Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on July 15, 2003

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Human Molecular Genetics, 2003, Vol. 12, No. 17 2145-2152
DOI: 10.1093/hmg/ddg230
© 2003 Oxford University Press

Development of a comparative genomic hybridization microarray and demonstration of its utility with 25 well-characterized 1p36 deletions

Wei Yu, Blake C. Ballif, Catherine D. Kashork, Heidi A. Heilstedt, Leslie A. Howard, Wei-Wen Cai, Lisa D. White, Wenbin Liu, Arthur L. Beaudet, Bassem A. Bejjani, Chad A. Shaw and Lisa G. Shaffer^*,

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

Received April 22, 2003; Accepted July 5, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS ELECTRONIC DATABASE INFORMATION REFERENCES

 
Chromosomal abnormalities, such as deletions and duplications, are characterized by specific and often complex phenotypes resulting from an imbalance in normal gene dosage. However, routine chromosome banding is not sensitive enough to detect subtle chromosome aberrations (<5–10 Mb). Array-based comparative genomic hybridization (array CGH) is a powerful new technology capable of identifying chromosomal imbalance at a high resolution by co-hybridizing differentially labeled test and control DNAs to a microarray of genomic clones. We used a previously assembled contig of large-insert clones that span 10.5 Mb of the most distal region of 1p36 to design a microarray. The array includes 97 clones from 1p36, 41 clones from the subtelomeric regions of all human chromosomes, and three clones from each of the X and Y chromosomes. We used this microarray to study 25 subjects with well-characterized deletions of 1p36. All array CGH results agree with the deletion sizes and locations of the breakpoints in these subjects as determined previously by FISH and microsatellite analyses. Terminal deletions, interstitial deletions, derivative chromosomes and complex rearrangements were also identified. We anticipate that array CGH will change the diagnostic approach to many congenital and acquired genetic diseases such as mental retardation, birth defects and cancer.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS ELECTRONIC DATABASE INFORMATION REFERENCES

 
Comparative genomic hybridization (CGH) was developed to screen the entire genome for DNA content differences by comparing a test sample to a control (1–3). Because metaphase chromosomes are used as the substrate for analysis, the detection of unbalanced alterations is limited to the resolution of the metaphase target (at the level of a 450 band karyotype, 5–10 Mb changes). Recently, DNA microarrays were developed that use either large-insert genomic clones (BACs/PACs) or smaller PCR products as targets for the hybridization (4–11). Array-based CGH (array CGH) offers many advantages over conventional CGH and other current diagnostic methods. It can be highly comprehensive (amenable to very high resolution), sensitive and fast. Because array CGH uses large-insert clones such as BACs or PACs with known map information, alterations are immediately linked to genetic markers, and the genomic resolution is determined by the map distances between markers (targets) or by the length of the clones used. Array CGH has been used successfully for genomic profiling in a wide variety of tumor samples (2,5,12–14). The application of array CGH was recently applied to constitutional chromosome abnormalities demonstrating single copy deletions or duplications (9,15,16). However, array CGH technology is still developing, and a number of challenges associated with the methodology remain unresolved.

Terminal deletions of 1p36 result in one of the most commonly observed mental retardation syndromes in humans (17,18). We previously reported the molecular characterization of 60 subjects with terminal deletions of 1p36, demonstrating that deletion sizes vary widely over the 10.5 Mb of 1p36 with no single common breakpoint (18). In addition, we have identified terminal deletions, interstitial deletions, derivative chromosomes, and complex rearrangements (18). Here, we have used a BAC/PAC contig of distal 1p36 (18) to develop a microarray to validate the application and accuracy of array CGH for detecting single-copy gains and losses and to more rapidly identify breakpoint locations in subjects with monosomy 1p36.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS ELECTRONIC DATABASE INFORMATION REFERENCES

