Human Reproduction Update

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Human Reproduction Update, Vol.10, No.3 pp.221-226, 2004
© European Society of Human Reproduction and Embryology 2004; all rights reserved

A cytogeneticist’s perspective on genomic microarrays

Lisa G. Shaffer¹ and Bassem A. Bejjani

Health Research and Education Center, Washington State University Spokane, Sacred Heart Medical Center, and Signature Genomic Laboratories, Spokane, Washington, USA ¹ To whom the correspondence should be addressed at: Health Research and Education Center, Washington State University Spokane, PO Box 1495, Spokane, WA 99210. e-mail: lshaffer{at}wsu.edu

	Abstract

TOP Abstract Introduction Advances in molecular... Molecular cytogenetics for the... Genomic microarrays Array CGH for clinical... Conclusions Acknowledgements References

 
The identification of cytogenetic imbalance is an important component of clinical genetics. About 1 in 154 newborns has a chromosome abnormality. Conventional cytogenetic analysis has enabled the identification of microscopic alterations of the chromosomes. The development of fluorescence in situ hybridization (FISH) and other molecular methodologies has made possible the identification of submicroscopic aberrations. An additional development was comparative genomic hybridization (CGH), a method that directly compares two genomes for DNA copy differences. As first developed, the substrate for CGH analysis is normal metaphase chromosomes. Recently, CGH has been applied to microarrays (array CGH) constructed from large insert clones to identify chromosome imbalance. Array CGH has many advantages over conventional cytogenetic and molecular cytogenetic techniques. Array CGH can be comprehensive (genome-wide), high resolution, amenable to automation, rapid, and sensitive. We anticipate that array CGH will be employed in the clinical cytogenetics laboratory in the near future and will lead to the identification of the chromosomal basis of new syndromes and existing genetic conditions.

Key words: comparative genomic hybridization/fluorescence in situ hybridization/genetic diagnostics/microarray

	Introduction

TOP Abstract Introduction Advances in molecular... Molecular cytogenetics for the... Genomic microarrays Array CGH for clinical... Conclusions Acknowledgements References

 
Chromosomal abnormalities often cause specific and complex phenotypes resulting from an imbalance in the normal dosage of genes located in a particular chromosomal segment. Many multiple malformation syndromes are caused by deletion (or duplication) of genomic regions. These have been identified through patients with large, visible deletions or through the rare patient carrying a translocation or other chromosomal rearrangement. The development of chromosomal banding techniques in the early 1970s facilitated the identification of chromosomal abnormalities. These techniques allowed for the unambiguous identification of each human chromosome and the detection of aneuploidy and many large structural rearrangements, including translocations, deletions and duplications. Banding techniques, particularly G-banding, are now routine procedures in all clinical cytogenetics laboratories. Subtle cytogenetic alterations are frequently not detectable by routine banding methods and require the application of high-resolution cytogenetic techniques. A typical, routinely prepared metaphase cell contains 400–500 bands per haploid genome. At this stage of chromosome condensation, deletions or duplications of >10 Mb can be detected reliably. High-resolution banding techniques can achieve 1000 bands per haploid genome, although this labour-intensive application is not used routinely in the clinical cytogenetics laboratory. At this level of resolution, an alteration of 3–5 Mb can be detected. Alterations <3 Mb are extremely difficult to detect reliably with these methods (reviewed in Shaffer et al., 2001). Chromosome analysis is relatively labour-intensive and has limitations that include the inconsistency with which band resolution can be routinely achieved and the difficulty in visualizing some rearrangements due to the staining properties of specific regions of the genome (e.g. duplication of a small band is likely to be missed). To overcome some of these limitations and to detect cryptic (submicroscopic) alterations, fluorescence in situ hybridization (FISH) was developed.

	Advances in molecular cytogenetics

TOP Abstract Introduction Advances in molecular... Molecular cytogenetics for the... Genomic microarrays Array CGH for clinical... Conclusions Acknowledgements References

