segmental copy - number variations ( 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 . molecular genetics analyses and cytogenetic analyses have provided significant information about these variations in the human genome , specifically as they relate to disease , such as cancer , and to congenital malformation ( see [ 1 , 2 ] , and review in ) . following the development of methodologies and the introduction of new research platforms [ 49 ] , information regarding the nature and pattern of cnvs from representative populations have accumulated . 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 [ 1013 ] , oligo - arrays [ 1416 ] , and others . the results are not always consistent and it is likely that different human populations bear different cnvs . the numbers of japanese individuals examined to date are not so large compared to the studies for other ethnicities . polymorphic cnvs have received considerable attention since they might play an important role in the etiology of common diseases . therefore , more data regarding cnvs should be accumulated from japanese populations . in this report , we focus on cnvs which were observed at a high frequency ( 5.0% of the individuals ) in the population residing in hiroshima and nagasaki , japan by acgh with bac - clones as targets . in the study , the population studies were conducted at two stages : stage ( 1 ) : 80 unrelated japanese individuals were examined using bac - acgh with an array having 2,241 bac clones , and stage ( 2 ) : 133 unrelated japanese individuals were examined using bac - acgh that contained 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 ) , in addition to bac clones used in stage ( 1 ) , an additional 381 bac clones were used , a majority of which were collaboratively obtained from dr . n. matsumoto of yokohama city university . the 2,241 clones of chromosomal fragments from chromosome 1 to chromosome 22 were used in stage ( 1 ) and 2,622 clones were used in stage ( 2 ) , respectively . that is , the additional 381 bac clones were examined for only 133 unrelated individuals in stage ( 2 ) . those clones are distributed every 1.2  mb across all of the human autosomes in stage ( 1 ) , and 1.1  mb in stage ( 2 ) , respectively . in addition to autosomal clones , four kinds of x - chromosomal clones were used as internal references . with respect to examination for stage ( 1 ) , three sets of arrays were constructed and imprinted : slide no.1 , consisted of 698 clones on chromosomes 1 to 4 ; slide no.2 consisted of 718 clones on chromosomes 5 to 10 , plus two bac clones on chromosome 3 , and six clones on chromosome 4 ; and slide no.3 consisted of 817 clones on chromosomes 11 to 22 . for stage ( 2 ) , all of the clones were printed onto one glass slid . the genomic dna samples used in this study were principally the same as those used in a previous study . the dna samples used for reference purposes were extracted from mononuclear cells of two physically and clinically normal volunteers ( a 57-year - old japanese male and a 54-year - old japanese female ) . the dna used for testing and analyses of this population was extracted from lymphoblastoid cell lines obtained from the offspring of atomic - bomb survivors . high molecular weight genomic dna was isolated using conventional methods as described in detail elsewhere . lymphoblastoid cell lines were derived from a cryopreserved archive of approximately 1000 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 of peripheral b - lymphocytes . the composition of the families has been reported elsewhere . three hundred five offspring were initially screened . since the offspring include some siblings , we selected one representative offspring to construct  unrelated individuals  to avoid double counting of polymorphic cnvs from families containing two or more siblings . we selected the offspring who first visited our institution for donating blood rather than other siblings . the 213 offspring selected as unrelated individuals included 124 offspring from hiroshima and 89 from nagasaki . cloned dnas for microarray targets were isolated from bacterial cultures using nucleobond bac 100 ( nippon genetics , tokyo ) . with respect to stage ( 1 ) , dna was digested by noti followed by phenol - chloroform - isoamyl alcohol ( 25:24:1 ) extraction and ethanol - precipitation . on the other hand , in stage ( 2 ) , cloned dna was digested with msei followed by phenol - chloroform - isoamyl alcohol ( 25:24:1 ) extraction and ethanol precipitation . the fragmented dnas were amplified by ligation - mediated pcr carried out as described by snijders et al . . 