 
Recently our laboratory developed a contig of the most distal 10.5 Mb of 1p36 and characterized in detail, using established methodology, the deletion sizes and types of rearrangements in 60 subjects with monosomy 1p36 (18). With these reagents, we were able to investigate the new technology of array CGH for identifying and characterizing cytogenetic imbalances. Different from a whole genome approach with BACs spaced every 1 Mb, we have used the adjacent clones of our contig to enhance the analysis of each chromosomal position and to increase the accuracy of the inferences. The clones used for the microarray consist of 97 BAC/PAC clones spanning 10.5 Mb of the most distal region of 1p36 with five sequence gaps and are essentially identical to the contig published recently (18). We end-sequenced and FISH mapped each of the 97 BAC/PAC clones from 1p36 prior to printing the array. Ninety-three clones map specifically to 1p36. However, the four most distal clones (RPCI5-857K21, RPCI11-206L10, GS-63M14 and RPCI11-34P13) map to the telomere-associated repeat (TAR) region of 1p36 and also to the telomeric regions on many other chromosome arms. Because the TAR region of most chromosomes consists of repetitive sequences shared by many chromosome ends, this result was not unexpected (19,20). Despite the FISH hybridization patterns, we included these distal clones to examine their performance on a microarray when hybridized with normal and hemizygous samples.

The microarray also included 41 BAC or PAC clones from the subtelomeric regions of human chromosomes, 32 of which were published previously (21), and nine novel clones were identified from the June 2002 draft sequence of the human genome (Table 1). We also included three BAC clones from each of the X and Y chromosomes (Table 1). We verified the chromosomal locations of these 47 clones by FISH. Each clone was printed selectively and in quadruplicate such that no contiguous BACs were printed adjacent to each other on the microarray. This layout controls for any areas of poor hybridization that might be erroneously interpreted as deletions.

View this table: [in this window] [in a new window]   Table 1. Subtelomeric clonesa and X- and Y-specific clones used on the microarray

 
We used this microarray to study 25 subjects with well-characterized deletions of 1p36 (18). A single-copy deletion of 1p36 was correctly identified in all 25 cell lines. Examples from four subjects are shown in Figure 1. In addition, the microarray CGH results confirmed the previous characterization of these cell lines using clones from the 1p36 contig with deletion sizes ranging from 1.5 to >10.5 Mb (18). This includes the correct identification of terminal and interstitial deletions and complex rearrangements. In comparing the FISH/microsatellite results (18) to the microarray results, excluding one breakpoint known to be located within the TAR region (subject 9 breakpoint B1) and one subject, 31, with a deletion of the entire 10.5 Mb of 1p36, 11 out of 23 subjects displayed the identical breakpoint locations. For the remaining rearrangements, the breakpoint locations differed by only one adjacent clone (Table 2). It is unclear from these data whether the FISH or the CGH-based microarray analysis is more accurate when analyzing clones that span the breakpoint. The results from either technology are probably dependent on the amount of DNA retained on the deleted chromosome that is complementary to the large-insert clone DNA and dependent on the particular sequence that is retained (e.g. repetitive or unique). Further analysis of the precise breakpoints of these deletions will probably reveal the various parameters necessary for detectable hybridization with either technology. Nevertheless, in a single experiment array CGH correctly narrowed the breakpoint junctions of each subject to within 100–300 kb of its precise breakpoint.

View larger version (62K): [in this window] [in a new window]   Figure 1. Shown are the plots of the hybridization results (top) and a diagram comparing the FISH results to the microarray results (bottom) for four study subjects. (A) Terminal deletion in subject 14. (B) Interstitial deletion in subject 18. (C) Derivative chromosome, der()t(;)(p36;q44) in subject 9. (D) Complex rearrangement in subject 29. In the plots of the hybridization results (top), the first 97 clones are those clones from the 1p36 contig (18) and are shown on the left. Clones 98–144 are shown to the right and represent the subtelomeric clones for chromosomes 1-X/Y and the X-specific and Y-specific clones. Each shows the individual signal ratios (scale shown on the left) for that study subject for each clone on the microarray. A ratio above 0.65 indicates a gain of DNA copy number, a ratio of less than -0.567 indicates a loss of DNA copy number, and a ratio of 0.005 indicates a normal DNA copy number. Comparison of FISH and microarray data is shown (bottom). The black line indicates the chromosome. Above the black line are microsatellite markers contained within the 1p36 contig (18). Black markers were not used in that particular patient, red markers showed deletion and green markers were normal. Below the black line is a representation of the contig clones. Gray clones were not used in FISH analysis, red clones were deleted by FISH, and green clones were not deleted by FISH analysis. Below the clones is the microarray result. The red line indicates deletion and green line indicates retention (normal).