 
Non-isotopic methods of in situ hybridization were developed in the 1980s and 1990s to detect subtle and submicroscopic alterations. The most common method is FISH. In this method, human DNA that is propagated in a variety of vectors, such as plasmids, cosmids, bacterial artificial chromosomes (BAC’s), P1-derived artificial chromosomes (PAC’s) or yeast artificial chromosomes (YAC’s), is labelled and used as a probe to hybridize to specific regions of human chromosomes. Applications of FISH include gene mapping and detection of cytogenetic rearrangements and aneuploidy. Specifically, probes to the common aneuploidies (13, 18, 21, X and Y) are used to rapidly screen pregnancies at risk (Ward et al., 1993). Many unique sequence or locus-specific probes were developed for particular microdeletions (e.g. Williams syndrome; Nickerson et al., 1995), multiple microdeletion targets (e.g. Ligon et al., 1997), fusion gene rearrangements in cancer (e.g. BCR/ABL; Dewald et al., 1993), and all telomeric regions (NIH and Institute of Molecular Medicine Collaboration, 1996). These probes to unique chromosome ends (telomere-region-specific probes) (NIH and Institute of Molecular Medicine Collaboration, 1996) have also been arrayed on a microscope slide for the simultaneous screening of all 41 unique telomeric regions on metaphase chromosomes (Knight et al., 1997, 2000). In addition to locus-specific FISH probes, other targeted probes include whole-chromosome painting probes, which can be produced in a 24-colour format for karyotyping. These technical advances allow for the detection of all 24 chromosomes (22 autosomes and the X and Y chromosomes) in 24 unique colour combinations (Schrock et al., 1996, 1997; Speicher et al., 1996). However, comparative genomic hybridization (CGH) is arguably the greatest achievement in the area of genome-wide screening strategies. In CGH, two genomes are directly compared for DNA content differences (Kallioniemi et al., 1992). The two genomes are differentially labelled in two distinct fluorochromes and compared by examining the ratio of the two fluorochromes on metaphase chromosomes. However, these techniques that rely on metaphase chromosomes for the detection of subtle rearrangements have a limited resolution that is defined by the metaphase chromosomes to a level of a 450-band karyotype (5–10 Mb changes). Although resolutions of 3–3.5 Mb have been reported (Kirchhoff et al., 2001), these resolutions are unattainable by most clinical cytogenetics laboratories. Additionally, 24-colour karyotyping cannot detect certain rearrangements, such as deletions and intrachromosomal duplications, and CGH cannot detect balanced rearrangements, such as reciprocal translocations, Robertsonian translocations, and inversions.

	Molecular cytogenetics for the diagnosis of submicroscopic and subtelomeric alterations

TOP Abstract Introduction Advances in molecular... Molecular cytogenetics for the... Genomic microarrays Array CGH for clinical... Conclusions Acknowledgements References

 
The development of a large number of locus-specific probes for known microdeletion syndromes and cancer loci has facilitated the rapid laboratory diagnosis of these disorders. The development of telomere-region-specific FISH probes has led to the identification of deletions and other unbalanced rearrangements in individuals with mental retardation and otherwise apparently normal karyotypes (reviewed in Knight and Flint, 2000). Examining the telomeric ends by FISH has increased the resolution of the analysis of these regions by 10-fold over conventional cytogenetics (NIH and Institute of Molecular Medicine Collaboration, 1996). Various publications have reported detection rates for telomeric deletions among patients with mental retardation to be between 0.5 and 23%, depending on the mode of ascertainment and study inclusion criteria (e.g. Knight et al., 1999). This has also led to the recognition of new terminal deletion syndromes such as monosomy 1p36 (Heilstedt et al., 2003a,b).

However, limitations of the established molecular cytogenetics methods include low resolution (e.g. CGH has essentially the same resolution of routine G-banding, alterations of 5–10 Mb), the need to have targeted FISH (e.g. FISH to known or suspected microdeletions), and fairly labour-intensive procedures (e.g. telomere FISH to 41 unique chromosome ends requires 8–23 hybridization reactions depending on the format used). It is likely that newer array-based methods of genome screening will allow the detection of submicroscopic genomic imbalance in a more comprehensive and higher resolution format.

	Genomic microarrays

TOP Abstract Introduction Advances in molecular... Molecular cytogenetics for the... Genomic microarrays Array CGH for clinical... Conclusions Acknowledgements References

 
Recently, genomic microarrays were developed for CGH applications. Array CGH is based on the same principles as traditional CGH, except that cloned DNA segments (e.g. BAC) are substituted for metaphase chromosomes as targets for the hybridization (Solinas-Toldo et al., 1997; Geschwind et al., 1998; Pinkel et al., 1998; Albertson et al., 2000; Bruder et al., 2001; Snijders et al., 2001; Yu et al., 2003) (Figure 1). Targets for array CGH can also be PCR-generated sequences (Snijders et al., 2001; Fiegler et al., 2003; Veltman et al., 2003a), cDNA clones (Pollack et al., 1999), or oligonucleotides (Albertson and Pinkel, 2003). Ratios between labelled genomes are compared with computer imaging and software analysis. Many types of microarrays exist; this review will focus on genomic microarrays.

