aortic dissection ( ad ) is an uncommon but highly lethal disease that leads to the gradual deterioration of the mechanical properties of the aortic wall . it is caused by weak aortic structure , incompetent repair mechanisms and powerful destructive forces . although genetic factors may play an important role in the pathogenesis of ad [ 1 - 3 ] , studies investigating the genetic determinants of ad have been superficial , and reports of genetic risk factors for ad in koreans are extremely rare . several chromosomal loci with evidence of linkage were found in studies of thoracic aortic diseases [ 4 - 6 ] , but the mode of genetic transmission for ad remains uncertain . the identification of genetic markers associated with an increased risk of ad formation and growth will improve the treatment outcomes of patients with ad . array comparative genomic hybridization ( cgh ) is a powerful technique , allowing the simultaneous quantitative analysis of entire genomes , including a higher resolution , more dynamic range , direct mapping of aberrations to genomic sequences , and higher throughput . therefore , array cgh has been used to detect chromosomal aberrations in mental retardation , epilepsy , and schizophrenia [ 9 - 12 ] . real - time polymerase chain reaction ( pcr ) has been used to empirically confirm the results of array cgh . moreover , quantitative real - time pcr has great advantages in measuring dna copy - number changes owing to the economy , simplicity , and flexibility of the technique . the aim of the present study was to identify possible chromosomal loci associated with the pathogenesis of ad in koreans using array cgh and to confirm the results with real - time pcr . the mean age of the patients was 54.17 years , ranging from 41 to 77 years . genomic dna was extracted from individual blood samples using the puregene dna isolation kit ( qiagen , hilden , germany ) . reference dna was pooled from ten gender - matched ( male ) , normal , healthy control subjects . approval was obtained from the institutional review board of the college of medicine of our university , and informed consent was provided according to the declaration of helsinki . array cgh was performed as described previously   using a commercial macarray-karyo 4k bac - chip ( macrogen , seoul , korea ) with 4,030 bacterial artificial chromosome ( bac ) clones duplicating the whole human genome with a resolution of 1 mbp . dna was labeled using the bioprime labeling kit ( invitrogen , carlsbad , ca ) . each dna ( target and reference dna ) sample ( 500~700 ng ) with random primers was boiled at 98~100 for 5 min for denaturation and then cooled on ice for 5 min . the denatured dna was differentially labeled with 3l of 1 mm cy3-dctp ( references dna ) , cy5-dctp ( target dna ) , respectively ( perkinelmer , boston , ma ) , and 1l klenow fragments were added to the mixture ( invitrogen , carlsbad , ca ) . , unincorporated nucleotides were removed using microspin g-50 columns ( amersham biosciences , buckinghamshire , england ) . cy3 and cy5 labeled dna ( target dna and reference dna , repectively ) was mixed with 50 g of human cot-1 dna for blocking of repeat sequences . after purification , the mixture was resolved in hybridization buffer containing yeast trna for blocking binding of non - specific nucleotides . after the chips were in hybridization buffer with salmon sperm dna for 1 h , the chips were hybridized with the purification mixture . then they were incubated for 72 h in the 37 hybridization chamber ( biomicro systems , salt lake city , ut ) . after hybridization was complete , the array chips were washed and dried and then the arrays were scanned with a genepix ( axon instruments , foster city , ca ) . the scanned images were analyzed to determine cy3:cy5 ratios for each array element using mac viewer v1.6.3 software ( macrogen , seoul , korea ) . data are presented as log2 ( cy3 intensity / cy5 intensity ratios ) plotted against the position of clones within the particular chromosome in the current version of the genome . ratios were normalized by using the median of fluorescence ratios computed as log2 values from the housekeeping dna control fragments linearly distributed across the genome . the information on each individual clone was obtained from the ucsc genome bioinformatics database ( may 2004 freeze , http://genome.ucsc.edu ) . then log2 ratios of chromosomal aberration regions were identified via t test analysis using the sigma plot program . all chromosome regions indicated as aberrant in table 2 were calculated via t test analysis . to validate genomic imbalances