BACKGROUND
there is considerable genomic variability among humans that is not associated with a recognizable clinical phenotype.
this variability is evident at both the chromosomal level   <cit>  and at the single nucleotide level )  <cit> .
the gains and losses of sub-microscopic dna segments larger than  <dig> kb are termed copy number variants   <cit> .
they represent a newly recognized class of dna variation, identified as a result of the introduction of comparative genomic hybridization  array technology that enables the study of variation in the number of copies of specific dna segments among individuals  <cit> .
the widespread presence of cnvs in normal individuals has now been documented using not only array cgh-containing bac clones  <cit>  but also oligonucleotide arrays at high resolution as well snp data analysis  <cit>  and dna sequence comparisons between individuals  <cit> .
the discovery of cnvs presents investigators with a number of challenges as cnvs complicate the interpretation of array data and efforts to attribute microdeletions and microduplications identified in individuals with constitutional disorders or in cancerous tissues to the disease processes.
the role of cnvs in causing or influencing the susceptibility to disease and genome evolution remains largely unknown.
a catalogue of published cnvs can be found in the public database  <cit> , and helps to guide the interpretation of array cgh findings.
however, the number and identity of polymorphic loci detected in different studies varies considerably  <cit> , likely because of differences across various array platforms, analytical methods and the populations investigated.
in addition, copy number differences for many of the cnvs listed in the above database are not confirmed using secondary independent quantification methods .
therefore, further work to identify and characterize cnvs in human populations and confirm copy number variability is essential in order to better understand the significance of cnvs and to determine their role in common disorders.
we studied  <dig> phenotypically normal individuals and detected  <dig> different sub-microscopic cnvs using the  <dig> mb resolution whole genome array-cgh.
we used real-time quantitative pcr  and fish to confirm cnvs.
the results of these studies are presented.
RESULTS
<dig>  array-cgh findings of cnvs in normal subjects
by studying  <dig> phenotypically normal, healthy individuals  using commercial array-cgh ,  <dig> different cnvs were identified and further classified into  <dig> subgroups.
group a contains  <dig> cnvs which were previously reported in the public database  <cit> , either as an identical clone entry or as part of a reported genomic cnv region.
the remaining  <dig> cnvs in group b of table  <dig> represent novel cnvs.
in total,  <dig> cnvs were observed in two or more individuals; the majority of recurrent cnvs  belonged to group a. segmental duplications were found in  <dig> cnvs .
of the  <dig> cnvs, only  <dig> were found to contain genes  listed in the omim database; 9/ <dig> genes were found in one cnv in group a .
in addition to genes primarily involved in functions of the human immune or sensory systems, signal transduction and metabolism, genes involved in transcription regulation, neurotransmitter transport, cell proliferation and differentiation or development were also identified .
note: * clone showing multiple sites based on e-fish.
aa: african american; h: hispanic; c: caucasian.
the three clones which showed copy number changes most frequently in our subjects were rp11-259n <dig> , rp11-100c <dig>  and rp11-79f <dig> , and were found in  <dig>   <dig> and  <dig> individuals respectively.
<dig>  fish and rt-qpcr analysis of polymorphic clones
a) fish
in order to establish the cellular copy number pattern of known and novel cnvs, we performed fish analysis of  <dig> cnvs  in subjects for whom a cell pellet was available.
for some clones, copy number changes could be confirmed by fish, while for others the fish patterns were discordant with array-cgh results.
for example, array analysis of clones rp5-1011o <dig> at 2q <dig>  and rp11-89m <dig> at 8p <dig> both showed a deletion upon array analysis, and both were subsequently confirmed by fish.
however, the fish signal pattern was different for these two clones: clone rp5-1011o <dig> had a complete loss of one copy, demonstrating a complete deletion while clone rp11-89m <dig> exhibited a diminished fish signal on one of the homologs suggestive of a partial deletion .
c = confirmed
nc = not confirmed
nt = not tested
conversely, fish analysis of clone rp11-100c <dig>  showed consistent normal signal patterns  in multiple subjects regardless of whether the clone was seen as a loss  or gain  on array analysis.
gains of clones rp11-125a <dig> and rp11-270m <dig> could not be confirmed by fish .