 

View this table: [in this window] [in a new window]   Table 2. Cell lines showing discrepancies of breakpoint locations detected by FISH and microarray CGH

 
Although the four most distal 1p36 clones (TAR clones) were known to cross-hybridize to other chromosome ends, they were included in the array to evaluate the performance of clones that represent duplicated regions of the genome. The microarray CGH analysis did not identify detectable copy number changes with any of the TAR clones. This suggests that clones from complex, duplicated regions of the genome do not perform well in array CGH due to the presence of multiple copies of the sequence at other chromosomal locations. However, duplicated regions of the genome are involved in a number of genomic disorders for which array CGH would be a valuable diagnostic or research tool (22). Additional studies, such as arraying PCR amplified unique sequence fragments instead of whole BAC/PAC clones, are therefore needed to address the important issue of the level of resolution in which array CGH can accurately detect copy number gains or losses in duplicated regions.

Known interstitial deletions and derivative chromosomes (18,23) were used to test the performance of subtelomeric clones in our microarray. All interstitial deletions (n=4) were identified correctly (Table 3). Four cell lines contained double segmental imbalances: deletion of 1p36, and addition of Xp (n=1) or addition of 1q (n=3). Two of three der(1)t(1;1)(p36;q44) were identified correctly (Table 3). The der(1)t(X;1) was not identified by the microarray (Table 3). When the cut-off threshold was lowered from 0.9 to 0.85, all three of the derivative t(1;1)(p36;q44) chromosomes were identified. The der(1)t(X;1) was still not identified. The Xp PAC clone GS-98-C4 on the microarray is not the same cosmid clone CY29 (21) that was used originally to detect the derivative chromosome in subject 31. FISH analysis with the Xp PAC clone GS-98-C4 that is on the microarray did not demonstrate the derivative 1 (data not shown). Therefore, this clone is not expected to show a DNA copy number change by microarray analysis in this subject. However, lowering the threshold also identified four false positive gains of subtelomeric clones for 3p (subject 13), 4q (subject 2), 16p (subject 11), and 19q (subject 11). For future diagnostic purposes, when setting the thresholds for detecting a ‘positive’ result for a single-copy gain or loss, one must weigh the detection of false positives and the additional FISH confirmation that would be needed with the false negatives that may result in a missed diagnosis.

View this table: [in this window] [in a new window]   Table 3. Comparison of subtelomeric FISH and Microarray CGH

 
We used array CGH to investigate chromosomal imbalance of 97 genomic clones from the human 1p36 region and 41 clones from the subtelomeric regions of most human chromosome arms in 25 cell lines with monosomy 1p36. Although molecular analyses of these cell lines were reported (18,23,24), those studies were based on the labor-intensive approaches of FISH and microsatellite analyses to determine the deletion size, breakpoint locations, and subtelomeric rearrangements. The use of the current microarray on these cell lines is equivalent to 144 FISH experiments performed twice on 25 cell lines, for a total of 7200 individual FISH experiments. Although we invested a large amount of time in developing the array and FISH mapping all 144 individual clones, in the future this array will drastically reduce the time required to characterize the deletion size in a subject with a suspected terminal deletion of 1p36. In addition, aneusomy for the subtelomeric regions can be screened in the same experiment, eliminating the expensive and time-consuming telomere-region specific FISH assays.

Our study illustrates the importance of several crucial steps in the development of a CGH microarray prior to clinical use. First, the chromosomal location of each clone on the array and its specificity must be verified by FISH and sequence analysis. Any BAC/PAC clone that hybridizes to multiple chromosomal locations should be excluded from the array because such clones cannot be used to accurately detect copy number changes. Second, the array must be designed to ensure proper normalization, particularly when copy number changes are expected with a large number of clones. This includes using an appropriate number of control clones on the array that are not expected to show copy number changes. In this study, subtelomeric clones, not expected to have dosage differences in most cell lines, were selected as control clones for normalization. Third, the use of multiple clones or overlapping clones for each region of interest is essential. This provides more information for the data analysis process than single clones. Adjacent and overlapping clones can be used together to create an efficient and highly accurate analysis.