identified by array cgh in this study , four genes with obvious genomic changes were analyzed using real - time pcr . for relative quantification , the reactions were performed in a total volume of 50l , including 25l of 2 iq sybr green supermix ( bio - rad , hercules , ca ) , 10 ng of dna , and 10 pmol of each primer . forty cycles of amplification , data acquisition , and data analysis were performed in an icycler ( bio - rad , hercules , ca ) . primers for four genes ( col6a2 , dgcr14 , pcsk6 , and sdha ) were selected and the position of each clone was obtained from the ucsc genome database ( may 2004 ) . the primer sequences used were as follows : col6a2 , forward , 5'-tccatgtctcaagcgttctg-3 ' ; reverse , 5'-tgggttctgccctatgactc-3 ' ; dgcr14 , forward , 5'-gacagttcagcaggctttcc-3 ' ; reverse , 5'-tgatcttggctcactgcaac-3 ' ; pcsk6 , forward , 5'-ggcaacaaaagaaagcaagc-3 ' ; reverse , 5'-tctcaacagctgggagaggt-3 ' ; sdha , forward , 5'-ctgagtgatgccctgcagta-3 ' ; reverse , 5'-ttccacgagcagaggaaact-3 ' . all real - time pcr procedures were performed three times and the normal control was different from each experiment ( all three normal samples ) . ct for each gene was determined using thermocycler software , and the average of three independent experiments was calculated . the copy number of the target gene was normalized to an endogenous reference , gapdh , which showed no significant copy number change in each genome . the fold change from normal samples was set at 1-fold and the ratio of the normalized fold change in tad compared to that of control samples was calculated . the mean age of the patients was 54.17 years , ranging from 41 to 77 years . genomic dna was extracted from individual blood samples using the puregene dna isolation kit ( qiagen , hilden , germany ) . reference dna was pooled from ten gender - matched ( male ) , normal , healthy control subjects . approval was obtained from the institutional review board of the college of medicine of our university , and informed consent was provided according to the declaration of helsinki . array cgh was performed as described previously   using a commercial macarray-karyo 4k bac - chip ( macrogen , seoul , korea ) with 4,030 bacterial artificial chromosome ( bac ) clones duplicating the whole human genome with a resolution of 1 mbp . dna was labeled using the bioprime labeling kit ( invitrogen , carlsbad , ca ) . each dna ( target and reference dna ) sample ( 500~700 ng ) with random primers was boiled at 98~100 for 5 min for denaturation and then cooled on ice for 5 min . the denatured dna was differentially labeled with 3l of 1 mm cy3-dctp ( references dna ) , cy5-dctp ( target dna ) , respectively ( perkinelmer , boston , ma ) , and 1l klenow fragments were added to the mixture ( invitrogen , carlsbad , ca ) . , unincorporated nucleotides were removed using microspin g-50 columns ( amersham biosciences , buckinghamshire , england ) . cy3 and cy5 labeled dna ( target dna and reference dna , repectively ) was mixed with 50 g of human cot-1 dna for blocking of repeat sequences . after purification , the mixture was resolved in hybridization buffer containing yeast trna for blocking binding of non - specific nucleotides . after the chips were in hybridization buffer with salmon sperm dna for 1 h , the chips were hybridized with the purification mixture . then they were incubated for 72 h in the 37 hybridization chamber ( biomicro systems , salt lake city , ut ) . after hybridization was complete , the array chips were washed and dried and then the arrays were scanned with a genepix ( axon instruments , foster city , ca ) . the scanned images were analyzed to determine cy3:cy5 ratios for each array element using mac viewer v1.6.3 software ( macrogen , seoul , korea ) . data are presented as log2 ( cy3 intensity / cy5 intensity ratios ) plotted against the position of clones within the particular chromosome in the current version of the genome . ratios were normalized by using the median of fluorescence ratios computed as log2 values from the housekeeping dna control fragments linearly distributed across the genome . the information on each individual clone was obtained from the ucsc genome bioinformatics database ( may 2004 freeze , http://genome.ucsc.edu ) . then log2 ratios of chromosomal aberration regions were identified via t test analysis using the sigma plot program . all chromosome regions indicated as aberrant in table 2 were calculated via t test analysis . to validate genomic imbalances identified by array cgh in this study , four genes with obvious genomic changes were analyzed using real - time pcr . for relative quantification , the reactions were performed in a total volume of 50l , including 25l of 2 iq sybr green supermix ( bio - rad , hercules , ca ) , 10 ng of dna , and 10 pmol of each primer . forty cycles of amplification , data acquisition , and data analysis were performed in an icycler ( bio - rad , hercules , ca ) . primers for four genes ( col6a2 , dgcr14 , pcsk6 , and sdha ) were selected and the position of each clone was obtained from the ucsc genome database ( may 2004 ) . the primer sequences used were as follows : col6a2 , forward , 5'-tccatgtctcaagcgttctg-3 ' ; reverse , 5'-tgggttctgccctatgactc-3 ' ; dgcr14 , forward , 5'-gacagttcagcaggctttcc-3 ' ; reverse , 5'-tgatcttggctcactgcaac-3 ' ; pcsk6 , forward , 5'-ggcaacaaaagaaagcaagc-3 ' ; reverse , 5'-tctcaacagctgggagaggt-3 ' ; sdha , forward , 5'-ctgagtgatgccctgcagta-3 ' ; reverse , 5'-ttccacgagcagaggaaact-3 ' . all real - time pcr procedures were performed three times and the normal control was different from each experiment ( all three normal samples ) . ct for each gene was determined using thermocycler software , and the average of three independent experiments was calculated . the copy number of the target gene was normalized to an endogenous reference , gapdh , which showed no significant copy number change in each genome . the fold change from normal samples was set at 1-fold and the ratio of the normalized fold change in tad compared to that of control samples was calculated . chromosomal copy - number changes were detected by array cgh in blood samples obtained from ad patients . the identified regions of genomic aberrations , ranging from 6 in 18 samples ( 33% frequency of chromosomal gain and loss ) to 10 in 18 samples ( 56% frequency of chromosomal gain at 8q and loss at 4q ) ( table 2 ) . overall , the individual chromosomal aberration pattern was not random and tended to be consistent , showing dna gains at 1p , 1q , 2p , 2q , 3q , 4q , 5p , 6p , 7p , 7q , 8p , 8q , 10p , 10q , 11q , 12p , 12q , 13q , 14q , 15q , 17q , 19p , 19q and 21q , and dna losses at 4q , 7q , 10q , 14q , 15q , 16p and 22q . dna gains were also frequently observed at 7q35 , 11q12.2 and 15q25.2 in 9 samples ( 50% ) , and in 2p13.1 , 4q13.3 , 7q11.23 and 19p13.11 in 8 samples ( 44% ) . regions with a 39% frequency of gains were 2q37.3 , 7p14.3 , 7q11.23 , 10q11.23 , 12p13.33 , 14q32.33 , 17q21.31 and 21q22.3 . other regions with 33% frequency of dna gains included 1p11.2 , 1q23.1 , 2q31.1 , 3q21.3 , 4q24 , 5p15.33 , 6p12.2 , 8p21.2 , 8p22 , 8p23.3 , 8q13.2 , 10p15.3 , 10q22.3 , 10q23.1 , 10q26.3 , 12q13.13 , 13q33.3 , 14q24.2 , 15q26.3 , 17q12 , 17q25.3 , 19q13.43 and 21q22.11 . regions with 44% frequency of losses were 14q32.33 and 15q11.2 . other regions with 33~39% frequency of dna loss included 7q22.1 , 10q26.3 , 16p12.3 , 16p13.11 , and 22q11.21 ( table 2 ) . to attempt to correlate a diagnosis with a specific region , we applied statistics for each potential loci . all aberrant chromosomal locations ( > 30% ) detected in array cgh ( table 2 ) were selected in t test analysis . although all 48 regions were calculated , only t test analysis of five regions is shown in table 3 . because a high association was detected in all regions , examples which indicated higher p - value ( > 50% frequency ) are shown in the tables . four genes with obvious genomic changes were analyzed using real - time pcr in order to assess the level of genomic imbalance identified by array cgh in this study . of the more than 30% frequency clones , those considered to have vascular related aberrations in the chromosome were selected for real - time pcr . gains were seen in 5p15.33 , 15q26.3 , and 21q22.3 , which contain the sdha , pcsk6 , and col6a2 genes ( a , b , and c ) , while a loss was observed at 22q11.21 , which contains the dgcr14 genes ( d ) . the fold difference obtained from the real - time pcr was in accordance with the array cgh results and the p - values of all samples was < 0.05 . the gain regions ( a~c ; col6a2 , pcsk6 , and sdha ) in the results from the real - time pcr were equal to all of the gain regions in the array cgh . chromosomal copy - number changes were detected by array cgh in blood samples obtained from ad patients . the identified regions of genomic aberrations , ranging from 6 in 18 samples ( 33% frequency of chromosomal gain and loss ) to 10 in 18 samples ( 56% frequency of chromosomal gain at 8q and loss at 4q ) ( table 2 ) . overall , the individual chromosomal aberration pattern was not random and tended to be consistent , showing dna gains at 1p , 1q , 2p , 2q , 3q , 4q , 5p , 6p , 7p , 7q , 