Gene dosage methods as diagnostic tools for the identification of chromosome abnormalities

Cytogenetics is the part of genetics that deals with chromosomes, particularly with numerical and structural chromosome abnormalities, and their implications in congenital or acquired genetic disorders. Standard karyotyping, successfully used for the last 50 years in investigating the chromosome etiology in patients with infertility, fetal abnormalities and congenital disorders, is constrained by the limits of microscopic resolution and is not suited for the detection of subtle chromosome abnormalities. The ability to detect submicroscopic chromosomal rearrangements that lead to copy-number changes has escalated progressively in recent years with the advent of molecular cytogenetic techniques. Here, we review various gene dosage methods such as FISH, PCR-based approaches (MLPA, QF-PCR, QMPSF and real time PCR), CGH and array-CGH, that can be used for the identification and delineation of copy-number changes for diagnostic purposes. Besides comparing their relative strength and weakness, we will discuss the impact that these detection methods have on our understanding of copy number variations in the human genome and their implications in genetic counseling.     

High‐resolution molecular karyotyping in patients with developmental delay and/or multiple congenital anomalies in a clinical setting

Wincent J, Anderlid B‐M, Lagerberg M, Nordenskjöld M, Schoumans J. High‐resolution molecular karyotyping in patients with developmental delay and/or multiple congenital anomalies in a clinical setting. Microarray‐based comparative genomic hybridization (array‐CGH) enables genomewide investigation of copy‐number changes at high resolution and has recently been implemented as a clinical diagnostic tool. In this study we evaluate the usefulness of high‐resolution arrays as a diagnostic tool in our laboratory and investigate the diagnostic yield in the first 160 patients who were clinically referred for investigation of developmental delay (DD)/multiple congenital anomalies (MCA). During this period both 38K BAC‐arrays and 244K oligonucleotide‐arrays were used. Copy‐number variations (CNVs) not previously reported as normal variants were detected in 22.5% of cases. In 13.1% the aberrations were considered causal to the phenotype and in 9.4% the clinical significance was uncertain. There was no difference in diagnostic yield between patients with mild, moderate or severe DD. Although the effective resolution of the 244K oligonucleotide‐arrays was higher than the 38K BAC‐array, the diagnostic yield of both platforms was approximately equal and no causal aberrations <300 kb were detected in this patient cohort. We experienced that increasing the resolution of a whole genome screen in the diagnostic setting has its drawback of detecting an increased number of CNVs with uncertain contribution to a phenotype. Based on our experiences, array‐CGH is recommended as the first‐step analysis in the genetic evaluation of patients with DD and/or MCA.     

A new diagnostic workflow for patients with mental retardation and/or multiple congenital abnormalities: test arrays first

High-density single-nucleotide polymorphism (SNP) genotyping technology enables extensive genotyping as well as the detection of increasingly smaller chromosomal aberrations. In this study, we assess molecular karyotyping as first-round analysis of patients with mental retardation and/or multiple congenital abnormalities (MR/MCA). We used different commercially available SNP array platforms, the Affymetrix GeneChip 262K NspI, the Genechip 238K StyI, the Illumina HumanHap 300 and HumanCNV 370 BeadChip, to detect copy number variants (CNVs) in 318 patients with unexplained MR/MCA. We found abnormalities in 22.6% of the patients, including six CNVs that overlap known microdeletion/duplication syndromes, eight CNVs that overlap recently described syndromes, 63 potentially pathogenic CNVs (in 52 patients), four large segments of homozygosity and two mosaic trisomies for an entire chromosome. This study shows that high-density SNP array analysis reveals a much higher diagnostic yield as that of conventional karyotyping. SNP arrays have the potential to detect CNVs, mosaics, uniparental disomies and loss of heterozygosity in one experiment. We, therefore, propose a novel diagnostic approach to all MR/MCA patients by first analyzing every patient with an SNP array instead of conventional karyotyping.     