Array CGH is a powerful tool for the identification of chromosomal changes associated with genetic disorders and cancer. The use of known BACs immediately links any DNA dosage imbalance to a precise genome location (25), provides clues to the underlying mechanisms (26), and can help identify potential genes or genetic markers linked to these disorders. We anticipate that the application of the array CGH methodology presented here, with its advantages of high-resolution, accuracy and speed, will change the diagnostic approach to many congenital and acquired genetic diseases such as mental retardation, birth defects and cancer.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS ELECTRONIC DATABASE INFORMATION REFERENCES

 
BAC/PAC DNA preparation
All 97 human 1p36 BAC/PAC clones used in this study have been verified by both FISH and end-sequencing. All 41 clones from the subtelomeric regions of all human chromosomes and three BAC clones from each of the X and Y chromosomes were verified by FISH. A standard alkaline lysis procedure was used to purify BAC/PAC DNA.

Patient specimens
Genomic DNA was isolated from lymphoblastoid cell lines from 25 patients with previously well-characterized 1p36 deletions (18) using a PureGene DNA purification kit (Gentra) and following the manufacturer's protocol. All subjects signed consent forms approved by the Baylor College of Medicine Institutional Review Board. Genomic DNAs from phenotypically normal individuals (one male and one female) were isolated from peripheral bloods and used as reference DNAs for array analysis. No prior cytogenetic or molecular data were provided to the individuals performing the experiments (W.Y.) and data analyses (C.S.). The 25 study subjects were selected from a total of 61 subjects on the basis of deletion size and complexity in order to validate the array and to evaluate the quantification and normalization software developed for data analysis in this study. The cell lines chosen were from study subjects 1–9, 11, 13–18, 20, 23–25, 29, 31, 33, 34 and 58 (18).

Chemical cross-linking of BAC/PAC DNA
The chemical cross-linking of BAC/PAC DNA was performed as described (8). Human BAC/PAC DNA was dissolved in 0.1 N NaOH at 37°C overnight, and 1/10 vol of 0.1 M 3-glycidoxypropyltrimethoxysilane (Sigma) in isopropanol was mixed with the DNA solution. The cross-linking reaction was allowed to proceed at 65°C for 3 h and then stopped by adding an equal volume of a neutralization buffer of 0.2 M Tris, 1 M NaCl and 10 mM EDTA with pH 4.3. The BAC/PAC DNA concentration was determined by fluorometer and adjusted to 70–100 ng/µl for printing.

Array printing and blocking
A total of 144 human BAC/PACs with 97 clones from 1p36, 41 subtelomeric clones from all 24 chromosomes, three clones from the X chromosome and three clones from the Y chromosome were used to construct a human 1p36 array. Chemical cross-linked DNAs were directly printed onto alkaline/acid-cleaned and Bis(Trichlorosilyl)octane-treated plain glass slides using OmniGrid Accent Microarrayer (GeneMachine). Each clone was printed in quadruplicate. Arrays were blocked in a Gene TAC (Genomic Solutions) automated hybridization chamber with 25 µg human Cot I (Invitrogen) in a hybridization buffer of 50% formamide, 2xSSC, 10% dextran sulfate and 0.2% SDS at 37°C for 4–5 h.

Probe labeling and hybridization
Patient and reference genomic DNAs were labeled by random priming using a BioPrime DNA labeling kit (Invitrogen) and following the described protocol (7) with some modification. All experiments included two-dye switch and two-array hybridizations to obtain an accurate ratio (13). About 250 ng genomic DNA was labeled in 25 µl volume containing 60 µM dA, dG, dTTP, 30 µM dCTP, 30 µM Cyanine 3 dCTP or Cyanine 5 dCTP (NEN). For array hybridization, 25 µl of reaction mixture from labeled patient DNA was co-precipitated with an equal volume of labeled reference DNA in the presence of 50 µg human Cot I and resuspended in 15.5 µl hybridization buffer of 50% formamide, 2xSSC, 10% dextran sulfate and 0.2% SDS or ULTRAhyb buffer (Ambion). The mixture was denatured at 75°C for 5 min, incubated at 37°C for 60 min, and added onto the microarray and covered with a 22x22 mm coverslip. The microarray was placed in a hybridization chamber (Corning) and incubated at 37°C for 22–26 h with shaking. After hybridization, the microarray was washed twice in 50% formamide and 2xSSC at 42°C for 10 min and once with 0.2xSSC at 42°C for 10 min.