8p , 8q , 10p , 10q , 11q , 12p , 12q , 13q , 14q , 15q , 17q , 19p , 19q and 21q , and dna losses at 4q , 7q , 10q , 14q , 15q , 16p and 22q . dna gains were also frequently observed at 7q35 , 11q12.2 and 15q25.2 in 9 samples ( 50% ) , and in 2p13.1 , 4q13.3 , 7q11.23 and 19p13.11 in 8 samples ( 44% ) . regions with a 39% frequency of gains were 2q37.3 , 7p14.3 , 7q11.23 , 10q11.23 , 12p13.33 , 14q32.33 , 17q21.31 and 21q22.3 . other regions with 33% frequency of dna gains included 1p11.2 , 1q23.1 , 2q31.1 , 3q21.3 , 4q24 , 5p15.33 , 6p12.2 , 8p21.2 , 8p22 , 8p23.3 , 8q13.2 , 10p15.3 , 10q22.3 , 10q23.1 , 10q26.3 , 12q13.13 , 13q33.3 , 14q24.2 , 15q26.3 , 17q12 , 17q25.3 , 19q13.43 and 21q22.11 . regions with 44% frequency of losses were 14q32.33 and 15q11.2 . other regions with 33~39% frequency of dna loss included 7q22.1 , 10q26.3 , 16p12.3 , 16p13.11 , and 22q11.21 ( table 2 ) . to attempt to correlate a diagnosis with a specific region , we applied statistics for each potential loci . all aberrant chromosomal locations ( > 30% ) detected in array cgh ( table 2 ) were selected in t test analysis . although all 48 regions were calculated , only t test analysis of five regions is shown in table 3 . because a high association was detected in all regions , examples which indicated higher p - value ( > 50% frequency ) are shown in the tables . four genes with obvious genomic changes were analyzed using real - time pcr in order to assess the level of genomic imbalance identified by array cgh in this study . of the more than 30% frequency clones , those considered to have vascular related aberrations in the chromosome were selected for real - time pcr . gains were seen in 5p15.33 , 15q26.3 , and 21q22.3 , which contain the sdha , pcsk6 , and col6a2 genes ( a , b , and c ) , while a loss was observed at 22q11.21 , which contains the dgcr14 genes ( d ) . the fold difference obtained from the real - time pcr was in accordance with the array cgh results and the p - values of all samples was < 0.05 . the gain regions ( a~c ; col6a2 , pcsk6 , and sdha ) in the results from the real - time pcr were equal to all of the gain regions in the array cgh . aortic dissection is a life - threatening disease that develops without warning . despite significant advances in diagnostic and therapeutic techniques , the overall in - hospital mortality was 27.4% according to the international registry of aortic dissection . it occurs in 2~9% of the population over 65 years old , and the prevalence may be increasing . modern diagnostic methods , such as computed or magnetic resonance tomography , are able to show an aortic wall hematoma at the acute onset of the disease . early recognition and management are crucial , and it is therefore important to be able to rapidly identify aortic dissection , initiate supportive therapy , and refer patients to appropriate specialty care . current research is aimed at determining the phenotypic consequences of specific copy number abnormalities in patients with ads . cytogenetic analyses have revealed that numerous somatic genetic changes in the pathogenesis of human diseases are involved in region - specific gains or losses of dna copy number ; however , such changes have not yet been reported in most ads . including classical cytogenetics , fluorescence in situ hybridization ( fish ) , southern blot analysis , quantitative pcr based assays , and cgh have been used to detect copy number changes in human diseases , but these techniques have several limitations . unlike chromosomal cgh , array cgh highly improves resolution and provides quantitative information of the level of chromosomal gain or loss . bac array cgh has been used in several diseases but has not been applied to ads . in this report , the dna copy number alterations frequently identified in 18 patients with ad were analyzed using array based cgh . the dna copy number alterations observed in the ads were not random ; they involved particular regions of the genome and most usually involved parts of or the entire dna . dna gains were found in regions 1p , 1q , 2p , 2q , 3q , 4q , 5p , 6p 7p , 7q , 8p , 8q , 10p , 10q , 11q , 12p , 12q , 13q , 14q , 15q , 17q , 19p , and 21q , and losses were found in 4q , 7q , 10q , 14q , 15q , 16p , and 22q ( table 2 ) . real - time pcr can be used to validate and quantify the genomic changes identified by bac array cgh . of the more than 30% frequency clones in array cgh results , those considered to have vascular related aberrations in the chromosome were selected for real - time pcr . frequently , gains of chromosomal dna at col6a2 were detected by cgh