No abstract

We report array-CGH screening of 95 syndromic patients with normal G-banded karyotypes and at least one of the following features: mental retardation, heart defects, deafness, obesity, craniofacial dysmorphisms or urogenital tract malformations. Chromosome imbalances not previously detected in normal controls were found in 30 patients (31%) and at least 16 of them (17%) seem to be causally related to the abnormal phenotypes. Eight of the causative imbalances had not been described previously and pointed to new chromosome regions and candidate genes for specific phenotypes, including a connective tissue disease locus on 2p16.3, another for obesity on 7q22.1→q22.3, and a candidate gene for the 3q29 deletion syndrome manifestations. The other causative alterations had already been associated with well-defined phenotypes including Sotos syndrome, and the 1p36 and 22q11.21 microdeletion syndromes. However, the clinical features of these latter patients were either not typical or specific enough to allow diagnosis before detection of chromosome imbalances. For instance, three patients with overlapping deletions in 22q11.21 were ascertained through entirely different clinical features, i.e., heart defect, utero-vaginal aplasia, and mental retardation associated with psychotic disease. Our results demonstrate that ascertainment through whole-genome screening of syndromic patients by array-CGH leads not only to the description of new syndromes, but also to the recognition of a broader spectrum of features for already described syndromes. Furthermore, on the technical side, we have significantly reduced the amount of reagents used and costs involved in the array-CGH protocol, without evident reduction in efficiency, bringing the method more within reach of centers with limited budgets.

Apoptosis is fundamental to the regulation of homeostasis of stem cells in vivo. Whereas the pathways underlying the molecular and biochemical details of nuclear breakdown that accompanies apoptosis have been elucidated, the precise nature of nuclear reorganization that precedes the demolition phase is not fully understood. Here, we expressed an inducible caspase-8 in human mesenchymal stem cells, and quantitatively followed the early changes in nuclear organization during apoptosis. We found that caspase-8 induces alteration of the nuclear lamina and a subsequent spatial reorganization of both centromeres, which are shifted towards a peripheral localization, and telomeres, which form aggregates. This nuclear reorganization correlates with caspase-3 sensitivity of lamina proteins, because the expression of lamin mutant constructs with caspase-3 hypersensitivity resulted in a caspase-8-independent appearance of lamina intranuclear structures and telomere aggregates, whereas application of a caspase inhibitor restrains these changes in nuclear reorganization. Notably, upon activation of apoptosis, we observed no initial changes in the spatial organization of the promyelocytic leukemia nuclear bodies (PML-NBs). We suggest that during activation of the caspase-8 pathway changes in the lamina structure precede changes in heterochromatin spatial organization, and the subsequent breakdown of lamina and PML-NB.

Array-based comparative genomic hybridization allows high-resolution screening of copy number abnormalities in the genome, and becomes an increasingly important tool to detect deletions and duplications in tumor and post-natal cytogenetics. Here we illustrate that genomic arrays can also provide novel clues regarding the structural basis of chromosome rearrangement, including instability and mechanisms of formation of ring chromosomes. We also showed that array results might impact the recurrence risks for relatives of affected individuals. Our data indicate that chromosome rearrangements frequently involve more breaks than current cytogenetic models assume.

Fluorescence in situ hybridization allows the enumeration of chromosomal abnormalities in interphase cell nuclei. This process is called dot counting. To estimate the distribution of chromosomes per cell, a large number of cells have to be analysed, particularly when the frequency of aberrant cells is low. Automation of dot counting is desirable because manual counting is tedious, fatiguing, and time consuming. We have developed a completely automated fluorescence microscope system that counts fluorescent hybridization dots for one probe in interphase cell nuclei. This system works with two fluorescent dyes-one for the DNA hybridization dots and one for the cell nucleus. A fully automated scanning procedure has been used for the image acquisition. After an image is acquired it has to be analysed in order to find the nuclei and to detect the dots. This article focuses upon the dot detection procedure. Three different algorithms are presented. The problems of 'overlapping' dots and split dots are discussed. The automated dot counter has been tested on a number of normal specimens where DAPI was used for the nucleus counter stain and a centromeric probe was used to mark the chromosome 12. The slides contained lymphocytes from cultured blood. The performance of the different algorithms has been evaluated and compared with manually obtained results. The automated counting results approximate the results of manual counting.