Image acquisition and analysis
Images were acquired using a GenePix 4000B (Axon Instruments) dual-laser scanner in combination with GenePix Pro 4.0 imaging software. Two simultaneous scans of each array were obtained at wavelengths of 635 and 532 nm, respectively. Microarray image quantification was performed using GLEAMS software (Nutec Sciences). Quantified array image files were subjected first to a single chip normalization to remove systematic artifacts such as spatial and intensity biases (27,28). Dye-reversed array pairs were then subjected to bi-chip scaling to bring the dye-reversed hybridizations to a common measurement scale to facilitate combining the data from two microarray experiments for each subject.

After data processing and the combination of data from dye-reversed pairs, inferences were made according to a clone-by-clone classification procedure to determine the gain/loss status of each clone for each subject. The clone-by-clone classification procedure we used was based on the integration of data from all 25 study subjects. To determine the status of each clone, a three-class cluster analysis of data from all 3600 clones (144 distinct clones, 25 subjects) was performed first. Clustering was performed by a dendrogram, a simple and highly utilized method for clustering both univariate and multivariate data. Dendrograms require specification of a distance measure; we used the absolute difference between mean normalized data, considering all clones for all patients (3600 data points) (29). The dendrogram analysis performed here used the hclust package implemented in the S-Plus stastistical programming environment. The complete linkage method was used to form the tree. The dendrogram was sliced to divide the data into three groups. After clustering the 3600 clones, the mean and variance of the single clone values in each class were determined. The ‘loss’ class has a mean of -0.567 and a variance of 0.02. The ‘normal’ class has a mean of 0.005 and a variance of 0.009. The ‘gain’ class has a mean of 0.65 and a variance of 0.073. To ascertain the gain/loss status of individual clones in a particular subject, we formed a ‘posterior class probability’ for each clone for each study subject. The class probabilities were calculated using Gaussian probability density functions conditioned on the group mean and variance values determined from clustering. The use of adjacent clones at each particular chromosomal position can make inference more accurate and statistically efficient. However, the amount of weight given to an adjacent clone in the inference is a topic of ongoing research, especially when a clone may contain a breakpoint. Thus, we took the most conservative approach and treated each clone independently. We required a class probability of greater than 0.9 to infer either gain or loss at any particular clone. The effective false positive rate using this criterion was less than 0.005 based on the known subtelomere FISH data.

Additionally, we calculated the mean variance between replicates for each clone across all 25 patients; with those values, we calculated the mean variance between replicates across all clones. For telomeric clones, the average variance between replicates is 0.01, and for the 1p clones across patients the mean variance between replicates is estimated to be 0.02. Coefficients of variation were also calculated; these statistics report the ratio of the standard deviation to the mean in absolute value and demonstrate the amount of variation observed in clones with very large or small normalized log-ratios. For clones that were inferred to change in our analysis, the mean coefficient of variation was 0.14. This value suggests that the relative variation is small among the clones for which a change was inferred.

	ACKNOWLEDGEMENTS

 
We thank Mack Neff and Sergio Rodriguez of the Baylor College of Medicine Microarray Core Facility for their help with the printing of the microarray. We are grateful to Jonathan Flint (Oxford, UK) for kindly providing the telomere clones used in this study. We thank Caron Glotzbach, Giuliana Gregato, Aaron Theisen, and Marzena Gajecka (Washington State University Spokane) for the FISH on subject 31 and their critical review of the manuscript. This study was supported in part by grants from the NIH National Institute of Deafness and Other Communication Disorders K08 DC00169 (H.A.H.), Baylor College of Medicine Mental Retardation Research Center NIH P30 HD24064 (L.G.S.) and NIH National Institute for Child Health and Development P01 HD39420 (L.G.S.).

	ELECTRONIC DATABASE INFORMATION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS ELECTRONIC DATABASE INFORMATION REFERENCES

 
UCSC genome browser: http://genome.ucsc.edu

	FOOTNOTES

 
^* To whom correspondence should be addressed at: Health Research and Education Center, Washington State University Spokane, Box 1495, Spokane, WA 99210-1495, USA. Tel: +1 5093686710; Fax: +1 5093587627; Email: lshaffer{at}wsu.edu

Present address: Health Research and Education Center, Washington State University, Spokane, Washington, USA.

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TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS ELECTRONIC DATABASE INFORMATION REFERENCES

 

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