[Microarray CGH: principle and use for constitutional disorders]. / La CGH microarray: principe et applications en pathologie constitutionnelle.

Chips technology has allowed to miniaturize process making possible to realize in one step and using the same device a lot of chemical reactions. The application of this technology to molecular cytogenetics resulted in the development of comparative genomic hybridization (CGH) on microarrays technique. Using this technique it is possible to detect very small genetic imbalances anywhere in the genome. Its usefulness has been well documented in cancer and more recently in constitutional disorders. In particular it has been used to detect interstitial and subtelomeric submicroscopic imbalances, to characterize their size at the molecular level or to define the breakpoints of translocation. The challenge today is to transfer this technology in laboratory medicine. Nevertheless this technology remains expensive and the existence of numerous sequence polymorphisms makes its interpretation difficult. Finally its is unlikely that it will make karyotyping obsolete as it does not allow to detect balanced rearrangements which after meiotic segregation might result in genome imbalance in the progeny.

[A preliminary study to assess the value of the DNA chips SpectralChip to detect subtle constitutional chromosome imbalances]. / Evaluation de la puce à ADN SpectralChip pour la détection des déséquilibres chromosomiques observés en pathologie constitutionnelle.

Comparative genomic hybridization on a microarray (microarray-CGH) allows to detect genomic chromosome imbalances. In order to assess its value to detect small chromosome imbalances observed in a clinical setting, using a DNA chip available commercially (Spectral Genomics, Houston, Texas, USA), we studied the DNA of 9 patients carrying a well characterized chromosome imbalance and the DNA of 11 patients where cytogenetic techniques such as high resolution banding karyotype, FISH using subtelomeric probes and comparative genomic hybridization on metaphase chromosomes conclude to a normal and/or balanced karyotype. A result was obtained for 19/20 patients. Failure of hybridization was observed for one patient. For all the other cases the sex of patients was correctly identified. Microarray-CGH was able to correctly diagnose the chromosome imbalance in 6/8 patients carrying such a defect i.e 9/11 imbalances (deletion or duplication) were detected. No chromosome imbalance was observed in 11 patients considered normal and/or balanced using cytogenetic techniques. Several clones were found to be polymorphic and required FISH studies to eliminate duplication or deletion. In conclusion, we think that this commercially available DNA chip might be useful to screen for chromosome imbalances. However, technical improvements are still necessary before using it in a clinical setting. Also, further studies are necessary to assess its sensitivity and specificity.

PMX2B, a new candidate gene for Hirschsprung's disease.

Hirschsprung's (HSCR) disease is a congenital intestinal malformation of the enteric nervous system. It is a multigenic malformation and until now, eight genes have been involved in the etiology of this disease: genes encoding proteins of the RET signaling pathway (RET, GDNF and NTN), genes participating in the endothelin (EDN) type B receptor pathway (EDNRB, EDN3 and ECE-1), the SOX10 gene and the SIP1 gene that is mutated in syndromic forms of HSCR. Mutations of these genes are found in not more than 50-60% of affected individuals. Here, we report on the results of a molecular cytogenetic study performed in a girl who presented with a syndromic short segment HSCR associated with a de novo t(4;8)(p13;p22) translocation. A comparative genomic hybridization (CGH) study found a 4p12p13 deletion. A molecular characterization of this rearrangement showed that the 4p13 deletion was 5 Mb in length and included the paired mesoderm homeobox gene (PMX2B) (MIM 603851), a gene expressed in the human embryonic gut and essential for the development of autonomic neural crest derivatives. The present observation suggests that PMX2B haploinsuffciency might predispose to HSCR.

Molecular characterization of partial trisomy 16q24.1-qter: clinical report and review of the literature.

We describe a 3(1/2)-year-old girl with psychomotor and mental retardation; dysmorphic features, including a high forehead with bitemporal narrowing; a broad nasal bridge and a broadened nose; downslanting palpebral fissures; abnormal ears; vertebral abnormalities; cardiac defect; genital hypoplasia; and anal abnormalities. The karyotype of our patient (550 bands) was normal. Molecular cytogenetic techniques, including comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH), revealed that this girl was a carrier of a de novo derivative chromosome 7 arising from a cryptic t(7;16)(p22.3;q24.1) translocation generating a trisomy 16q24.1-qter and a 7p22.3-pter deletion. FISH with a series of specific chromosome 7p and 16q probes allowed us to delineate the chromosome 7 breakpoint between YAC660G6 (WD7S517) and YAC848A12 (D7S521, D7S31, and WI-4829) and the chromosome 16 breakpoint between BAC457K7 (D42053) and BAC44201 (SGC30711). The comparison of the clinical features of our patient with those of 2 cases of pure terminal 7p deletion and 28 cases of trisomy 16q reported in the literature allowed us to establish the following phenotype-genotype correlation for trisomy of the long arm of chromosome 16: distinctive facies (high/prominent forehead, bitemporal narrowing, periorbital edema in the neonatal period); severe mental retardation; vertebral, genital, and anal abnormalities to 16q24; distal joint contractures and camptodactyly to 16q23; cleft palate and renal anomalies to 16q22; beaked nose and gall bladder agenesis to 16q21; gut malrotation; lung and liver anomalies to 16q13; and behavior abnormalities to band 16q11-q13.