View larger version (75K): [in this window] [in a new window]   Figure 1. (A) Large-insert clones, derived from a human chromosome, are printed onto a glass microscope slide (arrayed). The array can be stained to show the morphology and placement of each ‘spot’ of cloned DNA (far right). (B) Genomic DNA samples from a control (left) and test (right) are differentially labelled with two different fluorochromes. The labelled DNA is mixed and placed on the microarray. Computer imaging reveals a yellow hybridization colour for all clones that are in equal proportion between the control DNA and test DNA (middle and lower left). Those clones deficient in the test DNA, as compared to the control DNA, will appear, in this example, more green; those clones in excess in the test DNA, as compared to the control DNA, will appear, in this example, more red (middle). A plot of the ratio between control and test DNA for each clone (lower right) will reveal dosage differences, visualized as a deviation of the ratio from zero (horizontal red line).

 
Arrays have been developed for the analysis of whole chromosomes (Albertson et al., 2000; Bruder et al., 2001), portions of chromosomes (Yu et al., 2003), site-specific regions (i.e. telomeric regions; Veltman et al., 2002) and the entire genome (Snijders et al., 2001; Veltman et al., 2003b; Vissers et al., 2003). The methodologies involved in constructing and analysing genomic microarrays have been reviewed (Beheshti et al., 2002; Carter et al., 2002).

Array CGH has been applied to a number of malignancies including breast cancer (Daigo et al., 2001; Pollack et al., 2002; Albertson, 2003); glioblastoma (Hui et al., 2001); rhabdomyosarcoma (Pandita et al., 1999); nasopharyngeal carcinoma (Hui et al., 2002); ovarian cancer (Schraml et al., 2003); gastric cancer (Tay et al., 2003; Weiss et al., 2003); bladder tumours (Veltman et al., 2003b); lymphoma (Wessendorf et al., 2003); and sarcomas and adenocortical tumours (Fritz et al., 2002; Zhao et al., 2002). Array CGH has also been used for constitutional cytogenetic abnormalities through screening loci selected from the whole genome (Veltman et al., 2003b; Vissers et al., 2003), telomeric anomalies (Veltman et al., 2002) and rearrangements of 1p36 (Ballif et al., 2003; Yu et al., 2003; Figure 2).

View larger version (30K): [in this window] [in a new window]   Figure 2. A comparative genomic hybridization to a microarray constructed from a 10.5 Mb contig from 1p36 (Heilstedt et al., 2003a). (A) Hybridized microarray for comparison of DNA from a subject with a 1p36 deletion with a normal control sample. (B) Ratio of subject DNA to control DNA for each BAC on the microarray is plotted for each clone from the 1p telomere to the more proximal, centromeric clones (1–97). Clones 98–138 are single clones to the 41 subtelomeric regions. Note loss of single 1p telomere clone at position 98. Clones 139–144 are to the X (n = 3) and Y (n = 3) chromosomes. Data points below –0.5 indicate regions of deletion. Ratios clustering around 0.0 are DNA copy numbers in the normal range. Note that clones 1–4 cluster around zero. These clones map to the telomere-associated repeats and are contained in multiple copies on other chromosomes. Thus, array CGH with these clones does not show a significant difference between the deletion subject and the normal control. (C) Diagram showing the region of 1p36 that is deleted for this subject. Red indicates deletion and green indicates retention (normal). The 1p36 contig is shown as a thin horizontal line, with the BAC addresses and accession numbers listed below in their map positions. Microsatellite markers and individual loci used for FISH are listed above the thin contig line. Those markers in black were not tested on this patient. Those markers in red were deleted and those markers in green were normal. The thick red/green line below the contig line is the array CGH results. The array CGH results agree with the results obtained by FISH and microsatellite analysis. Note that the breakpoint in this subject falls between markers D1S2660 (deleted) and D1S1608 (retained). Clone RPI11-1096P7 contains the breakpoint as determined by microsatellite analysis, although the ratio for this clone as determined by array CGH indicated deletion. This illustrates the limits of array CGH when a breakpoint falls within a clone.

 
Array CGH offers a number of advantages over conventional cytogenetic analysis and FISH. Array CGH can be highly comprehensive, amenable to very high resolution, sensitive, and fast (Bruder et al., 2001). Because the arrays use BAC’s, alterations are immediately linked to genomic/genetic markers, and the genomic resolution is determined by the map distances between markers (targets) or by the length of the clones used (Pinkel et al., 1998). Recently, >8000 BAC’s that were each linked to a known target were mapped to individual chromosome bands (Cheung et al., 2001). The average density of these clones is 2.6 per Mb, and 86% of these BAC’s have a sequence that has been deposited in the public databases. This resource, in addition to the numerous large-insert clones that have been sequenced (Lander et al., 2001; Venter et al., 2001), provides a framework from which BAC CGH microarrays can be designed (Antonarakis, 2001).