finally, gain of the clone rp11-598f <dig> was seen as multiple signals mapping to several chromosomes, demonstrating the presence of homologous sequences within this clone in several regions of the genome .
this observation was also confirmed by the in silico efish simulation tool  <cit> .
b) rt-qpcr and fish comparisons
rt-qpcr analysis was performed for 3/ <dig> clones tested by fish in order to confirm the array results and to help resolve the array and fish discrepancies .
comparable results between fish and rt-qpcr were obtained for all  <dig> clones tested by both methods – i.e.
deletions of rp11-89m <dig> and pr5-1011o <dig> were confirmed by rt-qpcr and the lack of copy number change  for rp11-100c <dig> as seen by fish was also noted by rt-qpcr.
discussion
our study of  <dig> normal subjects revealed a total of  <dig> cnvs:  <dig> previously described cnvs  <cit>  and  <dig>  novel cnvs.
a higher number of known than novel cnvs showed recurrence in the subjects tested .
similarly, evidence of segmental duplications was found in 6/ <dig> previously described cnvs.
this confirms the observation that recurring cnvs are more prevalent in the human population, and tend to be associated with segmental duplications  <cit> , while our novel cnvs are possibly less frequent and individual specific.
the number of cnvs detected in our controls is comparable to the number obtained by other investigators using the same commercial array-cgh and cut-off levels  <cit> .
however, the number is significantly smaller than that observed by iafrate et al  <cit>  who reported  <dig> cnvs in  <dig> individuals.
although they used the same array-cgh platform, a different dynamic website-based analytical method was applied, instead of our fixed cut-off levels of  <dig>  for duplications and  <dig>  for deletions, suggesting that the analytical method used plays a significant role in the number of apparent cnvs detected among individuals.
recently, a number of studies addressed the question of global genomic variation using different approaches including tiling bac array  <cit> , snp polymorphisms and oligo arrays  <cit> .
the number of cnvs and chromosomal regions affected varied among studies even when the same array platform was used.
for example, the bac tiling arrays detected  <dig> autosomal segmental cnvs in  <dig> controls  <cit> ,  <dig> cnvs in  <dig> controls  <cit>  and  <dig> cnvs in  <dig> individuals with intellectual disability and their phenotypically normal parents  <cit> .
the differences in the populations studied may have contributed to the observed discrepancies, however, even when the same individuals were examined using a different approach  less than half  of the cnvs were detected on both platforms  <cit> .
it is now evident that none of the existing technologies can capture all human variation.
one of the pre-requisites for understanding global human variation is the confirmation of cnvs using alternative methods.
their recurrence and presence as detected using different platforms supports that these are true differences among individuals.
however, a large number of cnvs are still "unique", i.e.
specific for a control subject/family or study.
independent quantification methods such as fish or rt-qpcr should ideally be performed on many cnvs, particularly those appearing in one individual, as these are the most likely ones to be false positives  <cit> .
considering the large number of cnvs reported  the number of validated cnvs using independent quantification methods such as rt-qpcr or fish is still proportionally very small due to the time consuming or limited throughput of single locus analysis.
for example, in two recent larger studies reporting a total of > <dig> cnvs, less than  <dig> cnvs were validated using quantification methods  <cit> .
using fish we have confirmed array-detected copy number changes of 3/ <dig> selected cnvs , while for 3/ <dig> cnvs a normal two signal fish pattern was seen.
we tested two fish-confirmed cnvs using rt-qpcr, one partially and one fully deleted , and observed concordance between all  <dig> methods .
as two of the three fish non-confirmed cnvs  are recognized as being very common and recurrent on multiple platforms  <cit> , we further evaluated rp11-100c <dig> using rt-qpcr but failed to achieve confirmation of both gain and loss.
it is possible that this cnv is composed of tightly packed repeats which can be discerned only by fiber fish, as noted for clone rp11-259n <dig> from chromosome  <dig>  <cit> .
additional cause of the array-cgh vs. fish/rt-qpcr discrepancy may be due to the fact that array-cgh assays evaluate the relative ratio of segmental dna copy number in the test dna vs. reference dna, and do not provide an absolute number of copies, as explained in figure  <dig>
the information on new cnvs is expanding dramatically, and cataloguing clones for which independent quantification is performed are desirable, as only detailed analysis of a large number of cnvs will help better understand their basic structure, dna content and reasons for variability.