A CGH study of 27 patients with CHARGE association.

CHARGE association is a non-random occurrence of congenital malformations including coloboma, heart disease, choanal atresia, retarded growth and/or retarded development, genital hypoplasia, ear anomalies and/or deafness. The cause of this association remains unknown. Various genetic mechanisms have been proposed, including a contiguous gene syndrome but, so far, no recurrent locus has been identified. To address this question, we decided to perform a comparative genomic hybridization (CGH) study on a cohort of 27 patients with CHARGE association and a normal standard karyotype. We found two chromosomal anomalies: a der(9)t(9;13) derived from a paternal translocation and a der(6)t(4;6) of unknown origin. This suggests that chromosome imbalances may well mimic CHARGE association. Therefore patients with CHARGE association must be carefully tested with classical and molecular cytogenetic techniques to detect a potential chromosome imbalance. It is expected that more stringent diagnostic criteria of CHARGE association could define a more homogeneous group of patients where a single genetic cause might be identified.

Chromosome 10p11.2-p12.2 duplication: report of a patient and review of the literature.

We report on a young male with mental retardation, slightly upslanting palpebral fissures, strabismus, high-arched palate, retrognathia, and flat feet. Cytogenetic analysis in addition to fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH) showed the presence of a chromosome 10p11.2-->p12.2 duplication. Karyotypes of the parents were normal. Comparison of the clinical findings observed in the present patient with those observed in other reported cases with duplication 10p suggest that the presence of high arched/cleft palate and retrognathia may be related to the 10p11.2-->p12.2 duplication. Also, no critical region for the trisomy 10p syndrome has been delimited.

Comparative genomic hybridisation in mentally retarded patients with dysmorphic features and a normal karyotype.

Segmental aneusomy for small chromosomal regions has been shown to be a common cause of mental retardation and multiple congenital anomalies. A screening method for such chromosome aberrations that are not detected using standard cytogenetic techniques is needed. Recent studies have focused on detection of subtle terminal chromosome aberrations using subtelomeric probes. This approach however excludes significant regions of the genome where submicroscopic rearrangements are also liable to occur. The aim of the present study was to evaluate the efficiency of comparative genomic hybridisation (CGH) for screening of submicroscopic chromosomal rearrangements. CGH was performed in a cohort of 17 patients (14 families) with mental retardation, dysmorphic features and a normal karyotype. Five subtle unbalanced rearrangements were identified in 7 patients. Subsequent FISH studies confirmed these results. Although no interstitial submicroscopic rearrangement was detected in this small series, the study emphasises the value of CGH as a screening approach to detect subtle chromosome rearrangements in mentally retarded patients with dysmorphic features and a normal karyotype.

Chromosome 7q22-q31 duplication: report of a new case and review.

We report on a girl with psychomotor retardation, growth retardation, microcephaly, frontal bossing, large ears, small nose, high arched and narrow palate, short neck, and generalized hirsutism. Cytogenetic analysis in addition to fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH) showed the presence of a chromosome 7q22-->q31.3 duplication. Comparison with other reported cases shows some resemblance but insufficient to enable us to establish a definite syndrome with specific clinical manifestations. The importance in better analyzing further cases by new molecular cytogenetics techniques is raised.

Identification of the binding site for a novel class of CCR2b chemokine receptor antagonists: binding to a common chemokine receptor motif within the helical bundle.

