Three new cases of terminal deletion of the long arm of chromosome 7 and literature review to correlate genotype and phenotype manifestations.

Partial monosomy of the long arm of chromosome 7 has been characterized by wide phenotypic manifestations, but holoprosencephaly (HPE) and sacral agenesis have frequently been associated with this chromosomal deletion. A clear relationship between genotype and phenotype remains to be defined in the 7q deletion syndrome. Three patients (1, 2, and 3) were investigated with 7q terminal deletion and compared with similar deletion cases in the literature in order to stratify the phenotypes associated with 7q35 and 7q36 terminal deletion patients. Patients 1, 2, and 3 were carrying a de novo terminal deletion at bands 7q36.2, 7q35, and 7q36.1, respectively. In patient 3, a small Xq28 duplication was also identified by array-CGH. Our patients presented with heterogeneous phenotypic manifestations, which could imply the possible role of environmental factors (multifactorial inheritance), structural variations in the non-coding regions, penetrance, and/or polymorphism. The varying length of deletion was also taken into account. Growth retardation was the most frequent symptom found in both 7q35 and 7q36 patients we reviewed. The occurrence of HPE and sacral malformation together was seen in less than 10% of the reviewed cases in both kinds of deletion. HPE was associated mainly in cases with an unbalanced translocation.

Efficiency of manual scanning in recovering rare cellular events identified by fluorescence in situ hybridization: simulation of the detection of fetal cells in maternal blood.

Fluorescence in situ hybridization (FISH) and manual scanning is a widely used strategy for retrieving rare cellular events such as fetal cells in maternal blood. In order to determine the efficiency of these techniques in detection of rare cells, slides of XX cells with predefined numbers (1-10) of XY cells were prepared. Following FISH hybridization, the slides were scanned blindly for the presence of XY cells by different observers. The average detection efficiency was 84% (125/148). Evaluation of probe hybridization in the missed events showed that 9% (2/23) were not hybridized, 17% (4/23) were poorly hybridized, while the hybridization was adequate for the remaining 74% (17/23). In conclusion, manual scanning is a relatively efficient method to recover rare cellular events, but about 16% of the events are missed; therefore, the number of fetal cells per unit volume of maternal blood has probably been underestimated when using manual scanning.

Partial trisomy of chromosome 22 resulting from a supernumerary marker chromosome 22 in a child with features of cat eye syndrome.

Small supernumerary marker chromosomes are present in about 0.05% of the human population. In approximately 28% of persons with these markers (excluding the approximately 60% derived from one of the acrocentric chromosomes), an abnormal phenotype is observed. We report on a 3-month-old girl with intrauterine growth retardation, craniofacial features, hypotonia, partial coloboma of iris and total anomalous pulmonary venous return. Cytogenetic analysis showed the presence of a supernumerary marker chromosome, identified by fluorescence in situ hybridization as part of chromosome 22, and conferring a proximal partial trisomy 22q22.21, not encompassing the DiGeorge critical region (RP11-154H4 + , TBX1-). This observation adds new information relevant to cat eye syndrome and partial trisomy of 22q.

Application of multi-PRINS to simultaneously identify chromosomes 18, X, and Y in prenatal diagnosis.

Since its discovery by Koch in 1989, primed in situ labeling (PRINS) reaction provides an alternative approach for direct detection of human chromosomes. The multiple color (multi)-PRINS technique can simultaneously and specifically display different chromosomes with different colors in the same metaphase or interphase nucleus by using sequential labeling of different chromosome targets. We developed a triple-PRINS reaction on uncultured amniotic cells by omitting the blocking step and taking advantage of mixing two fluorochromes (fluorescein and rhodamine) to create a third color for simultaneous detection in the same amniocytes of three different chromosome targets, e.g., chromosomes 18, X, and Y. Fluorescent signals corresponding to chromosomes 18, X, and Y were shown as yellow, red, and green color spots, respectively. Multi-PRINS is as accurate and reliable as multicolor fluorescent in situ hybridization (multi-FISH) for the detection of aneuploidies involving chromosomes 18, X, and Y. Furthermore, multi-PRINS represents a faster and more cost-effective alternative to FISH for prenatal testing of aneuploidy in uncultured amniocytes.

New rapid multicolor PRINS protocol.

In the multiple-color primed in situ labeling (multi-PRINS) technique, using ddNTPs between two PRINS reactions can block the free 3'-end generated in the previous PRINS reaction, thus avoiding the next PRINS reaction, using it as a primer to perform spurious elongation at nondesired sites. However, by omitting the blocking step and taking advantage of the color mixing, we developed a simple and rapid multi-PRINS technique to simultaneously detect three chromosomes in the same cell. With this protocol, one can create a third color using the two most common forms of labeled dUTP (biotin- and digoxigenin-labeled dUTP) and two fluorochromes (fluorescein and rhodamine). The signals at the centromeres of three different chromosomes displayed perfect yellow, red, and green colors, respectively. The entire procedure could be completed in less than 90 min because the blocking step was omitted. This protocol is practical and efficient for multi-PRINS so that even more than three chromosome targets could be detected in the same cell.