The development of a CGH-based microarray will (i) increase the resolution (e.g. the resolution is dependent on the number of BAC’s placed on the array), (ii) eliminate the need for targeted FISH experiments (i.e. needing to know where to FISH), and (iii) substantially decrease the labour involved (e.g. FISH using BAC’s covering 5 Mb of each telomeric region would require >2000 individual hybridizations and analyses).

	Array CGH for clinical diagnosis

TOP Abstract Introduction Advances in molecular... Molecular cytogenetics for the... Genomic microarrays Array CGH for clinical... Conclusions Acknowledgements References

 
With the development of microarrays that contain loci selected from the entire genome (Snijders et al., 2001; Vissers et al., 2003), much interest has been generated in providing this format for cytogenetic diagnosis. However, the whole-genome approach that researchers are using is likely to generate data that may be difficult to interpret. Alterations in regions of the genome that do not have established clinical relevance will also be burdensome on the clinical cytogeneticists for useful interpretation. Based on our experience with the subtelomeric FISH probes, which hybridize to only 41 regions in the genome, we found several polymorphisms (Shaffer et al., 1999; Ballif et al., 2000). Thus, with a whole genome approach, polymorphisms are expected to be abundant.

Our recent experience in building a microarray for 1p36 (Yu et al., 2003; Ballif et al., 2003) and a whole chromosome 16 microarray (Gregato et al., 2003) has taught us important lessons that need to be considered when designing and implementing a diagnostically useful microarray. First and foremost is the necessity of FISH-mapping all clones that will be used on the array. FISH-mapping will uncover mismapped clones and those that cross-hybridize to either multiple sites within a chromosome or to multiple chromosomes. Either situation is not suitable for diagnostics. Second, because the microarray is essentially a simultaneous FISH experiment with several hundred or thousand probes, all probes destined for the microarray should work equally well under the same experimental FISH conditions. Third, the array design should include reference clones that are used for data normalization. Finally, the building of the 10.5 Mb contig microarray for 1p36 (Yu et al., 2003) clearly demonstrated that the presence of multiple clones on the microarray to the same locus provides a necessary redundancy for accurate interpretation of results. Thus, a diagnostically useful microarray must be reliable, must accurately detect the chromosome abnormalities assayed, and must provide interpretable results. Until such an array is constructed and clinical confidence is established, we advocate FISH confirmation of microarray results followed by karyotypic analysis to delineate the abnormalities. As with the identification of any chromosome rearrangement in a child or fetal sample, parental array CGH, FISH or karyotyping should be performed to exclude a parental balanced rearrangement or familial polymorphism. Caution should be exercised when interpreting single clone imbalances for regions of unknown clinical significance. Until clinical relevance is established, ‘whole genome’ microarrays should not be used for clinical applications, particularly prenatal diagnosis. Additionally, the smallest amount of DNA needed for array CGH from the subject has not been fully delineated. It remains to be determined if array CGH can be performed on direct (uncultured) prenatal specimens or single cells for preimplantation genetic diagnosis (PGD). Finally, array CGH will not detect balanced rearrangements (inversions and reciprocal and Robertsonian translocations). Thus, karyotype analysis should be performed on all normal microarray results when a chromosome anomaly is still suspected based on the clinical phenotype because ‘balanced’ rearrangements may disrupt a gene or alter gene expression through a position effect, which will be undetectable on a genomic microarray.

	Conclusions

TOP Abstract Introduction Advances in molecular... Molecular cytogenetics for the... Genomic microarrays Array CGH for clinical... Conclusions Acknowledgements References

 
Array CGH can detect cytogenetic anomalies at a resolution unachievable by conventional techniques. Imbalances as small as the size of the clones used will identify new syndromes and will elucidate the molecular basis of clinically recognized syndromes. Given the great achievements in the past few years, 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. Diagnostic guidelines should be established to enable the reliable and accurate detection of chromosome imbalances by array CGH in the cytogenetics laboratory.

	Acknowledgements

TOP Abstract Introduction Advances in molecular... Molecular cytogenetics for the... Genomic microarrays Array CGH for clinical... Conclusions Acknowledgements References

 
We thank Aaron Theisen, Washington State University Spokane, for assistance with the literature review and his critical editing of the manuscript and Drs Blake Ballif, Wei Yu and Chad Shaw, Baylor College of Medicine, Houston, TX, for images for Figures 1 and 2.

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