Kada Krabchi

Quantification of all fetal nucleated cells in maternal blood in different cases of aneuploidies

We quantified all fetal nucleated cells (FNCs) per unit volume of maternal blood in different aneuploid pregnancies using molecular cytogenetic techniques. Seven cases of male trisomy 18, two triploidies (69,XXX), two 47,XXX, one 47,XXY, one 47,XYY, one male trisomy 13, and one case of 47,XY,r(22),+r(22) were analyzed. Whole blood samples were obtained from 15 women between 17 and 29 gestational weeks and harvested without using fetal cell enrichment procedures. Fluorescence in situ hybridization and primed in situ labeling were performed to identify the FNCs. All slides were manually scanned to quantify those cells. We have identified 4–20 FNCs/ml of maternal blood in the cases of trisomy 18; 10 and 25 FNCs/ml in the two cases of triploidy; 16 and 14 FNCs/ml, respectively, in the two X trisomies; 19 FNCs/ml in the 47,XXY; 26 FNCs/ml in the 47,XYY; nine FNCs/ml in the trisomy 13; and 10 FNCs/ml in the case of r(22). To detect all FNCs in all aneuploid pregnancies, we have used a very simple method that minimizes the manipulation steps to avoid losing fetal cells. The number of FNCs identified in aneuploid pregnancies was 2–5 times higher than in normal pregnancies. This higher number of FNCs will favor the design of a non‐invasive pre‐natal test.

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.

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 ∼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.

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.

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.

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.

Simultaneous identification of chromosomes 18, X and Y in uncultured amniocytes by using multi-primed in situ labelling technique.

The aim of this study is to validate the multi‐PRINS (primed in situ labelling) technique for simultaneous detection of chromosomes 18, X and Y in uncultured amniocytes for prenatal diagnosis of aneuploidy. The sites of the newly synthesized DNA sequences were showed as fluorescent signals by using immunochemistry. A multi‐PRINS technique was specifically performed for simultaneous detection in the same cells of three chromosome targets, e.g. chromosomes 18, X and Y. Fluorescent signals corresponding to chromosomes 18, X and Y were showed as yellow, red and green colour spots, respectively. A multi‐FISH technique using chromosome 18, X and Y probes was performed for comparison. Sixty cases were analysed using both multi‐PRINS and multi‐FISH. Fifty to two hundred nuclei were scored for each case for each technique. In all cases, there was no significant difference in the detection of chromosomes 18, X and Y regarding the sensitivity, the specificity and the efficiency; multi‐PRINS and multi‐FISH yield a similar distribution of the number of spots per nucleus. Both techniques were able to identify aneuploid cases without any ambiguity. Both multi‐PRINS and multi‐FISH can accurately and reliably detect aneuploidies involving chromosomes 18, X and Y in uncultured amniocytes. Finally, multi‐PRINS represents a faster and more cost‐effective alternative to FISH for prenatal testing of aneuploidy in uncultured amniocytes.

Prenatal Diagnosis and Molecular Characterization of Two Constitutional Rings Derived From One Chromosome 22

Prenatal Diagnosis and Molecular Characterization of Two Constitutional Rings Derived From One Chromosome 22 Macoura Gadji, Kada Krabchi, Paul Langis, Azeddine Aboura, Martine P erigny, St ephanie Côt e, M elissa Ferland, and R egen Drouin* Division of Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Universit e de Sherbrooke, Sherbrooke, Quebec, Canada Division of Radiology, Hôpital Saint-François d’Assise, CHUQ, Laval University, Quebec, Quebec, Canada Cytogenetics Laboratory, Hôpital Robert Debr e, Paris, France Division of Pathology, Hôpital Saint-François d’Assise, CHUQ, Laval University, Quebec, Quebec, Canada Cytogenetics Laboratory, CHUL, CHUQ, Quebec, Quebec, Canada