This study is the largest series of patients reported who have been screened for chromosomal imbalances with a 1 Mb resolution BAC array. In a total of 140 patients, 28 chromosomal imbalances were detected (20%). These included seven duplications, 18 deletions, and three unbalanced translocations. To determine the causal role of these chromosomal aberrations, parents were investigated in 24 of 28 patients. In addition, the Toronto database of normal variants was consulted. About three quarters (17/24) of the observed chromosomal aberrations were de novo and not reported before as a normal variant. In one patient for whom the parents could not be tested, available phenotypic data for similar published cases indicated that the genotype could explain the observed phenotype, and in one patient with inherited deletion the mother was equally affected. This brings the total of clinically relevant imbalances to 19. Taking into account these data and excluding those subtelomeric imbalances that could have been detected by FISH or MLPA/MAPH analysis, our study has identified 11 clinically relevant imbalances (8%) undetectable by karyotyping and subtelomeric screening. This is in accordance with previous findings of 10–15% causal interstitial submicroscopic imbalances in patients with MCA/MR.10,11,12,13,14
Imbalances identified thus far in MCA/MR patients have been positioned on the human genome map in order to assess their genomic distribution and to detect overlapping regions. This map further confirms that most imbalances are scattered across the genome.
From our data and data from other published reports it has become clear that the clinical application of array CGH poses new challenges. While it is assumed that de novo alterations result in the observed phenotype, only the recurrent association of imbalances with specific phenotypic features will reinforce this causal relation. Hence, it will be essential to collect genotypic and phenotypic information on a large number of MCA/MR patients. In contrast to de novo alterations, many chromosomal imbalances are inherited. Although it is likely that frequently occurring genomic CNVs may not have major disease causing phenotypic effects, rare variants, such as the six familial inherited imbalances detected in this study, should be evaluated with care. In particular, imbalances of regions which are recurrently involved in familial transmission from a normal parent to affected children will pose specific problems for genetic counselling, as illustrated by the 22q11.2 duplication. This is in line with previous observations that 22q11 duplications result in diverse phenotypes from normal to mild to severe, and sharing a tendency for velopharyngeal insufficiency with DiGeorge/VCFS (velo‐cardio‐facial syndrome) but with other distinctive characteristics as well.24,25
The 22q11 duplication syndrome may hallmark a novel paradox encountered by molecular karyotyping, as the causal relation between a chromosomal anomaly and an associated phenotype becomes blurred. Hence, imbalances inherited from phenotypical normal parents may contribute to the phenotype through variable penetrance or expressivity, or both, through epigenetic effects, or by uncovering a recessive mutation on the non‐deleted allele. To understand the involvement of these variations in the observed phenotypes, it will be necessary not only to collect benign variation in the genome and information on de novo imbalances associated with disease phenotypes, but also to collect both genotype and phenotype information from patients with familial inherited imbalances and phenotypically normal parents. To start this data collection, both genotype and phenotype data from all patients who consented was submitted at the DECIPHER database (http://www.sanger.ac.uk/Postgenomics/decipher/
Segmental chromosomal imbalances in mosaic state are causal in several MCA/MR syndromes.26
The present study illustrates that array CGH may detect segmental chromosomal imbalances which may be overlooked in standard karyotyping when a small number of cells is analysed or when the abnormality is too small to arouse suspicion. A remarkable observation in one of the mosaics was that phytohaemagglutinin stimulation of lymphocytes and subsequent short culture apparently induced a selective growth advantage for the normal cells. Clearly, such culture effects can bias the final cytogenetic observations, as was observed in patient 14. Presently a theoretical model is being developed which should enhance the sensitivity for the detection of low grade mosaicism. Clearly, the presence of a large deletion present in as few as 5% of cells can easily be detected. The ability to detect low grade mosaics will allow the detection of chromosomal aneuploidies in highly contaminated specimens such as aborted fetuses27
and in the analysis of tumours and leukaemias.28
In all reports, including this study, the number of deletions (57) was greater than the number of duplications (24). This may have both a technical and a biological component. Technically, most threshold algorithms may favour more false negatives for duplication events as compared with deletion events. Most threshold algorithms determine cut offs for both deletions and duplications at equal distance from the mean of all intensity ratios. As the intensity ratios for chromosomal deletions are more distant from the mean (ratio of 1/2) as compared with the intensity ratios observed for duplications (ratio of 3/2), inevitably there is a greater chance that some duplications may be missed. Second, there may be a biological bias. Duplications generally result in a milder phenotype; therefore there may be a selection bias in this patient population. In addition, the frequency of random duplication events in the human genome may be lower than the frequency of deletion events. Van Ommen29
estimated the frequency of deletion events to be one in every eight births, and the duplication frequency one in every 50 births. This suggests that the number of deletion events is about sixfold greater than the number of duplication events. In patients with MCA/MR, deletions outnumber duplications by approximately twofold.
In conclusion, we confirm that a high percentage of MCA/MR cases hitherto considered idiopathic is caused by submicroscopic chromosomal imbalances. Consequently, screening of selected patients with normal karyotypes seems desirable and feasible. The availability of commercial platforms and improved hybridisation schemes resulting in reduction of costs for these analyses opens the way for implementing array CGH in routine diagnostic analysis. At present it remains unclear what resolution of the array will be optimal for screening MCA/MR patients. Higher resolution arrays may reveal larger numbers of small chromosomal imbalances. However, the finding of only 10% of de novo imbalances in a cohort of 100 patients by a full coverage array may indicate that higher resolution does not necessarily increase the diagnostic yield. More studies using high resolution arrays are needed to compare the incidence of small imbalances in different patient populations. Nevertheless, using a 1 Mb resolution array, some imbalances smaller than 1 Mb are being missed. In addition, the false positive rate may be lowered, especially if the identification of imbalances is based on intensity alterations of three or more aberrant flanking clones.12
Considering the large percentage of inherited chromosomal imbalances, establishing both benign copy number variations in the human genome as well as developing a comprehensive morbid map of the human genome will be of major importance for understanding which imbalances are causative.