Density matters: comparison of array platforms for detection of copy-number variation and copy-neutral abnormalities

PurposeA combination of oligonucleotide and single-nucleotide polymorphism probes on the same array platform can detect copy-number abnormalities and copy-neutral aberrations such as uniparental disomy and long stretches of homozygosity. The single-nucleotide polymorphism probe density in commercially available platforms varies widely, which may affect the detection of copy-neutral abnormalities.MethodsWe evaluated the ability of array platforms with low (Oxford Gene Technology CytoSure ISCA uniparental disomy), mid-range (Agilent custom array), and high (Affymetrix CytoScan HD) single-nucleotide polymorphism probe density to detect copy-number variation, mosaicism, uniparental isodisomy, and absence of heterozygosity in 50 clinical samples.ResultsAll platforms reliably detected copy-number variation, mosaicism, and uniparental isodisomy; however, absence-of-heterozygosity detection varied significantly. The low-density array called absence-of-heterozygosity regions not confirmed by the other platforms and also overestimated the length of true absence-of-heterozygosity regions. Furthermore, the low- and mid-density platforms failed to detect some small absence-of-heterozygosity regions that were identified by the high-density platform.ConclusionVariation in single-nucleotide polymorphism density can lead to major discrepancies in the detection of and confidence in copy-neutral abnormalities. Although suitable for uniparental disomy detection, copy-number plus single-nucleotide polymorphism arrays with 30,000 or fewer unique single-nucleotide polymorphism probes miscall absence-of-heterozygosity regions due to identity by descent.Genet Med15 9, 706–712.     

Screening for copy-number alterations and loss of heterozygosity in chronic lymphocytic leukemia--a comparative study of four differently designed, high resolution microarray platforms

Screening for gene copy-number alterations (CNAs) has improved by applying genome-wide microarrays, where SNP arrays also allow analysis of loss of heterozygozity (LOH). We here analyzed 10 chronic lymphocytic leukemia (CLL) samples using four different high-resolution platforms: BAC arrays (32K), oligonucleotide arrays (185K, Agilent), and two SNP arrays (250K, Affymetrix and 317K, Illumina). Cross-platform comparison revealed 29 concordantly detected CNAs, including known recurrent alterations, which confirmed that all platforms are powerful tools when screening for large aberrations. However, detection of 32 additional regions present in 2-3 platforms illustrated a discrepancy in detection of small CNAs, which often involved reported copy-number variations. LOH analysis using dChip revealed concordance of mainly large regions, but showed numerous, small nonoverlapping regions and LOH escaping detection. Evaluation of baseline variation and copy-number ratio response showed the best performance for the Agilent platform and confirmed the robustness of BAC arrays. Accordingly, these platforms demonstrated a higher degree of platform-specific CNAs. The SNP arrays displayed higher technical variation, although this was compensated by high density of elements. Affymetrix detected a higher degree of CNAs compared to Illumina, while the latter showed a lower noise level and higher detection rate in the LOH analysis. Large-scale studies of genomic aberrations are now feasible, but new tools for LOH analysis are requested.     

Defining the impact of maternal cell contamination on the interpretation of prenatal microarray analysis

PurposeTo understand the ability of microarray-based comparative genomic hybridization to detect copy-number variation in the presence of maternal cell contamination.MethodsTo simulate maternal cell contamination, normal female DNA was mixed at various levels with DNA carrying known copy-number variations. Mixtures were run on a whole-genome 135K oligonucleotide-based array. Data were analyzed with custom analysis software.ResultsThe array and software design allowed detection of larger copy-number variations at higher levels of maternal cell contamination than smaller copy-number variations. The smallest duplications and deletions were obscured at 22–31% and 55–58% maternal cell contamination, respectively. With male fetal samples, the sex chromosome ratios started showing observable shifts at ~10% maternal cell contamination.ConclusionAs knowledge of the maternal cell contamination level aids in interpretation of array results, we recommend concurrent, independent maternal cell contamination studies for all fetal samples for accurate and timely results. With male fetal samples in our laboratory, interfering levels of maternal cell contamination can be excluded when the sex chromosome plots appear normal. Thus, reportable male microarray-based comparative genomic hybridization results may be occasionally achieved without maternal cell contamination studies. Because the effects of maternal cell contamination on microarray results are dependent on array platforms, experimental techniques, and software algorithms, each laboratory should perform its own analysis to determine acceptable levels of maternal cell contamination for its assays.Genet Med 2012:14(11):914–921     

Detection of mosaicism for genome imbalance in a cohort of 3,042 clinical cases using an oligonucleotide array CGH platform

