genomes vary from one another in multifarious ways , and the totality of this genetic variation underpins the heritability of human traits . various recent studies show the landscape of genetic variation and allow estimation of the relative contributions of sequence ( base substitutions ) and structural variation ( indels , insertions , or deletions ; copy - number variations [ cnvs ] , and inversions ) . among dna sequence variations in the human genome , cnvs directly contribute to gene expression changes through gene dosage effects ; the distribution of cnvs is now considered to be wider than previously thought [ 16 ] . genome resequencing studies have shown that cnvs , involving the gain or loss of several hundreds of bases to several hundred kilobases ( kb ) of the genome , can be an important source of genetic variation among human populations of different ethnic groups as well as among individuals . following the development of methodologies and the introduction of new research platforms [ 712 ] , information regarding the nature and pattern of cnvs from representative populations has been accumulated . the functional impact of cnvs has been demonstrated across the full range of biology , from cellular phenotypes , such as gene expression , to all classes of human disease with an underlying genetic basis : sporadic , mendelian , complex , and infectious ( reviewed in ) . clinical geneticists need to discriminate pathogenic from benign cnvs in their patients and have made extensive use of data from cnv surveys of apparently healthy individuals . the mere presence or absence of a variant in such control data sets is only partially informative , as determination of the pathogenicity of inherited cnvs is at present limited by a lack of information on cnv frequency and combinations in apparently healthy individuals . based on these considerations , examinations of a relatively large number of individuals from various specific ethnic groups have recently been conducted using different array platforms , such as bac arrays [ 6 , 17 , 18 ] , oligo arrays [ 1921 ] , and others . the results are not always consistent , and it is likely that different human populations bear different inherited cnvs . the numbers of japanese individuals examined to date are smaller than those in studies of other ethnicities . polymorphic cnvs have received considerable attention since they might play an important role in the etiology of common diseases . we summarized the available data by focusing on highly polymorphic cnvs ( in 5.0% of the individuals ) in a previous paper . however , rare variations have recently received attention from scientists who espouse a hypothesis called  common disease and rare variants ,  in contrast to the  common disease - common variants  hypothesis that underpins most genome - wide association studies . in this paper , we focus on cnvs observed at relatively low frequency ( < 5.0% of the individuals ) in a population residing in hiroshima and nagasaki , japan , using cgh with bac clones as targets . the study was conducted in two stages : stage ( 1 ) 80 unrelated japanese individuals were examined by bac - acgh with an array having 2,241 bac clones ; stage ( 2 ) 133 unrelated japanese individuals were examined by bac - acgh containing 2,622 bac clones . the majority of the clones used in stage 1 of this study were selected from the set of cytogenetically mapped p1 artificial chromosome ( pac ) clones and bacterial artificial chromosome ( bac ) clones reported by the bac resource consortium   and obtained from either the children 's hospital oakland research institute ( oakland , ca , usa ) or from invitrogen inc . , co. ( carlsbad , ca , usa ) . in stage 2 , together with the bac clones in stage 1 , additional 381 bac clones were used , a majority of which were collaboratively obtained from dr . a total of 2,241 clones of chromosomal fragments from chromosome 1 to chromosome 22 were used in stage 1 and 2,622 clones in stage 2 . the clones were distributed every 1.2 mb across all human autosomes in stage 1 and every 1.1 mb in stage 2 . in addition to autosomal clones , four kinds of x - chromosomal clones were used as internal references . with respect to the examinations in stage 1 , three sets of arrays the genomic dna samples used in this study were reported in a previous study [ 23 , 27 ] . in brief , the dna used for the population studies and the family studies was extracted from lymphoblastoid cell lines obtained from the offspring of atomic - bomb survivors and their family members . lymphoblastoid cell lines were derived from a cryopreserved archive of families consisting of father , mother , and offspring from hiroshima and nagasaki for whom permanent cell lines have been established by epstein - barr ( eb ) virus transformation . since the offspring in some cases included siblings , we selected one representative offspring to create a group of  