of paternal mutations doubling every 16.5 years , is striking . even more so is the fraction of the variation it explains , which limits the possible contribution by other factors , such as the environment and the genetic and non - genetic differences between individuals , to mutation rate on a population level . given the results , it may no longer be meaningful to discuss average mutation rate in a population without consideration of father s age . also , even though factors other than father s age do not appear to contribute substantially to the mutation rate diversity in our data , it does not mean that hazardous environmental conditions could not cause a meaningful increase in mutation rate . rather , the results indicate that , to estimate such an effect for a specific incident , it is crucial to take the father s age into account . it is well known that demographic characteristics shape the evolution of the gene pool through the forces of genetic drift , gene flow and natural selection . with the results here , it is now clear that demographic transitions that affect the age at which males reproduce can also have a considerable impact on the rate of genomic change through mutation . recent transition of icelanders from a rural agricultural to an urban industrial way of life , which engendered a rapid and sequential drop in the average age of fathers at conception from 34.9 years in 1900 to 27.9 years in 1980 , followed by an equally swift climb back to 33.0 years in 2011 , primarily due to the impact of higher education and the increased use of contraception ( fig 4 ) . based on the fitted linear model , while individuals born in 1900 carried on average 73.7 de novo mutations , those born in 1980 carried on average only 59.7 such mutations ( a decrease of 19.1% ) , while the mutational load of individuals born in 2011 has increased by 17.2% to 69.9 . demographic change of this kind and magnitude is not unique to iceland , and it raises the question of whether the reported increase in asd diagnosis lately is at least partially due to an increase in the average age of fathers at conception . also , the observations here are likely to have important implications for the use of genetic variation to estimate divergence times between species or populations , since the mutation rate can not be treated as a constant scaling factor , but rather must be considered along with the paternal generation interval as a time - dependent variable . whole genome sequence data for this study were generated using the illumina gallx and hiseq2000 instruments . the sequencing reads were aligned to the hg18 reference genome with bwa and duplicates were marked with picard [ http://picard.sourceforge.net/ ] . quality score recalibration , indel realignment and snp / indel discovery was then performed on each sample separately , using gatk 1.2 ( ref . ) . likelihoods presented are based on the normalized phred - scaled likelihoods that are calculated by the gatk variant calling . statistical analysis was performed in part using the r statistical package . estimates and confidence intervals for the fraction of variance explained after accounting for poisson variation were calculated using monte carlo simulations ( supplementary information ) . variants were annotated using snp effect predictor ( snpeff2.0.5 , database hg36.5 ) and genome analysis toolkit 1.4 - 9-g1f1233b with only the highest - impact effect ( cingolani , p.  snpeff : variant effect prediction  , http://snpeff.sourceforge.net , 2012 ) .
mutations generate sequence diversity and provide a substrate for selection . 
 the rate of de novo mutations is therefore of major importance to evolution . 
 we conducted a study of genomewide mutation rate by sequencing the entire genomes of 78 icelandic parent - offspring trios at high coverage . here 
 we show that in our samples , with an average father s age of 29.7 , the average de novo mutation rate is 1.20108 per nucleotide per generation . 
 most strikingly , the diversity in mutation rate of single - nucleotide polymorphism ( snp ) is dominated by the age of the father at conception of the child . 
 the effect is an increase of about 2 mutations per year . 
 after accounting for random poisson variation , father s age is estimated to explain nearly all of the remaining variation in the de novo mutation counts . 
 these observations shed light on the importance of the father s age on the risk of diseases such as schizophrenia and autism .