Mosaicism for chromosome imbalance has traditionally been detected by karyotype analysis. The introduction of array CGH into clinical diagnostic laboratories and routine clinical practice has raised concerns as to the ability of this new test to detect the presence of more than one cell line. We present our validation data on the detection of chromosome mosaicism by oligonucleotide array CGH, and the cases detected in a cohort of 3042 clinical referrals. Using an artificial mosaicism series, we found that oligonucleotide array CGH using specific analysis parameters could accurately measure levels of mosaicism down to 10% and that the degree of mosaicism could be predicted from fluorescence ratios. We detected 12 cases of mosaicism in our clinical cohort, in 9 of which there was no previous indication of mosaicism. In two cases, G-banded chromosome analysis had been carried out previously, and had failed to detect the abnormal cell line. Three cases had mosaicism for the X chromosome and 9 involved autosomes, of which 4 were mosaic for whole chromosome trisomies, one for whole chromosome monosomy, and four were mosaic for segmental imbalances. We conclude that oligonucleotide array CGH has the power to detect a range of mosaic abnormalities in clinical diagnostic samples.     

Enhanced detection of clinically relevant genomic imbalances using a targeted plus whole genome oligonucleotide microarray

PurposeArray comparative genomic hybridization is rapidly becoming an integral part of cytogenetic diagnostics. We report the design, validation, and clinical utility of an oligonucleotide array which combines genome-wide coverage with targeted enhancement at known clinically relevant regions.MethodsProbes were placed every 75 kb across the entire euchromatic genome to establish a chromosomal “backbone” with a resolution of ∼500 kb, which is increased to ∼50 kb in targeted regions.ResultsFor validation, 30 samples showed 100% concordance with previous G-banding and/or fluorescence in situ hybridization results. Prospective array analysis of 211 clinical samples identified 33 (15.6%) cases with clinically significant abnormalities. Of these, 23 (10.9%) were detected by the “targeted” coverage and 10 (4.7%) by the genome-wide coverage (average size of 3.7 Mb). All abnormalities were verified by fluorescence in situ hybridization, using commercially available or homebrew probes using the 32K bacterial artificial chromosome set. Four (1.9%) cases had previously reported imbalances of uncertain clinical significance. Five (2.4%) cases required parental studies for interpretation and all were benign familial variants.ConclusionsOur results highlight the enhanced diagnostic utility of a genome-wide plus targeted array design, as the use of only a targeted array would have failed to detect 4.7% of the clinically relevant imbalances.     

A male newborn with VACTERL association and Fanconi anemia with a FANCB deletion detected by array comparative genomic hybridization (aCGH)

We report on a male newborn with multiple congenital abnormalities consistent with the diagnosis of VACTERL association (vertebral, anal, cardiac, tracheo-esophageal fistula, renal, and limb anomalies), who had Fanconi anemia (complementation group B) recognized by the detection of a deletion in chromosome Xp22.2 using an oligonucleotide array. The diagnosis of Fanconi anemia was confirmed by increased chromosomal breakage abnormalities observed in cultured cells that were treated with cross-linking agents. This is the first report in the literature of Fanconi anemia complementation group B detected by oligonucleotide array testing postnatally.     

Detection of chromosomal imbalances in children with idiopathic mental retardation by array based comparative genomic hybridisation (array-CGH)

Chromosomal aberrations are a common cause of multiple anomaly syndromes that include growth and developmental delay and dysmorphism. Novel high resolution, whole genome technologies, such as array based comparative genomic hybridisation (array-CGH), improve the detection rate of submicroscopic chromosomal abnormalities allowing re-investigation of cases where conventional cytogenetic techniques, Spectral karyotyping (SKY), and FISH failed to detect abnormalities. We performed a high resolution genome-wide screening for submicroscopic chromosomal rearrangements using array-CGH on 41 children with idiopathic mental retardation (MR) and dysmorphic features. The commercially available microarray from Spectral Genomics contained 2600 BAC clones spaced at approximately 1 Mb intervals across the genome. Standard chromosome analysis (>450 bands per haploid genome) revealed no chromosomal rearrangements. In addition, multi-subtelomeric FISH screening in 30 cases and SKY in 11 patients did not detect any abnormality. Using array-CGH we detected chromosomal imbalances in four patients (9.8%) ranging in size from 2 to 14 Mb. Large scale copy number variations were frequently observed. Array-CGH has become an important tool for the detection of chromosome aberrations and has the potential to identify genes involved in developmental delay and dysmorphism. Moreover, the detection of genomic imbalances of clinical significance will increase knowledge of the human genome by performing genotype-phenotype correlation.     