unrelated individuals ,  thereby avoiding any duplication of cnvs from families containing two or more siblings , in accordance with the rules described in a previous report . in this study , 80 individuals ( 40 from each city ) were included in examinations in stage 1 and 133 ( 84 from hiroshima , 49 from nagasaki ) in stage 2 . the arrays were prepared as described in the previous paper . in brief , with respect to stage 1 , cloned dna was digested by noti . on the other hand , in stage 2 , cloned dna was digested with msei , and the fragmented dnas were amplified by ligation - mediated pcr carried out as described by snijders et al . . in both stages , the target dnas ( 0.5  g/l ) were dissolved in 50% dimethylsuloxide and printed in triplicate onto the glass slides ( matsunami glass co. ltd . ) using the affymetrix 417 arrayer ( affymetrix ) . the screenings of both stages were conducted following the procedures described previously . in brief , for labeling dna , test and reference genomic dna ( 1.25  g each ) was cut by bamhi and labeled by a random - priming method with cyanine-5- and cyanine-3-labeled dutp ( cy5- and cy3-dutp ; perkinelmer life sciences , wellesley , ma , usa ) . prehybridization was conducted to block repetitive sequence binding of target dna on the arrays and to prevent nonspecific binding of probe dna to the targets . following the initial incubation , the prehybridization solution was removed , and hybridization solution with cy - labeled dna ( prepared as described above ) was added . all of the procedures were conducted using the genetac hybridization station ( genomic solutions inc . after washing procedures , fluorescent images of the hybridized arrays were obtained using a scanarray 5000 confocal laser scanner ( perkinelmer life sciences ) . , fairfax , va , usa ) in stage 1 and gene pix ( axon instruments , sunnyvale , ca ) in stage 2 were used to quantify the fluorescence of each spot on the array images . the population studies were conducted in accordance with accepted procedures as described in the previous paper . in brief , hybridization was performed as follows : mixtures of ( a ) cy5-labeled reference dna and cy3-test dna , and ( b ) inverse labeling with cy3-reference dna and cy5-test dna were applied to the slides . the two complementary hybridizations ( i.e. , ( a ) and ( b ) ) were conducted for each individual . the variations in a given sample from a single individual that were identified consistently in both complementary hybridizations ( ( a ) and ( b ) ) were regarded as putative cnvs . since only dna segments detected as putative cnvs in two individuals or more were regarded as true cnvs , further analyses were not conducted . on the contrary , when cnvs were detected in only one individual , all were confirmed with repeated examinations using arrays . the qpcr procedure was performed using sybr premix ex taq ( takara - bio , ohtsu , japan ) and the light cycler system ( roche diagnostics japan , tokyo , japan ) , according to the procedures described in the previous paper . the results were analyzed with light cycler data analysis software using a second derivative maximum model . the relative copy number of each site was normalized using quantity of the amplified segment of a portion of a different chromosome ( chromosome 16:7888854178888690 ; this position empirically showed the smallest deviation among many samples in our experiments ) as a standard ( relative copy number = 2 ) and was indicated as mean  sd ( n = 3 ) . for the cnvs identified in only one offspring , further examinations were carried out for confirmation of inheritance . the family studies using qpcr for the cnvs were conducted using dnas from the mother , the father and sibling of offspring , if available . the main purpose of this paper is to report the data accumulated about cnvs found in < 5.0% of the individuals . the results obtained from the population studies are shown in tables 1 and 2 . in table 1 , the cnvs identified in two individuals or more are summarized . on the other hand , in table 2 , a total of 126 cnvs were identified at 52 different bac regions in the genome . the cnvs observed at 27 of 52 bac regions were found in only one unrelated individual , and all of them were confirmed by repetitive analysis using the same array system as in the population studies . the family studies by qpcr were conducted for 23 of 27 cnvs , and the results revealed that those 23 cnvs were inherited from one of the parents . the inheritance of cnvs from the remaining two offsprings was not confirmed , since dna from the parents was not obtained . on the other hand , the cnvs on two bac clones ( rp11 - 88b18 , rp11 - 86b6 ) were identified in only one unrelated individual . in fact , however , they were observed in individuals who were not selected as the numbers of individuals who expressed the cnvs are described in the parentheses in table 2 . for