currently, the significance of cnvs remains puzzling, as many of these genomic regions contain genes and coding sequences associated with known genetic disorders  <cit> .
in our subjects 13/ <dig> different cnvs were associated with omim genes; the number was usually not higher than  <dig> genes/cnv, except for cnv rp11-144o <dig> which had  <dig> genes involved in sensory perception, cell adhesion-mediated signaling, immune and defense processes .
this clone was noted in one of our hispanic individuals and was reported as one of the more divergent clones in the  <dig> populations reported by redon et al  <cit> .
many of the genes in cnvs are described as "environmental sensor genes" and are associated with mechanisms mediating immune responsiveness , cellular metabolism , and membrane surface interactions .
it is now established that the copy number variability of some genes can influence susceptibility to some diseases  <cit> .
for example, it has been reported that people with fewer copy numbers of ccl3l <dig>  a gene involved in immunity, are more susceptible to hiv infection  <cit> .
the extent of associations of cnvs with disease susceptibility will become clearer as we learn more about the distribution of well characterized cnvs in individuals whose health and medical histories are fully evaluated.
CONCLUSIONS
submicroscopic cnvs are a common form of human genomic variation, which can be readily identified by array-cgh technology in phenotypically normal individuals.
the number of cnvs detected in each study is influenced by several factors, especially the array platform and method of analysis.
our results confirm the wide distribution of cnvs in three different ethnic populations and expand the number of recognized cnvs.
cataloguing of confirmed cnvs, quantified using independent methods, would facilitate their interpretation and understanding of their significance in the future.

 there is considerable genomic variability among humans that is not associated with a recognizable clinical phenotype. this variability is evident at both the chromosomal level and at the single nucleotide level ). the gains and losses of sub-microscopic dna segments larger than kb are termed copy number variants. they represent a newly recognized class of dna variation, identified as a result of the introduction of comparative genomic hybridization array technology that enables the study of variation in the number of copies of specific dna segments among individuals. the widespread presence of cnvs in normal individuals has now been documented using not only array cgh-containing bac clones but also oligonucleotide arrays at high resolution as well snp data analysis and dna sequence comparisons between individuals. the discovery of cnvs presents investigators with a number of challenges as cnvs complicate the interpretation of array data and efforts to attribute microdeletions and microduplications identified in individuals with constitutional disorders or in cancerous tissues to the disease processes. the role of cnvs in causing or influencing the susceptibility to disease and genome evolution remains largely unknown. a catalogue of published cnvs can be found in the public database, and helps to guide the interpretation of array cgh findings. however, the number and identity of polymorphic loci detected in different studies varies considerably, likely because of differences across various array platforms, analytical methods and the populations investigated. in addition, copy number differences for many of the cnvs listed in the above database are not confirmed using secondary independent quantification methods. therefore, further work to identify and characterize cnvs in human populations and confirm copy number variability is essential in order to better understand the significance of cnvs and to determine their role in common disorders. we studied phenotypically normal individuals and detected different sub-microscopic cnvs using the mb resolution whole genome array-cgh. we used real-time quantitative pcr and fish to confirm cnvs. the results of these studies are presented.  array-cgh findings of cnvs in normal subjects by studying phenotypically normal, healthy individuals using commercial array-cgh, different cnvs were identified and further classified into subgroups. group a contains cnvs which were previously reported in the public database, either as an identical clone entry or as part of a reported genomic cnv region. the remaining cnvs in group b of table represent novel cnvs. in total, cnvs were observed in two or more individuals; the majority of recurrent cnvs belonged to group a. segmental duplications were found in cnvs. of the cnvs, only were found to contain genes listed in the omim database; 9/ genes were found in one cnv in group a. in addition to genes primarily involved in functions of the human immune or sensory systems, signal transduction and metabolism, genes involved in transcription regulation, neurotransmitter transport, cell proliferation and differentiation or development were also identified. note: * clone showing multiple sites based on e-fish. aa: african american; h: hispanic; c: caucasian. the three clones which showed copy number changes most frequently in our subjects were rp11-259n, rp11-100c and rp11-79f, and were found in and individuals respectively. fish and rt-qpcr analysis of polymorphic clones a) fish in order to establish the cellular copy number pattern of known and novel cnvs, we performed