Monocyte chemoattracant-1 (MCP-1) stimulates leukocyte chemotaxis to inflammatory sites, such as rheumatoid arthritis, atherosclerosis, and asthma, by use of the MCP-1 receptor, CCR2, a member of the G-protein-coupled seven-transmembrane receptor superfamily. These studies identified a family of antagonists, spiropiperidines. One of the more potent compounds blocks MCP-1 binding to CCR2 with a K(d) of 60 nm, but it is unable to block binding to CXCR1, CCR1, or CCR3. These compounds were effective inhibitors of chemotaxis toward MCP-1 but were very poor inhibitors of CCR1-mediated chemotaxis. The compounds are effective blockers of MCP-1-driven inhibition of adenylate cyclase and MCP-1- and MCP-3-driven cytosolic calcium influx; the compounds are not agonists for these pathways. We showed that glutamate 291 (Glu(291)) of CCR2 is a critical residue for high affinity binding and that this residue contributes little to MCP-1 binding to CCR2. The basic nitrogen present in the spiropiperidine compounds may be the interaction partner for Glu(291), because the basicity of this nitrogen was essential for affinity; furthermore, a different class of antagonists, a class that does not have a basic nitrogen (2-carboxypyrroles), were not affected by mutations of Glu(291). In addition to the CCR2 receptor, spiropiperidine compounds have affinity for several biogenic amine receptors. Receptor models indicate that the acidic residue, Glu(291), from transmembrane-7 of CCR2 is in a position similar to the acidic residue contributed from transmembrane-3 of biogenic amine receptors, which may account for the shared affinity of spiropiperidines for these two receptor classes. The models suggest that the acid-base pair, Glu(291) to piperidine nitrogen, anchors the spiropiperidine compound within the transmembrane ovoid bundle. This binding site may overlap with the space required by MCP-1 during binding and signaling; thus the small molecule ligands act as antagonists. An acidic residue in transmembrane region 7 is found in most chemokine receptors and is rare in other serpentine receptors. The model of the binding site may suggest ways to make new small molecule chemokine receptor antagonists, and it may rationalize the design of more potent and selective antagonists.

Analysis of uncultured amniocytes by comparative genomic hybridization: a prospective prenatal study.

Comparative genomic hybridization (CGH) is a new molecular cytogenetic technique which can detect and map whole and partial aneuploidies throughout a genomic specimen DNA without culturing specimen cells. Thus, CGH may be used as a comprehensive and rapid screening test in prenatal unbalanced chromosomal abnormalities detection. We report the results of the first prospective study to evaluate the use of the CGH technique on uncultured amniocytes. Seventy-one amniotic fluid samples, obtained by transabdominal amniocentesis between the 14th and 35th weeks of gestation, were simultaneously investigated using CGH and conventional cytogenetics. Amniocentesis were done for advanced maternal age (21.1%), fetal ultrasound anomalies (73.3%) and high level of biochemical markers in maternal serum (5.6%). Sixty-six (93%) informative results were generated on a total of 71 analysed specimens. Fifty-nine samples were reported as disomic for all autosomes with a normal sex chromosome constitution using CGH and conventional cytogenetics. Among them, three pericentromeric chromosomal inversions were undetected by CGH analysis. Seven numerical aberrations were characterized, including one case of trisomy 13, one case of trisomy 18 and five cases of trisomy 21. Advantages and limitations of CGH for a rapid prenatal screening of unbalanced chromosomal aberrations are discussed.

Cytogenetic analysis from DNA by comparative genomic hybridization.

Comparative genomic hybridization (CGH) is a modified in situ hybridization technique which allows detection and mapping of DNA sequence copy differences between two genomes in a single experiment. In CGH analysis, two differentially labelled genomic DNA (study and reference) are co-hybridized to normal metaphase spreads. Chromosomal locations of copy number changes in the DNA segments of the study genome are revealed by a variable fluorescence intensity ratio along each target chromosome. Since its development, CGH has been applied mostly as a research tool in the field of cancer cytogenetics to identify genetic changes in many previously unknown regions. CGH may also have a role in clinical cytogenetics for detection and identification of unbalanced chromosomal abnormalities.

De novo inverted duplication 9p21pter involving telomeric repeated sequences.

We report on clinical and cytogenetic findings in a boy with partial 9p duplication, dup(9)(p21pter). Clinical manifestations included facial and hand anomalies and mental retardation. Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) were used to characterize further and confirm the conventional banding data. Investigation by FISH using whole chromosome 9 paint probe showed that the additional material was derived from chromosome 9. Using CGH, a region of gain was found in the chromosome segment 9p21pter. YACs and telomeric probes confirmed the duplicated region. Using the all-human telomeric sequences probe, intrachromosomal telomeric signal was noted on the short arm of the abnormal chromosome 9. Mechanism of formation of the duplication, including intrachromosomal telomeric sequences, is discussed.

Comparison of comparative genomic hybridization with conventional karyotype and classical fluorescence in situ hybridization for prenatal and postnatal diagnosis of unbalanced chromosome abnormalities.