0.5 Mb Array as a First‐Line Prenatal Cytogenetic Test in Cases without Ultrasound Abnormalities and Its Implementation in Clinical Practice

Using whole‐genome array testing instead of karyotyping in prenatal diagnosis for all indications may be desirable because of the higher diagnostic yield and shorter reporting time. The goal of this research was finding the optimal array resolution that could replace routine prenatal karyotyping in cases without ultrasound abnormalities, for example, referred for advanced maternal age or abnormal first trimester screening. As variants of unknown clinical significance (VOUS), if reported, might complicate decision‐making about continuation of pregnancy, such an optimal array resolution should have a high abnormality detection rate and reveal a minimal amount of VOUS. The array data of 465 fetuses were retrospectively evaluated with several resolution levels, and the Decipher microdeletion/microduplication syndrome list was reviewed to assess what could be theoretically missed with a lower resolution. A 0.5‐Mb resolution showed a high diagnostic yield potential and significantly minimized the number of VOUS. Based on our experience, we recommend genomic SNP array as a first‐tier test in prenatal diagnosis. The resolution should be chosen based on the indication. In cases of fetal ultrasound abnormalities or intrauterine fetal death (IUFD), high‐resolution analysis should be done. In other cases, we advise replacing karyotyping by SNP array analysis with 0.5 Mb resolution.     

BAC-based PCR fragment microarray: high-resolution detection of chromosomal deletion and duplication breakpoints

The introduction of molecular techniques in conjunction with classical cytogenetic methods has in recent years greatly improved the diagnostic potential for chromosomal abnormalities. In particular, microarray-comparative genomic hybridization (CGH) based on the use of BAC clones promises a sensitive strategy for the detection of DNA copy-number changes on a genomewide scale, offering a resolution as high as >30,000 "bands" (as defined by the number of BACs within the currently highest-density BAC array) [Ishkanian et al., 2004]. We have tested the possibility of further increasing this resolution using PCR fragments generated from individual BAC clones. Using this approach, we have efficiently defined the proximal and distal breakpoints in two cytogenetic cases, one duplication and one deletion, to within 5-20 kb. The results support the potential use of BAC-based PCR fragments to further improve the resolution of the microarray-CGH strategy by an order of magnitude.     

Systematic evaluation of variability in ChIP-chip experiments using predefined DNA targets

The most widely used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. We found that microarray platform choice is not the primary determinant of overall performance. In fact, variation in performance between labs, protocols, and algorithms within the same array platform was greater than the variation in performance between array platforms. However, each array platform had unique performance characteristics that varied with tiling resolution and the number of replicates, which have implications for cost versus detection power. Long oligonucleotide arrays were slightly more sensitive at detecting very low enrichment. On all platforms, simple sequence repeats and genome redundancy tended to result in false positives. LM-PCR and WGA, the most popular sample amplification techniques, reproduced relative enrichment levels with high fidelity. Performance among signal detection algorithms was heavily dependent on array platform. The spike-in DNA samples and the data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated.     

Construction of a high-density and high-resolution human chromosome X array for comparative genomic hybridization analysis

The human chromosome X is closely associated with congenital disorders and mental retardation (MR), because it contains a significantly higher number of genes than estimated from the proportion in the human genome. We constructed a high-density and high-resolution human chromosome X array (X-tiling array) for comparative genomic hybridization (CGH). The array contains a total of 1,001 bacterial artificial chromosome (BACs) throughout chromosome X except pseudoautosomal regions and two BACs specific for Y. In four hybridizations using DNA samples from healthy males, the ratio of each spotted DNA was scattered between −3SD and 3SD, corresponding to a log₂ ratio of −0.35 and 0.35, respectively. Using DNA samples from patients with known congenital disorders, our X-tiling array was proven to discriminate one-copy losses and gains together with their physical sizes, and also to estimate the percentage of a mosaicism in a patient with mos 45,X[13]/46,X,r(X)[7]. Furthermore, array-CGH in a patient with atypical Schinzel-Giedion syndrome disclosed a 1.1-Mb duplication at Xq22.3 including a part of the IL1RAPL2 gene as a likely causative aberration. The results indicate our in-house X-tiling array to be useful for the identification of cryptic copy-number aberrations containing novel genes responsible for diseases such as congenital disorders and X-linked MR.     