fish analysis of cnvs in subjects for whom a cell pellet was available. for some clones, copy number changes could be confirmed by fish, while for others the fish patterns were discordant with array-cgh results. for example, array analysis of clones rp5-1011o at 2q and rp11-89m at 8p both showed a deletion upon array analysis, and both were subsequently confirmed by fish. however, the fish signal pattern was different for these two clones: clone rp5-1011o had a complete loss of one copy, demonstrating a complete deletion while clone rp11-89m exhibited a diminished fish signal on one of the homologs suggestive of a partial deletion. c = confirmed nc = not confirmed nt = not tested conversely, fish analysis of clone rp11-100c showed consistent normal signal patterns in multiple subjects regardless of whether the clone was seen as a loss or gain on array analysis. gains of clones rp11-125a and rp11-270m could not be confirmed by fish. finally, gain of the clone rp11-598f was seen as multiple signals mapping to several chromosomes, demonstrating the presence of homologous sequences within this clone in several regions of the genome. this observation was also confirmed by the in silico efish simulation tool. b) rt-qpcr and fish comparisons rt-qpcr analysis was performed for 3/ clones tested by fish in order to confirm the array results and to help resolve the array and fish discrepancies. comparable results between fish and rt-qpcr were obtained for all clones tested by both methods – i.e. deletions of rp11-89m and pr5-1011o were confirmed by rt-qpcr and the lack of copy number change for rp11-100c as seen by fish was also noted by rt-qpcr. discussion our study of normal subjects revealed a total of cnvs: previously described cnvs and novel cnvs. a higher number of known than novel cnvs showed recurrence in the subjects tested. similarly, evidence of segmental duplications was found in 6/ previously described cnvs. this confirms the observation that recurring cnvs are more prevalent in the human population, and tend to be associated with segmental duplications, while our novel cnvs are possibly less frequent and individual specific. the number of cnvs detected in our controls is comparable to the number obtained by other investigators using the same commercial array-cgh and cut-off levels. however, the number is significantly smaller than that observed by iafrate et al who reported cnvs in individuals. although they used the same array-cgh platform, a different dynamic website-based analytical method was applied, instead of our fixed cut-off levels of for duplications and for deletions, suggesting that the analytical method used plays a significant role in the number of apparent cnvs detected among individuals. recently, a number of studies addressed the question of global genomic variation using different approaches including tiling bac array, snp polymorphisms and oligo arrays. the number of cnvs and chromosomal regions affected varied among studies even when the same array platform was used. for example, the bac tiling arrays detected autosomal segmental cnvs in controls, cnvs in controls and cnvs in individuals with intellectual disability and their phenotypically normal parents. the differences in the populations studied may have contributed to the observed discrepancies, however, even when the same individuals were examined using a different approach less than half of the cnvs were detected on both platforms. it is now evident that none of the existing technologies can capture all human variation. one of the pre-requisites for understanding global human variation is the confirmation of cnvs using alternative methods. their recurrence and presence as detected using different platforms supports that these are true differences among individuals. however, a large number of cnvs are still "unique", i.e. specific for a control subject/family or study. independent quantification methods such as fish or rt-qpcr should ideally be performed on many cnvs, particularly those appearing in one individual, as these are the most likely ones to be false positives. considering the large number of cnvs reported the number of validated cnvs using independent quantification methods such as rt-qpcr or fish is still proportionally very small due to the time consuming or limited throughput of single locus analysis. for example, in two recent larger studies reporting a total of > cnvs, less than cnvs were validated using quantification methods. using fish we have confirmed array-detected copy number changes of 3/ selected cnvs, while for 3/ cnvs a normal two signal fish pattern was seen. we tested two fish-confirmed cnvs using rt-qpcr, one partially and one fully deleted, and observed concordance between all methods. as two of the three fish non-confirmed cnvs are recognized as being very common and recurrent on multiple platforms, we further evaluated rp11-100c using rt-qpcr but failed to achieve confirmation of both gain and loss. it is possible that this cnv is composed of tightly packed repeats which can be discerned only by fiber fish, as noted for clone rp11-259n from chromosome. additional cause of the array-cgh vs. fish/rt-qpcr discrepancy may be due to the fact that array-cgh assays evaluate the relative ratio of segmental dna copy number in the test dna vs. reference dna, and do not provide an absolute number of copies, as explained in figure the information on new cnvs is expanding dramatically, and cataloguing clones for which independent