The comparative genomic hybridization (CGH) technique was initially used for detection of chromosomal imbalances in tumor cells. CGH can also be used as a supplementary method to karyotypic analysis in clinical cytogenetic cases. In order to evaluate CGH usefulness in prenatal and postnatal analysis of whole chromosome and segmental aneusomies, we investigated 13 clinical samples from blood, cultured chorionic villi, cultured amniotic fluids and uncultured amniotic fluids. These specimens, initially analyzed by conventional cytogenetics, included 5p monosomy, 9p duplication, add 6p, unbalanced translocation between chromosomes 5 and 10, mosaic tetrasomy 12p (50%), unbalanced (X;X) translocation and Prader-Willi deletion (15q11-13). In addition, six numerical chromosome aberrations (tetrasomy X, trisomies 13, 18, 21 and monosomy X) were analysed. All the chromosomal abnormalities, except the Prader-Willi deletion, were correctly detected by CGH. Here, we have demonstrated that the CGH technique is an alternative to classical fluorescence in situ hybridization using specific probes for detection of the unbalanced chromosomal aberrations in prenatal and postnatal diagnosis and could be used for rapid prenatal screening for unbalanced aberrations.

Identification and characterization of a de novo partial trisomy 10p by comparative genomic hybridization (CGH).

We report the characterization of a de novo unbalanced chromosome rearrangement by comparative genomic hybridization (CGH) in a 15-day-old child with hypotonia and dysmorphia. We describe the combined use of CGH and fluorescence in situ hybridization (FISH) to identify the origin of the additional chromosomal material on the short arm of chromosome 6. Investigation with FISH revealed that the excess material was not derived from chromosome 6. Identification of unknown unbalanced aberrations that could not be identified by traditional cytogenetics procedures is possible by CGH analysis. Visual analysis of digital images from CGH-metaphase spreads revealed a predominantly green signal on the telomeric region of chromosome 10p. After quantitative digital ratio imaging of 10 CGH-metaphase spreads, a region of gain was found in the chromosome band 10p14-pter. The CGH finding was confirmed by FISH analysis, using a whole chromosome 10 paint probe. These results show the usefulness of CGH for a rapid characterization of de novo unbalanced translocation, unidentifiable by karyotype alone.

Altered patterns of DNA methylation on chromosomes from leukemia cell lines: identification of 5-methylcytosines by indirect immunodetection.

An immunodetection technique has been developed to map with high resolution the methylated sites of human chromosomes. We have used this method to define the methylated areas of chromosomes from normal donors and from leukemia cell lines. The chromosomes were exposed for a short time to UV light to induce mild denaturation. The methylated sites were detected in situ by using monoclonal antibodies against 5-methylcytosine (prepared in mouse), and fluorescein-conjugated antimouse immunoglobulins. The chromosomes from normal cells exhibited a fluorescent pattern with RCT banding, although some differences from previously reported patterns could be detected. With this method we have been able to show the presence of two types of R-bands: High fluorescence R-band (HFR) and low fluorescence R-band (LFR). Chromosomes from leukemia cell lines exhibited low global staining with disrupted RCT banding of the chromosomes. The decreased level of the methylation status of the chromosomes from leukemia cells was confirmed by detection of 5-methylcytosines on total immobilized DNA. Thus, we have shown that this method can be used to determine the methylated status of chromosomes and, in turn, to map not only the structural (banding) but also the functional (methylation status) properties of the different chromosome domains in normal and pathologic human cells.

Comparative genomic hybridization: technical development and cytogenetic aspects for routine use in clinical laboratories.

Comparative genomic hybridization (CGH) offers a new global approach for detection of chromosomal material imbalances of the entire genome in a single experiment without cell culture. In this paper, we discuss the technical development and the cytogenetic aspects of CGH in a clinical laboratory. Based only on the visual inspection of CGH metaphase spreads, the correct identification of numerical and structural anomalies are reported. No commercial image analysis software was required in these experiments. We have demonstrated that this new technology can be set up easily for routine use in a clinical cytogenetics laboratory.

Localization of the CDKN4/p27Kip1 gene to human chromosome 12p12.3.

CDKN4/p27Kip1 is a cyclin-dependent kinase (Cdk) inhibitor implicated in G1 phase arrest, which negatively regulates G1 phase progression in response to TGF beta, and might represent a tumor suppressor gene. We report here the chromosomal assignment of the human CDKN4 gene to chromosome 12p12.3 in close proximity to highly polymorphic microsatellite markers.

Prenatal diagnosis of a (X;X) translocation by fluorescence in situ hybridization and laser scanning image cytometry.

A de novo structural abnormality of one X chromosome was prenatally detected in a female fetus. This chromosomal abnormality has been analyzed by conventional cytogenetic methods, fluorescence in situ hybridization, and laser scanning image cytometry. The association of these techniques has demonstrated that this anomaly corresponds to a (X;X) translocation. Analysis of hybridization signals by laser scanning image cytometry allowed to localize that the breakpoints were at the X-centromeric region and Xp11.3, respectively. These results show the usefulness of image analysis and fluorescence in situ hybridization for a rapid characterization of de novo structural chromosome anomalies in prenatal diagnosis.