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 ) . the labeled probes were mixed and centrifuged with microcon column ( millipore co. , bedford , ma , usa ) to purify the probes . subsequently , human coti dna ( 120  g ; roche diagnostic gmbh , mannheim , germany ) was added to the column , and recentrifuged . after the volume of the mixture became less than 20  l , it was transferred to microtubes with 100  l of hybridization solution ( 50% formamide , 10% dextran sulfate , 1% tween 20 , 2  ssc , 10  mm tris - hcl [ ph 7.4 ] and 800  g of yeast t - rna [ invitrogen , carlsbad , ca , usa ] ) . the hybridization mixture was then denatured at 70c for 10 minutes , and subsequently incubated at 37c for at least five hours to block repetitive sequences of the labeled probes . prehybridization was conducted in order 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 ( overnight at 37c ) , the prehybridization solution was removed , and fresh hybridization solution with cy - labeled dna ( prepared as described above ) was added . again , hybridization processes were carried out . all of the procedures were conducted using the genetac hybridization station ( genomic solutions inc . , ann arbor , mi , usa ) . 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 ) , respectively , were used to quantify the fluorescence of each spot on the array images . fluorescent ratios of the total integrated cy3 and cy5 intensities for each target ( triplicate spots for each target ) were calculated , along with the mean ratios of the triplicate spots ( if the raw ratio of one of the triplicate spots differed by more than 10% from the other two , the value was automatically excluded from the mean ratio calculation ) . normalized ratios were computed by dividing each raw ratio by the mean raw ratios of every autosomally mapped target ( therefore , after normalization , the averaged ratio for all targets in one array was 1.0 ) . the spots whose ratios were 2.58 standard deviations ( sd ) below the mean ( 1.0 ) , or 2.58 sd above the mean , were marked  cnv .  to confirm the quality of each analysis , the ratio of x - linked clones was verified . dna in the plugs was cleaved by restriction enzymes ( paci , or sse8387i ) . the resulting fragments were then separated by pulse field gel electrophoreses ( pfge ) on 1% pulse field certified agarose ( biorad , hercules , ca , usa ) with  0.5 tbe ( tris - borate - ethylenediaminetetraacetic acid ) . using the chef - dr ii system ( biorad ) , electrophoresis was carried out using 6  v / cm at 14c for 22  hrs . the angle of pulse was 120. the switch time was used : ramped from 0.3 seconds to 15 seconds . southern blot analyses were carried out using conventional , well - described procedures . in brief , after completion of pfge , the dna in the gel was cleaved by uv irradiation and blotted onto nitrocellulose filters ( schleiche & schuell , dassel , germany ) . the filters were prehybridized with human coti dna ( 48  g / ml , roche diagnostic gmbh ) and salmon testis dna ( 14  g / ml , sigma - aldrich ) to decrease the background due to repetitive sequences . subsequently , the filters were hybridized with whole bac - dna as a probe . dna probes were labeled with [ -32p ] dctp ( amersham biosciences , piscataway , nj , usa ) and preannealed with human coti dna and salmon testis dna ( 10.5  g / ml ) . prehybridization and hybridization were performed overnight at 37c in a solution containing 50% formamide , 10% dextran sulfate , 1% tween 20 , 2  ssc , and 10 mm tris - hcl ( ph 7.4 ) . after hybridization , the filters were washed at 65c with 1.0  ssc containing 0.1% sds ( sodium dodecyl sulfate ) and 0.5  ssc containing 0.1% sds . banding patterns were obtained by either exposure to x - ray film ( fuji film , tokyo , japan ) or through use of the molecular imager fx ( biorad ) . the qpcr was performed using sybr premix ex taq ( takara - bio ) and the light cycler system ( roche diagnostics ) , according to the manufacturers ' protocols . primers were designed with primer3 software ( http://primer3.sourceforge.net ) , and the size of pcr products was confirmed by the pattern of restriction enzyme digested fragments using the labchip dna 500 kit on the 2100 bioanalyzer ( agilent technologies , waldbronn , germany ) . two , 0.5 , and 0.125  ng of genome dna from an individual with cnv were used and the quantification of each amplicon was carried out at 45 cycles of pcr . the results were analyzed with light cycler data analysis software using a second derivative maximum model . the main purpose of this paper is to report the accumulation of the data about highly polymorphic cnvs found in 5.0% of the individuals . ( the number of cnvs was 11 or more in each bac - spot . ) as shown in table 1 , 680 polymorphic cnvs were observed on 16 bac - regions . as described before , the results of two bac ( rp11 - 259n12 and rp11 - 121a8 ) were obtained from 133 unrelated individuals examined in stage ( 2 ) . southern blot analyses followed by pfge were carried out for the highly polymorphic