that reason , we did not conduct family studies for those cnvs . as typical examples of family studies , the results of qpcr carried out for the cnvs observed in three bac - clone regions are described in figure 1 ( rp11 - 81h5 ) , figure 2 ( rp11 - 90m13 ) , and figure 3 ( rp11 - 90o18 and rp11 - 90c3 ) . for the first case , a single 180,264  bps deletion was observed in an offspring . the bac - clone ( rp11 - 90m13 ) contains two genes , protein phosphatase 2 , regulatory subunit b , alpha ( ppp2r2a ) and early b - cell factor 2 ( ebf2 ) . the deletion - type cnv was confirmed by qpcr , and the cnv was found to be inherited from the father . the deletion covered part of intron 7 of ppp2r2a and the promoter region of ebf2 . for the last case , as shown in figure 3 , the cnv was covered by two bac clones ( rp11 - 90o18 and rp11 - 90c3 ) . this result was revealed by qpcr , and the cnv was found to be inherited from the father . the cnv contained the series of zinc finger protein families ( znf 107 , 138 , 273 , 117 , 92 ) . many segmental duplications have already been summarized in public databases , such as the database of genomic variants ( dgv database ; http://projects.tcag.ca/variation/ ) , ucsc human genome browser ( ucsc database . when the cnvs reported in the databases or papers were too small to be detected by our bac - acgh methods , we assumed that they were different from our cnvs . the cnv data obtained in our studies , including an indication of the presence or absence of cnvs already reported in other databases , are summarized in tables 1 and 2 . compared to caucasian populations , very little information about cnvs in japanese populations has been systematically screened . we compared our data with the data of japanese populations from the hapmap project as reported by redon et al . , in addition to the data by bac array , three groups , including redon et al . , reported the data of japanese populations based on examinations conducted using oligo - array methods , although each group used a different array platform [ 24 , 25 ] . these results are also summarized in tables 1 and 2 . in the case where a multiple number of cnvs were identified in our study ( table 1 ) , moreover , regarding one individual , the cnvs identified in nine of 27 bac regions were reported in the data reported by the others , although we should emphasize again that the cnvs identified in our bac region are not exactly the same as those reported by the others . the majority of cnvs identified in our study tend not to be identified in the other reports . we summarized some of the genes and disease - related genes that overlap with the bac - clone region for our cnvs ( table 3 ) . in addition , mrnas also have been reported in the databases , but they are too numerous to describe here . all bac - clone regions contained at least one mrna , although the functions of a majority of the mrnas are not yet known ( data not shown ) . we examined 213 unrelated japanese individuals using bac - acgh and found a total of 126 cnvs on 52 different bac regions in the genomes . the cnvs observed on 27 of the 52 bac - regions were found in only one unrelated individual . the family studies were conducted for 23 cnvs , and the results demonstrated that these were inherited from one of the parents . a fraction of the regions involved in the cnvs observed in our study ( i.e. , 34 of 126 or about 27% ; tables 1 and 2 ) were not reported previously in other studies listed in the database . thus , they are considered novel cnvs . in contrast , the data of highly polymorphic cnvs   demonstrated that only 8% ( 55 of 680 ) had not been reported . this result showed that a significantly large number of novel cnvs have been identified among relatively less frequent cnvs (  = 36.64 , p = 5.10  10 ) . this finding suggests that the structural rearrangements for creating less frequent cnvs are evolutionarily recent . we thus paid attention to differences in the number of cnvs identified in hiroshima compared to nagasaki . with respect to the cnvs observed multiple times , 12 of 25 were identified in one city . these cnvs appear to be so - called  private polymorphisms  occurring more recently than the others . on the other hand , quite a small number ( 21 of 126 ; about 17% ) of the cnvs in this report were found on the bac clones that overlapped with segmental duplication ( tables 1 and 2 ) . a majority ( about 63% ) of highly polymorphic cnvs , however , were observed on the bac - clone overlapping with segmental duplication ( see table 1 in ) . this result suggests that segmental duplication might play a significant role in the creation of highly polymorphic cnvs . these observations are supported by previous data from sharp et al . , who reported the sharing of cnvs among several populations , meaning that the specific genomic imbalances either predated the migration of modern humans from africa or arose