This article explores the feasibility of the use of automated microscopy and image analysis to detect the presence of rare fetal nucleated red blood cells (NRBCs) circulating in maternal blood. The rationales for enrichment and for automated image analysis for "rare-event" detection are reviewed. We also describe the application of automated image analysis to 42 maternal blood samples, using a protocol consisting of one-step enrichment followed by immunocytochemical staining for fetal hemoglobin (HbF) and FISH for X- and Y-chromosomal sequences. Automated image analysis consisted of multimode microscopy and subsequent visual evaluation of image memories containing the selected objects. The FISH results were compared with the results of conventional karyotyping of the chorionic villi. By use of manual screening, 43% of the slides were found to be positive (>=1 NRBC), with a mean number of 11 NRBCs (range 1-40). By automated microscopy, 52% were positive, with on average 17 NRBCs (range 1-111). There was a good correlation between both manual and automated screening, but the NRBC yield from automated image analysis was found to be superior to that from manual screening (P=.0443), particularly when the NRBC count was >15. Seven (64%) of 11 XY fetuses were correctly diagnosed by FISH analysis of automatically detected cells, and all discrepancies were restricted to the lower cell-count range. We believe that automated microscopy and image analysis reduce the screening workload, are more sensitive than manual evaluation, and can be used to detect rare HbF-containing NRBCs in maternal blood.

In situ hybridization techniques allow the enumeration of chromosomal abnormalities and form a great potential for many clinical applications. Although the use of fluorescent labels is preferable regarding sensitivity and colormultiplicity, chromogenic labels can provide an excellent alternative in relatively simple situations, e.g., where it is sufficient to use a centromere specific probe to detect abnormalities of one specific chromosome. When the frequency of chromosomal aberrations is low, several hundreds or even thousands of cells have to be evaluated to achieve sufficient statistical confidence. Since manual counting is tedious, fatiguing, and time consuming, automation can assist to process the slides more efficiently. Therefore, a system has been developed for automated spot counting using brightfield microscopy. This paper addresses both the hardware system aspects and the software image analysis algorithms for nuclei and spot detection. As a result of the automated slide analysis the system provides the frequency spot distribution of the selected cells. The automatic classification can, however, be overruled by human interaction, since each individual cell is stored in a gallery and can be relocated for visual inspection. With this system a thousand cells can be automatically analyzed in approximately 10 min, while an extra 5–10 min is necessary for visual evaluation. The performance of the system was analyzed using a model system for trisomy consisting of a mixture of male and female lymphocytes hybridized with probes for chromosomes 7 and Y. The sensitivity for trisomy detection in the seeding experiment was such that a frequency of 3% trisomic cells could be picked up automatically as being abnormal according to the multiple proportion test, while trisomy as low as 1.5% could be detected after interaction. © 1996 Wiley‐Liss, Inc.

S U M M A R Y Segregation of mitochondrial DNA (mtDNA) is an important underlying pathogenic factor in mtDNA mutation accumulation in mitochondrial diseases and aging, but the molecular mechanisms of mtDNA segregation are elusive. Lack of high-throughput singlecell mutation load assays lies at the root of the paucity of studies in which, at the single-cell level, mitotic mtDNA segregation patterns have been analyzed. Here we describe development of a novel fluorescence-based, non-gel PCR restriction fragment length polymorphism method for single-cell A3243G mtDNA mutation load measurement. Results correlated very well with a quantitative in situ Padlock/rolling circle amplification-based genotyping method. In view of the throughput and accuracy of both methods for single-cell A3243G mtDNA mutation load determination, we conclude that they are well suited for segregation analysis.