High-throughput analysis of chromosome abnormality in spontaneous miscarriage using an MLPA subtelomere assay with an ancillary FISH test for polyploidy

Chromosome analysis of spontaneous miscarriages is clinically important but is hampered by frequent tissue culture failure and relatively low-resolution analysis. We have investigated replacement of conventional karyotype analysis with a quantitative subtelomere assay performed on uncultured tissue samples, which is based on Multiplex Ligation-Dependent Probe Amplification. This assay is suitable for this purpose as approximately 98% of all observed karyotype abnormalities in spontaneous miscarriages involve copy-number change to one or more subtelomere regions. A pilot study has compared karyotyping and subtelomere analysis on 78 samples. Extensive tissue necrosis accounted for failure of both karyotyping and subtelomere testing in four (5.1%) samples. Excluding these, there were no (0/74) subtelomere test failures compared to 9.5% (7/74) karyotype failures. Twenty-two (30%) whole chromosome aneuploidies and five (6.8%) structural abnormalities were detected using the subtelomere assay. With the exception of three cases of triploidy, all karyotype abnormalities were detected by the subtelomere assay. Following on from this study, a further 100 samples were tested using the subtelomere assay in conjunction with a simple ancillary FISH test using uncultured cells to exclude polyploidy in the event of a normal subtelomere assay result. Except for three necrotic samples, tests results were obtained for all cases revealing 18 abnormalities including one case of triploidy. Taking into consideration the high success rate for the combined MLPA and FISH test results, and the very significant additional advantages of cost-effective, high-throughput batching, and automated, objective analysis, this approach greatly facilitates routine investigation of chromosome abnormalities in spontaneous miscarriage.     

A 400kb duplication, 2.4Mb triplication and 130kb duplication of 9q34.3 in a patient with severe mental retardation

The presence of a duplication as well as a triplication in one chromosome is a rare rearrangement and not easy to distinguish with routine chromosomal analysis. Recent developments in array technologies, however, not only allow screening of the whole genome at a higher resolution, but also make it possible to characterize complex chromosomal rearrangements in more detail. Here we report a molecular cytogenetic analysis of a 16-year old female with severe mental retardation and an abnormality at the end of the long arm of chromosome 9. Subtelomeric multiplex ligation-dependent probe amplification (MLPA) analysis revealed that the extra material originated from the telomeric end of chromosome 9q. Fine mapping using a high-resolution single nucleotide polymorphism (SNP) array detected a duplication of approximately 400kb upstream of a approximately 2.4Mb triplication followed by a duplication of approximately 130kb of chromosome 9q34.3. This study underscores the value of combining conventional karyotyping with novel array technologies to unravel complex chromosomal alterations in order to study their phenotypic impact.     

Detection of pathogenic gene copy number variations in patients with mental retardation by genomewide oligonucleotide array comparative genomic hybridization

Genomic imbalance is a major cause of developmental disorders. Microarray-based comparative genomic hybridization (aCGH) has revealed frequent imbalances associated with clinical syndromes, but also a large number of copy number variations (CNVs), which have complicated the interpretation of results. We studied 100 consecutive patients with unexplained mental retardation and a normal karyotype using several platforms of CGH arrays. A genomewide array with 44,290 oligonucleotide probes (OaCGH44K) detected imbalances in 15% of cases studied with sizes ranged from 459 kb to 19 Mb while revealing a small number of CNVs (0.72/individual). Another platform with approximately 240,000 oligonucleotide probes (OaCGH244K) revealed a large number of CNVs (20/individual) in selected cases and their normal parents. We used a comprehensive approach for interpreting the results of aCGH, including consideration of the size, inheritance and gene content of CNVs, and consultation with an online Database of Genomic Variants (DGV) and Online Mendelian Inheritance in Men (OMIM) for information on the genes involved. Our study suggests that genomewide oligonucleotide arrays such as the OaCGH44K platform can be used as a powerful diagnostic tool for detection of genomic imbalances associated with unexplained mental retardation or syndromic autism spectrum disorders. It is interesting to note that a small number of common variants were revealed by OaCGH244K in some study subjects but not in their parents and that some inherited CNVs had altered breakpoints. Further investigations on these alterations may provide useful information for understanding the mechanism of CNVs.     