independently in different populations . on the other hand , the data representing japanese populations are based on reports from three groups . we compared our cnv data to that of these other reports [ 4 , 24 , 25 ] . the cnvs found in 27 of 52 bac regions ( about 52% ) were not identified by the other groups . speculation as to the reason for this includes the possibility that since the cnvs were private polymorphisms as mentioned above , they were not identified in the japanese populations used in the other works . in humans , it is estimated that around 23% of currently known cnvs reside in known or putative genes . many cnvs that lie outside genic regions of the genome may still have a significant influence on gene expression by affecting gene regulatory elements . another important area of exploration is linkage disequilibrium ( ld ) , that is , nonrandom association between alleles at different loci , between cnvs and other genomic variants ( especially snps ) , which may provide important insight into the genomic evolution ( e.g. , the recombination patterns ) of cnvs [ 31 , 32 ] . however , studies of ld between cnvs and snps are hindered by the fact that cnvs generally reside in genomic regions associated with segmental duplications and/or repeat - rich regions , which are difficult to sequence and use for detection of snps . recent association studies have attempted to link cnvs with expression profiles , diseases , and known phenotypic differences . on the other hand , distribution of cnvs differs depending on genetic background , and population - specific cnvs might account for the divergence of some physiological traits and disease prevalence among populations . like snps , cnvs also show variability among populations ; therefore , comprehensive cnv information is needed when applying cnvs as genetic markers in disease or trait studies . for the reasons cited above , we made plans to accumulate japanese data in which we provide information about the relation between japanese cnv data and phenotypic differences correlated with reported diseases . in our previous paper , we summarized such data by focusing on highly polymorphic cnvs found in 5.0% of the individuals . more recent studies based on advanced molecular technologies , such as genome - wide association studies [ 33 , 34 ] and next generation sequencing [ 35 , 36 ] , reported that many genes appear to play important roles in the etiology of common diseases . depending on newly developed technologies , rare variations have recently received attention from scientists who espouse a hypothesis called  common disease and rare variants . in this paper , we summarized cnvs that were identified in fewer than 10 individuals in our previous population study . we focused on genes reported in the dgv , ucsc , and ncbi databases ( table 3 ) , although many mrnas were also listed in them . since these genes are good candidate markers for enabling us to examine the etiology of common diseases and phenotypical heterogeneities among individuals , our cnvs have the potential to become useful markers in future studies . in this study , 34 cnvs were new , indicating that cnv coverage of the human genome is still incomplete and that there is diversity between the japanese and other ethnic populations . moreover , the cnvs found in about two - thirds of the bac regions examined in our study were not identified in the studies in which three groups examine japanese populations using different array platforms . the newly identified cnvs extend the coverage of cnvs in the human genome ( also the general japanese population ) . moreover , it is expected that the cnvs could now be taken into consideration when genetic studies , for example , cnv association studies , are conducted .
copy - number variations ( cnvs ) may contribute to genetic variation in humans . 
 reports regarding existence and characteristics of cnvs in a large apparently healthy japanese cohort are quite limited . 
 we report the data from a screening of 213 unrelated japanese individuals using comparative genomic hybridization based on a bacterial artificial chromosome microarray ( bac acgh ) . in a previous paper 
 , we summarized the data by focusing on highly polymorphic cnvs ( in 5.0 % of the individuals ) . 
 however , rare variations have recently received attention from scientists who espouse a hypothesis called  common disease and rare variants .  here , we report cnvs identified in fewer than 10 individuals in our study population . 
 we found a total of 126  cnvs at 52 different bac regions in the genome . 
 the cnvs observed at 27 of the 52 bac - regions were found in only one unrelated individual . the majority of cnvs found in this study 
 were not identified in the japanese who were examined in the other studies . 
 family studies were conducted , and the results demonstrated that the cnvs were inherited from one parent in the families .