Dual-color PRINS for in situ detection of fetal cells in maternal blood.

Fetal nucleated cells circulating in the peripheral blood during pregnancy are potential targets for noninvasive genetic testing. Fluorescence in situ hybridization (FISH) frequently is used to quantify the total number of fetal cells in peripheral blood of pregnant women. We describe an alternative molecular cytogenetic procedure that is the primed in situ labeling (PRINS). This technique consists of annealing oligonucleotides specific to individual chromosome targets and in situ elongation using Taq DNA polymerase to incorporate labeled dUTPs. The sites of the newly synthesized DNA sequences were revealed as fluorescent signals using an immunochemical reaction. The dual-color PRINS was specifically performed for simultaneous detection of two chromosome targets, X and Y. The fluorescent signals corresponding to chromosomes X and Y were displayed as red and green color spots, respectively. The sensitivity and specificity of PRINS are similar to FISH and allow us to efficiently and reliably detect fetal cells in maternal blood. Moreover, dual-color PRINS is faster and more cost-effective than FISH.

[Dominant negative activity of mutated p53 proteins]. / Activité dominante négative des protéines p53 mutées.

Tumor suppressor gene inactivation as proposed by the Knudson model implies a sequential inactivation of two alleles of a gene. For example, the first allele is inactivated by a missense mutation, and the second one is inactivated by a deletion or insertion. The alteration of the p53 tumor suppressor gene is far to correspond only to this model. In the great majority of cancers, the mutated allele of p53 coexists with the normal allele. It is well known that the transcriptional activity is one of the most important functions of p53. The p53 protein is active as a tetramer (this complex activates the expression of targeted genes by binding to its consensus DNA sequence called the p53 response element). Experimental evidence shows that wild-type p53 interacts with mutant proteins to form heterotetramers. In association with wild-type proteins, mutant proteins drive the wild-type subunits into a mutant conformation. This association leads to a loss of trans-activating function. The capacity of mutant subunits to form heterotetramers with wild-type subunits and to commit them into a mutant conformation is called << dominant negative effect >>. Many p53 mutant proteins possess this dominant negative activity. Recently, several factors, which are implicated in the control of the dominant negative activity of p53 mutants, have been identified. The elucidation of these complex molecular functions, which are implicated in the dominant negative activity of the p53 mutated protein represents an important aspect in the comprehension of the biological mechanisms involved in carcinogenesis.

Quantification of fetal nucleated cells in maternal blood of pregnant women with a male trisomy 21 fetus using molecular cytogenetic techniques.

BACKGROUND: Prenatal diagnosis of trisomy 21 is based on fetal karyotyping generally obtained using invasive methods. During pregnancy, the circulating fetal cells in maternal blood constitute a potential source for development of a noninvasive prenatal diagnosis. The objective of this study was the identification and quantification of all fetal nucleated cells per unit volume of peripheral blood of pregnant women carrying male fetuses with trisomy 21 using molecular cytogenetic techniques. METHODS: Peripheral blood samples were obtained from 16 women carrying male fetuses with trisomy 21. We used a simple and rapid method of harvesting blood without recourse to any enrichment procedures or cell-separation techniques. To evaluate the potential of this method, 16 specimens were analyzed by molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) using specific probes to chromosomes X, Y and 21. RESULTS: The number of fetal cells varied between 6 and 32 per mL of maternal blood. This number is 3-5 times higher than that from normal pregnancies. CONCLUSIONS: Our current results are in agreement with the results previously reported by other groups showing that the number of fetal cells in maternal blood in trisomic 21 pregnancies is higher than in normal pregnancies. This high number of fetal cells is regarded as an advantage for the development of a noninvasive prenatal diagnostic test.

[From the conception of the PRINS to its coronation]. / De la conception du PRINS à son couronnement.

As a non-isotopic molecular cytogenetic technique, the primed in situ (PRINS) labelling reaction represents a major technological progress achieved in the past decade. It has become a routine technique for the microscopic visualization of specific DNA sequences in cells and nuclei and constitutes a good alternative to the fluorescence in situ hybridization (FISH) procedure. Among the multiple advantages that characterize the PRINS technique, specificity, rapidity, reliability, reproducibility, and cost-effectiveness can be mentioned. PRINS can be in addition associated with other techniques like FISH, indirect immunofluorescence, and nick translation. The most recent developments show the great potential of this technique. Now PRINS can be used to study single-copy genes and, consequently, can be routinely used to investigate deletions associated with microdeletion syndromes. Therefore, the PRINS technique has the potential to become a widely used molecular cytogenetic tool in clinics and research. This short review presents how the PRINS technique contributed to further the understanding of biological phenomena and describes the different possibilities and applications of the PRINS method in several biological and clinical fields (pre-implantation testing, prenatal, constitutional and oncologic genetic diagnosis).