cnvs . as shown by the typical cases in figures 1 and 2 , the patterns of these two bac clones ( rp11 - 79f15 and rp11 - 88l18 ) are shown in figures 1 ( rp11 - 79f15 ) and 2 ( rp11 - 88l18 ) as the typical examples . since each individual contained a different number of core segmental duplication units , each individual showed bands having different motilities . the results of qpcr conducted for two bac clone regions are described in figure 3 ( rp11 - 89b15 ) and figure 4 ( rp11 - 79o18 ) . for the former case , a part of a gene ( meox2 ) was deleted . on the contrary , for the latter case , the copy number of a part of the gene ( nsf ) increased , but the copy number of the gene ( wnt3 ) did not change . there are many segmental duplications which have already been summarized in a public data base , such as human genome segmental duplication database ( tcag database ; http://projects.tcag.ca/cgi-bin/variation/gbrowse ) , ucsc human genome browser ( ucsc database ; http:/genome.ucsc.edu / index.html ) , and ncbi map viewer ( ncbi database ; http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi ) . the cnv data obtained in our studies are summarized in table 1 in addition to the presence or absence of cnvs already reported in the databases . one bac clone , named  rp11 - 115g22 ,  was mapped on two chromosomes , 6 and 15 in tcag database , so it is likely that this clone is present on two discrete chromosomes . on the contrary , however , we accept the reports from the latter two databases and described that this clone was mapped on only chromosome no.15 . with respect to segmental duplications , 10 out of 16 ( about 63% ) were present in the above databases ( ucsc database , and ncbi database ) . it was noteworthy that the majority of bac clones containing our cnvs were known to overlap to at least one cnv reported in the database . however , that does not mean that our highly polymorphic cnvs are exactly the same as those reported in previous reports , since precise comparisons between our study and the other studies were not carried out . as mentioned before , there was very little information about the cnvs of 45 japanese individuals for which relatively large sizes of population have been systematically screened . we compared our data with the data of japanese including the hapmap project as reported by redon et al . ten out of 16 cnvs identified in our study were reported in the data reported by redon et al . , although we should emphasize again that the cnvs identified in our bac region are not exactly the same as those reported by redon et al . . cnvs identified in our study with lower numbers tend to not be identified in redon 's report . on the other hand , when our data are compared with redon 's oligo - data from affymetrix 500ea array conducted for japanese , only two cnvs were overlapped to our cnvs . as redon et al . mentioned in their report , the reason appears to be that oligo arrays have some limitations for a complicated genome , such as segmental duplication areas . we summarized the genes and disease - related genes in omim which overlap to the bac - clone region with our cnvs ( table 2 ) . in addition to those two categories , mrnas have been also reported in the database , but they are too many to describe here . all bac - clone regions contained at least one mrna , although the functions of a majority of those mrnas are not known yet ( data not shown ) . we examined 213 unrelated japanese using bac - acgh and found a total of 680 cnvs on 16 bac clones . a large fraction of the regions involved in the cnvs observed in our study ( i.e. , 625 out of 680 ( 92% ) , table 1 ) have been reported previously in other studies listed in the database . a majority (  63% ) of the cnvs had been found on the bac clones that overlapped with segmental duplication , suggesting the notion that segmental duplication might play a significant role in the creation of cnvs ( table 1 ) . in which they reported the sharing of cnvs among several populations , meaning those specific genomic imbalances either predated the dispersal of modern humans out of africa or arose independently in different populations . on the other hand , our cnvs , especially those showing high frequencies , were also identified by redon 's work . on the contrary , our cnvs showing low frequency , such as less than about 10% of individuals , were not observed in their work ( table 1 ) . that result suggests that those cnvs might be identified if the number of individuals examined by redon et al . were increased to the level of our study ( about 200 individuals ) . moreover , as described before , when our data compared with redon 's oligo - data conducted for japanese , only two of their cnvs were overlapped to our cnvs . although the oligo - based method tends to detect smaller cnvs ( about a few