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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.

Thirty-four families with a child or fetus with Turner's syndrome were studied using a series of polymorphic DNA probes. Analysis of the origin of the normal X chromosome was possible in all cases. In 16 families with 45,X (four fetuses and 12 livebirths), the observed X was maternal in each case, indicating a preferential loss of the paternal sex chromosome at, or before, conception. In the remaining 18 families with a variety of karyotypes, but especially in those where the child had an isochromosome of Xq or a ring X, there was again a strong tendency for the normal X to be maternal. Analysis of parental ages was performed with known origin of each abnormality, but no evidence for an increased or decreased parental age effect was detected.Turner's syndrome is one of the most common chromosomal abnormalities with an estimated frequency at conception of 1-5%. Most affected fetuses are, however, spontaneously aborted and the residual birth frequency is 1 in 2500 to 5000 females.' In a large consecutive study the majority of fetuses were found to have monosomy X (45,X) with 45,X/46,XX in the remainder, whereas in the same study, among liveborn Turner's syndrome patients, 53% were 45,X, 14-8% were mosaic 45,X/46,XX, 90/o were 46,Xi(Xq)/ 45,X, 4 90/o were mosaic 47,XXX/45,X, and the remaining 9-7% showed five other karyotypes (46, 46, mosaic applicable to cultured cells from affected fetuses, were only informative in a proportion (31%) of families, and were biased in that informative families were more frequent for 45,Xm than for 45,XP.3 4 The development of multiple polymorphic DNA markers for the X chromosome offers an alternative approach which should avoid these difficulties. Hassold et a15 6 used this alternative approach to study 40 families with Turner's syndrome where the majority were spontaneously aborted fetuses, and were able to identify the parental origin in 35 (88%) of their families. We describe our results from a study of 34 families in which the majority were live born and where the origin of the abnormality has been determined for a variety of karyotypic abnormalities. Patients and methods ASCERTAINMENT OF CASESFamilies for this study were recruited from the records of the West of Scotland Regional Genetics Service and with the assistance ofthe United Kingdom Child Growth Foundation. CYTOGENETIC ANALYSISChromosomal analysis was undertaken using standard cytogenetic techniques on peripheral blood lymphocytes or cultured products of conception. Two families where the proband had Turner's syndrome but 45,X/46,XY (that is, a paternal defect) were excluded from DNA analysis.DNA ANALYSIS DNA was extracted from cultured fetal cells in the case of an affected fetus or from peripheral blood lymphocytes of affected children and of both parents.DNA samples were digested with the appropriate restriction enzymes under the conditions specified by the manufacturers. The resulting fragments were fractionated on 0-8% agarose gels and Southern blotted onto Hybond-N (Amersham) followed by hybridisation wi...

A series of Y recombinants have been isolated from Y-specific DNA libraries and regionally located on the Y chromosome using a Y deletion panel constructed from individuals carrying structural abnormalities of the Y chromosome. Of twenty recombinants examined twelve have been assigned to Yp and eight to Yq. Five of the Yp recombinants map between Yp11.2 and Ypter and one can only be assigned to Yp. Of the former, four detect homologies on the X chromosome between Xq13 and Xq24 and the latter one between Xp22.3 and Xpter. The sixth recombinant detects autosomal homologous sequences. The six remaining Yp probes are located between Ycen and Yp11.2. One of these detects a homology on the X chromosome at Xq13-Xq24 and a series of autosomal sequences, two detect uniquely Y-specific sequences and three a complex pattern of autosomal homologies. The remaining eight recombinants have been assigned to three intervals on Yq. Of three recombinants located between Ycen and Yq11.21 two detect only Y sequences and one additional autosomal homologies. Two recombinants lie in the interval Yq11.21-Yq11-22, one of which detects only Y sequences and the other an Xp homology between Xp22.3 and Xpter. Finally, the three remaining Yq recombinants all detect autosomal homologies and are located between Yq11.22 and Yq12. The divergence between homologies on different chromosomes has been examined for three recombinants by washing Southern Blots at different levels of stringency. Additionally, Southern analysis of DNA from flow sorted chromosomes has been used to identify autosomes carrying homologies to two of the Y recombinants.

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