Fryns syndrome phenotype caused by chromosome microdeletions at 15q26.2 and 8p23.1

Background: Fryns syndrome (FS) is the commonest autosomal recessive syndrome in which congenital diaphragmatic hernia (CDH) is a cardinal feature. It has been estimated that 10% of patients with CDH have FS. The autosomal recessive inheritance in FS contrasts with the sporadic inheritance for the majority of patients with CDH and renders the correct diagnosis critical for accurate genetic counselling. The cause of FS is unknown. Methods: We have used array comparative genomic hybridisation (array CGH) to screen patients who have CDH and additional phenotypic anomalies consistent with FS for cryptic chromosome aberrations. Results: We present three probands who were previously diagnosed with FS who had submicroscopic chromosome deletions detected by array CGH after normal karyotyping with G-banded chromosome analysis. Two female infants were found to have microdeletions involving chromosome band 15q26.2 and one male had a deletion of chromosome band 8p23.1. Conclusions: We conclude that phenotypes similar to FS can be caused by submicroscopic chromosome deletions and that high resolution karyotyping, including array CGH if possible, should be performed prior to the diagnosis of FS to provide an accurate recurrence risk in patients with CDH and physical anomalies consistent with FS.     

Exonic duplication CNV of NDRG1 associated with autosomal-recessive HMSN-Lom/CMT4D

PurposeCopy-number variations as a mutational mechanism contribute significantly to human disease. Approximately one-half of the patients with Charcot–Marie–Tooth (CMT) disease have a 1.4 Mb duplication copy-number variation as the cause of their neuropathy. However, non-CMT1A neuropathy patients rarely have causative copy-number variations, and to date, autosomal-recessive disease has not been associated with copy-number variation as a mutational mechanism.MethodsWe performed Agilent 8 × 60K array comparative genomic hybridization on DNA from 12 recessive Turkish families with CMT disease. Additional molecular studies were conducted to detect breakpoint junctions and to evaluate gene expression levels in a family in which we detected an intragenic duplication copy-number variation.ResultsWe detected an ~6.25 kb homozygous intragenic duplication in NDRG1, a gene known to be causative for recessive HMSNL/CMT4D, in three individuals from a Turkish family with CMT neuropathy. Further studies showed that this intragenic copy-number variation resulted in a homozygous duplication of exons 6–8 that caused decreased mRNA expression of NDRG1.ConclusionExon-focused high-resolution array comparative genomic hybridization enables the detection of copy-number variation carrier states in recessive genes, particularly small copy-number variations encompassing or disrupting single genes. In families for whom a molecular diagnosis has not been elucidated by conventional clinical assays, an assessment for copy-number variations in known CMT genes might be considered.     

Chromosomal copy number changes in patients with non-syndromic X linked mental retardation detected by array CGH

Several studies have shown that array based comparative genomic hybridisation (CGH) is a powerful tool for the detection of copy number changes in the genome of individuals with a congenital disorder. In this study, 40 patients with non‐specific X linked mental retardation were analysed with full coverage, X chromosomal, bacterial artificial chromosome arrays. Copy number changes were validated by multiplex ligation dependent probe amplification as a fast method to detect duplications and deletions in patient and control DNA. This approach has the capacity to detect copy number changes as small as 100 kb. We identified three causative duplications: one family with a 7 Mb duplication in Xp22.2 and two families with a 500 kb duplication in Xq28 encompassing the MECP2 gene. In addition, we detected four regions with copy number changes that were frequently identified in our group of patients and therefore most likely represent genomic polymorphisms. These results confirm the power of array CGH as a diagnostic tool, but also emphasise the necessity to perform proper validation experiments by an independent technique.