kilobases ) , this approach is less effective in tracking cnvs in genomic regions of complex structure , like segmental duplications , that are not sufficiently tagged by oligo targets . on the contrary , the bac platform can only identify larger cnvs ( > 40  kb ) , but this method has some advantage for detecting cnvs present in the regions of segmental duplications . the cnvs in the human genome are often associated with developmental disorders and susceptibility to diseases . large duplications and deletions have been known to be present within the human genome based initially on cytogenetic observations in the course of etiological studies of congenital malformations ( e.g. , [ 1 , 2 ] ) . the frequency of those duplications and deletions was presumed to be low and , for the most part , directly related to specific genetic disorders . a limited number of studies reported the presence of specific large duplications and deletions that were not apparently related to diseases ( e.g. , ) . reported that cgh on a bac - dna - based microarray could reliably detect single - copy gene decreases or increases from normal diploidy . following this , other array platforms , such as cdna , and oligo - nucleotide , have been developed and many data from them have been reported . as we mentioned before , many cnvs were reported to be closely related to disease phenotypes , and recent studies based on advanced molecular technologies , such as genomewide association studies [ 2729 ] and next generation sequencing [ 30 , 31 ] , reported that many genes appear to play important roles in the etiology of common diseases . we report our highly polymorphic cnvs in bac clones , which were reported to contain genes , expected to be related to phenotypic heterogeneity of each individual , based on the tcag database . we focused on genes reported in the above database , although many mrna were listed in the database in addition to genes ( table 2 ) . bac clone ( rp11 - 90a9 ) contains two genes : ankyrin repeat domain 34b ( ankrd34b ) and dihydrofolate reductase ( dhfr ) . a phosphoprotein encoded by ankrd34b is induced during bone marrow commitment to dendritic cells which play an important role in vertebrate immunity . dhfr genes were reported to be related to various malignancies including lymphoproliferative disorders such as systemic non - hodgkin 's lymphoma , primary central nervous system lymphoma ( pcnsl ) , and childhood acute lymphoblastic leukemia . those two genes are fully overlapped to the bac clone ( rp11 - 90a9 ) . it is likely that the cnvs might affect the copy number of those genes . one bac clone ( rp11 - 89b15 ) contains a gene mesenchyme homeobox 2 ( meox2 ) . the analyses by qpcr ( figure 3 ) demonstrated that the copy number of gene ( meox2 ) is deleted in the individual having cnvs detected by this bac clone . meox2 suppressed epithelial cell proliferation in cooperation with tgf - beta1 , and mediated induction of the cell - cycle inhibitor gene p21 . finally , the data from genome wide association study ( gwas ) reported that this is one of the candidate genes that might be associated with ischaemic stroke . another bac clone ( rp11 - 115g22 ) was mapped on the chromosomes 15 . ( chrna7 ) . that gene was used as a candidate target for examining interactions on the severity of adult attention deficit hyperactivity disorder ( adhd ) . moreover , it was reported that the gene is one of the candidates for alzheimer 's disease . the gene chrna7 is overlapped to the segmental duplication region of bac clone ( rp11 - 115g22 ) . many cnvs were listed in the databases mentioned as above , and those are overlapped to the gene . for those reasons , our cnv appears to be overlapped to the genes , and it might affect the copy number of gene chrna7 . a bac clone ( rp11 - 79o18 ) contains two genes which are n - ethylmaleimide - sensitive factor ( nsf ) and wingless - type mmtv integration site family , member 3 ( wnt3 ) . the qpcr results ( figure 4 ) demonstrated that the copy number of a gene ( nsf ) increased but no change was observed in the gene ( wnt3 ) . that result suggested that the cnv might affect the nsf gene , but not the wnt3 gene . however , there may be an opportunity for the cnvs to become a surrogate marker of wnt3 for the future association study between the cnv and some disease phenotype . the nsf gene is one of the essential components of membrane fusion machinery which is an important homeostatic process in eukaryotic cells . a recent study showed that the nsf gene is a good candidate marker for association studies for genetic risk underlying parkinson 's disease . the wnt3 gene 's single nucleotide polymorphisms ( snps ) were used as candidate markers for association studies of hemorrhagic stroke , hypertension , and chronic kidney disease . upregulation of the wnt gene family , including wnt3 , suggested involvement of the wnt 's canonical and/or noncanonical signaling pathway in chronic lymphocytic leukemia . a bac clone ( rp11 - 79f15 ) contains two genes : methyl - cpg binding domain protein 3-like 1 ( mbd3l1 ) and cell surface associated mucin 16 ( muc16 ) . the protein is localized to discrete areas in the nucleus , and expression appears to be restricted to round spermatids , suggesting that the protein plays a role in the postmeiotic stages of male germ cell development . on the other hand , muc16 is a member of the mucin gene family and encodes cancer antigen 125 ( ca125 ) which is a blood biomarker routinely used to monitor the progression of human epithelial ovarian cancer ( eoc ) , although its potential role in eoc is poorly understood . maintenance of an intact mucosal barrier , one of whose components is a gene product of muc16 , is critical to preventing damage and infection of wet - surfaced epithelia . as we demonstrated by southern blot analysis ( figure 1 ) , the polymorphism was caused by segmental duplications in the bac clone . the series of segmental duplications were on the 5-region of two genes ( mbd3l1 and muc16 ) . the cnvs do not affect the expression pattern of the genes , but these cnvs might be useful surrogate markers for future studies . glutamate receptor , ionotropic , ( ampa 2 ) .  ampas are ligand - activated cation channels that mediate the fast component of excitatory postsynaptic currents in neurons of the central nervous system . since the size of the gene ( ampa 2 ) is larger than that of the bac clone ( rp11 - 231j7 ) , it is likely that the cnvs affect the copy number of the gene . a bac clone ( ctd-2100f13 ) contains two genes : rio kinase 3 ( yeast ) ( riok3 ) and niemann - pick disease , type c1 ( npc1 ) . npc1 can have a function in the egress of certain membrane - impermeable lysosomal cargo . the membrane - bound npc1 and soluble npc2 play an important role for the release of cholesterol from lysosomes . as a result of that mechanism , the gene is associated with obesity [ 5658 ] . the bac clone ( ctd-2100f13 ) is fully overlapped to both of the two genes ( riok3 and npc1 ) . the size of cnvs summarized in the databases shows that the reported cnvs were overlapped to both genes . we assumed that our cnvs may reflect the change of copy number of the genes themselves . since the genes mentioned are good candidate markers for enabling us to examine the etiology of common diseases and phenotypical heterogeneities among individuals , our highly polymorphic cnvs should be able to become good markers in future studies . we are currently planning to examine the same population using a high - density oligo - array platform to accumulate more cnv data for japanese . the reason , as mentioned before , is that the bac platform and oligo methods complement each other . the oligo platform is known to be more effective in detecting smaller cnvs ( around a few kilobases ) , even though this approach is less effective in tracing cnvs in genomic regions of complex structure that are covered by the bac approach conducted in this study . we expect to construct a more definite japanese cnv database by the combination of bac- and oligo - platform arrays . we conducted population screening for 213 unrelated japanese , and observed 680 highly polymorphic cnvs . the majority of the polymorphic cnvs presented on bac clones that overlapped with regions of segmental duplication , and had been previously reported in other publications . moreover , it is expected that the cnvs might be good surrogate markers for detecting etiological genes , even if cnvs did not directly affect the genes themselves .
segmental copy - number variations ( cnvs ) may contribute to genetic variation in humans . 
 reports 
                   of the existence and characteristics of cnvs in a large japanese cohort are quite limited . 
 we report the data from a large japanese population . 
 we conducted population screening for 213 unrelated japanese individuals using comparative genomic hybridization based on a bacterial artificial 
                   chromosome microarray ( bac - acgh ) . 
 we summarize the data by focusing on highly polymorphic cnvs in 5.0% of the individual , 
                   since they may be informative for demonstrating the relationships between genotypes and their phenotypes . 
 we found a total of 680 cnvs at 16 
                   different bac - regions in the genome . 
 the majority of the polymorphic cnvs presented on bac - clones that overlapped with regions of segmental 
                   duplication , and the majority of the polymorphic cnvs observed in this population had been previously reported in other publications . 
 some of the cnvs contained genes which might be related to phenotypic heterogeneity among individuals .