in patients with ads . the collagen , type vi , alpha-2 ( col6a2 ) gene encodes 1 of the 3 subunits that comprise the full collagen vi protein . the finding that collagen vi is a structural protein supports that collagen vi is a candidate gene involved in the development of congenital heart disease in down syndrome . the most frequent locus alteration was a gain at the 15q26.3 region containing the pcsk6 gene . pcsk6 ( pace4 ) is expressed at high levels in the anterior pituitary , central nervous system , the developing olfactory bulb , heart , and liver . moreover , sdha is complex ii of the mitochondrial respiratory chain , also known as succinate dehydrogenase or succinate ubiquinone oxidoreductase , and it consists of four nuclear - encoded polypeptides . bourgeron et al . identified a homozygous mutation in the sdha gene in two siblings with a mitochondrial complex ii deficiency presenting as leigh 's syndrome . their study results demonstrated that the flavoprotein subunit gene is duplicated in the human genome and located on chromosome 5p15 . chromosome arm 22q , which includes the comt and dgcr14 genes , was frequently under - represented . chromosome 22q11.21 includes the catechol o - methyltransferase ( comt ) gene , and its deletion syndrome is a common microdeletion syndrome associated with a markedly elevated risk of several diseases . in addition to this region , digeorge syndrome critical region gene 14 ( dgcr14 ) has been associated with an elevated risk of several diseases . a group of developmental disorders , including digeorge syndrome ( dgs ) , velocardiofacial syndrome ( vcfs ) , conotruncal anomaly face syndrome , and some familial or sporadic conotruncal cardiac defects have been associated with microdeletion of 22q11.2 . the four altered regions ( 5p15.33 , 15q26.3 , 21q22.3 and 22q11.21 ) that showed frequent genomic alteration were confirmed by real - time pcr . in summary , array based cgh analysis of 18 patients with ad showed that dna copy number alterations are common in ad . real - time pcr confirmed that array cgh properly detected gains in ad related genes . aortic dissection is believed to develop through a chronic degeneration of the aortic wall associated with aging , atherosclerosis , or hypertension . in this study , we were able to delineate discrete regions of dna copy number alterations in several regions of the chromosomes that are likely to harbor some relevant genes . these data support the utility of array cgh for the identification of genomic alterations in ad . although our results suggest a new aspect of genomic alteration in patients with ads , our study was limited by a small sample number ( n=18 ) and the fact that it only included individuals of korean descent . therefore , further studies should be carried out using cgh techniques to validate our results . information on chromosomal variability can be generated by comparing the results of array cgh with information from the human structural variation database and increasing patient numbers . this study is the first to use array cgh to search for candidate regions in korean aortic dissection patients , these findings might contribute to the understanding of important chromosomal regions and the identification of candidate genes , and may be used to investigate the pathogenesis of aortic dissections .
backgroundthe aim of the present study was to identify chromosomal loci that contribute to the pathogenesis of aortic dissection ( ad ) in a korean population using array comparative genomic hybridization ( cgh ) and to confirm the results using real - time polymerase chain reaction ( pcr).materials and methodseighteen patients with ads were enrolled in this study . 
 genomic dna was extracted from individual blood samples , and array cgh analyses were performed . 
 four corresponding genes with obvious genomic changes were analyzed using real - time pcr in order to assess the level of genomic imbalance identified by array cgh.resultsgenomic gains were most frequently detected at 8q24.3 ( 56% ) , followed by regions 7q35 , 11q12.2 , and 15q25.2 ( 50% ) . 
 genomic losses were most frequently observed at 4q35.2 ( 56% ) . 
 real - time pcr confirmed the results of the array cgh studies of the col6a2 , dgcr14 , pcsk6 , and sdha genes.conclusionthis is the first study to identify candidate regions by array cgh in patients with ads . 
 the identification of genes that may predispose an individual to ad may lead to a better understanding of the mechanism of ad formation . 
 further multicenter studies comparing cohorts of patients of different ethnicities are warranted .