A method for high-resolution analysis of the human karyotype by flow cytometry has been developed. Metaphase chromosomes are prepared from short-term peripheral blood cultures, stained with ethidium bromide, and analyzed on a standard fluorescence-activated cell sorter (FACS-iI). Flow karyotypes with up to 20 peaks can be obtained with coefficients of variation in the range 1-2%. At this level of resolution the contribution of many of the human chromosomes can be evaluated separately. Significant and reproducible differences between normal individuals have been detected and have been correlated with differences in the centric heterochromatin of certain chromosomes as revealed in their C-banded karyotypes.In recent years, flow cytometry has shown great potential for automated analysis of the human karyotype. In principle, the development of such an approach could offer the clinician an objective assessment of chromosome abnormalities, thus complementing the more traditional forms of karyotype analysis. However, there have been two main obstacles to the development of flow cytometry as a cytogenetic tool. First, conventional methods of chromosome preparation (1) may damage interphase nuclei, producing fragments of chromatin which cannot be distinguished from chromosomes by a flow cytometer. For this reason, most flow karyotypes have been derived from cells grown in monolayer (2, 3) and it has not been possible to use peripheral blood lymphocytes as a source. The second limitation is that commercially available flow cytometers have not been shown to produce adequate flow karyotypes. Only specially built, and therefore expensive, systems have been used (4, 5).We report here an extension of a previously developed procedure (6) which yields high-resolution flow karyotypes from short-term peripheral blood cultures with a standard fluorescence-activated cell sorter (FACS-II). At this level ofresolution it is possible to detect some of the commonly occuring centric, satellite, and Y polymorphisms (7).MATERIALS AND METHODS Short-Term Lymphocyte Culture. A 20-ml sample of heparinized blood was obtained from each subject and diluted with an equal volume ofphosphate-buffered saline. This was divided equally into four sterile tubes and each aliquot was carefully underlayered with 7.5 ml of Lymphoprep (Nyegaard, Oslo, Norway). Centrifugation (400 x g at the interface) for 30 min at 20°C gave a well-separated band oflymphocytes which were removed and washed twice in medium to dilute out any remaining Lymphoprep which might inhibit stimulation. The cells in an aliquot were counted in a hemocytometer, and the lymphocyte concentration was adjusted to between 0.3 X 106 and 0.4 x 106 cells per ml, typical culture volumes being between 20 and 30 ml in sterile glass bottles with a 5-cm-diameter base. The culture medium was chromosome medium 1A (GIBCO) containing phytohemagglutinin.After 24 hr at 37C, leuko-agglutination could be observed, with clumps of approximately 20 cell diameters increasing in size to about 50 cell diame...
Cryptic unbalanced chromosome rearrangements in the telomeric bands of human chromosomes constitute a significant cause of "idiopathic" mental retardation. Here, we have described a new strategy based upon comparative genomic hybridisation (CGH) to screen for these abnormalities.. A modified CGH analysis showed three unbalanced cryptic rearrangements in five patients from three families. These chromosome abnormalities and their balanced forms in the relatives were then confirmed by fluorescence in situ hybridisation (FISH). This study describes a new approach to the diagnosis of cryptic translocations between the G band negative ends of chromosomes and confirms the significant contribution ofcryptic telomeric rearrangements to idiopathic mental retardation. (aMed Get 1998;35:225-233)
Critical illness in COVID-19 is an extreme and clinically homogeneous disease phenotype that we have previously shown1 to be highly efficient for discovery of genetic associations2. Despite the advanced stage of illness at presentation, we have shown that host genetics in patients who are critically ill with COVID-19 can identify immunomodulatory therapies with strong beneficial effects in this group3. Here we analyse 24,202 cases of COVID-19 with critical illness comprising a combination of microarray genotype and whole-genome sequencing data from cases of critical illness in the international GenOMICC (11,440 cases) study, combined with other studies recruiting hospitalized patients with a strong focus on severe and critical disease: ISARIC4C (676 cases) and the SCOURGE consortium (5,934 cases). To put these results in the context of existing work, we conduct a meta-analysis of the new GenOMICC genome-wide association study (GWAS) results with previously published data. We find 49 genome-wide significant associations, of which 16 have not been reported previously. To investigate the therapeutic implications of these findings, we infer the structural consequences of protein-coding variants, and combine our GWAS results with gene expression data using a monocyte transcriptome-wide association study (TWAS) model, as well as gene and protein expression using Mendelian randomization. We identify potentially druggable targets in multiple systems, including inflammatory signalling (JAK1), monocyte–macrophage activation and endothelial permeability (PDE4A), immunometabolism (SLC2A5 and AK5), and host factors required for viral entry and replication (TMPRSS2 and RAB2A).
We report male and female siblings with extreme microcephaly and mental retardation, growth retardation, and multiple chromosome mosaicism. Mental retardation associated with chromosome mosaicism does not always carry a low recurrence risk.
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