quantification is performed are desirable, as only detailed analysis of a large number of cnvs will help better understand their basic structure, dna content and reasons for variability. currently, the significance of cnvs remains puzzling, as many of these genomic regions contain genes and coding sequences associated with known genetic disorders. in our subjects 13/ different cnvs were associated with omim genes; the number was usually not higher than genes/cnv, except for cnv rp11-144o which had genes involved in sensory perception, cell adhesion-mediated signaling, immune and defense processes. this clone was noted in one of our hispanic individuals and was reported as one of the more divergent clones in the populations reported by redon et al. many of the genes in cnvs are described as "environmental sensor genes" and are associated with mechanisms mediating immune responsiveness, cellular metabolism, and membrane surface interactions. it is now established that the copy number variability of some genes can influence susceptibility to some diseases. for example, it has been reported that people with fewer copy numbers of ccl3l a gene involved in immunity, are more susceptible to hiv infection. the extent of associations of cnvs with disease susceptibility will become clearer as we learn more about the distribution of well characterized cnvs in individuals whose health and medical histories are fully evaluated.  submicroscopic cnvs are a common form of human genomic variation, which can be readily identified by array-cgh technology in phenotypically normal individuals. the number of cnvs detected in each study is influenced by several factors, especially the array platform and method of analysis. our results confirm the wide distribution of cnvs in three different ethnic populations and expand the number of recognized cnvs. cataloguing of confirmed cnvs, quantified using independent methods, would facilitate their interpretation and understanding of their significance in the future. 
fish analysis of cnvs in normal subjects resulted in the confirmation of copy number changes for of novel cnvs and of known cnvs. a selection of cnvs was further analyzed by fish and real-time quantitative pcr. genomic copy number variants involving > kb of dna have recently been found to be widely distributed throughout the human genome. this included the lack of copy number change by both rt-qpcr and fish for clone rp11-100c one of the most common known copy number variants, as well as confirmation of deletions for clones rp11-89m and rp5-1011o in order to understand their potential significance and to facilitate interpretation of array-cgh findings in constitutional disorders and cancers, we studied normal individuals using commercially available mb resolution bac array. further study of a small selection of cnvs indicated concordant and discordant array vs. fish/rt-qpcr results. a total of different cnvs were detected in normal subjects. although a large number of cnvs has been reported to date, quantification using independent methods and detailed cellular and/or molecular assessment has been performed on a very small number of cnvs. we have described novel cnvs in individuals. they represent a newly recognized form of dna variation in normal populations, discovered through screening of the human genome using high-throughput and high resolution methods such as array comparative genomic hybridization. this information is, however, very much needed as it is currently common practice to consider cnvs reported in normal subjects as benign changes when detected in individuals affected with a variety of developmental disorders.
 genomic copy number variants involving > kb of dna have recently been found to be widely distributed throughout the human genome. they represent a newly recognized form of dna variation in normal populations, discovered through screening of the human genome using high-throughput and high resolution methods such as array comparative genomic hybridization. in order to understand their potential significance and to facilitate interpretation of array-cgh findings in constitutional disorders and cancers, we studied normal individuals using commercially available mb resolution bac array. a selection of cnvs was further analyzed by fish and real-time quantitative pcr.  a total of different cnvs were detected in normal subjects. sixteen were not previously reported. thirteen of the cnvs contained genes listed in omim. fish analysis of cnvs in normal subjects resulted in the confirmation of copy number changes for of novel cnvs and of known cnvs. three cnvs tested by fish were further validated by rt-qpcr and comparable data were obtained. this included the lack of copy number change by both rt-qpcr and fish for clone rp11-100c one of the most common known copy number variants, as well as confirmation of deletions for clones rp11-89m and rp5-1011o  we have described novel cnvs in individuals. further study of a small selection of cnvs indicated concordant and discordant array vs. fish/rt-qpcr results. although a large number of cnvs has been reported to date, quantification using independent methods and detailed cellular and/or molecular assessment has been performed on a very small number of cnvs. this information is, however, very much needed as it is currently common practice to consider cnvs reported in normal subjects as benign changes when detected in individuals affected with a variety of developmental disorders. 