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Preimplantation Genetic Testing for Structural Rearrangements
Published in Carlos Simón, Carmen Rubio, Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
Inmaculada Campos-Galindo, Vanessa Peinado
Cytogenetics is the study of chromosome structure and properties that capitalizes on the constant morphology and size of normal chromosomes to detect abnormalities [9]. Karyotyping is a useful cytogenetic tool that uses in vitro culture and banding techniques to perform structural and numerical chromosomal analyses in metaphases to describe normal or abnormal chromosome complement of an individual, tissue, or cell line [10]. Currently, karyotyping of peripheral blood samples remains the first-line method for detecting chromosomal abnormalities in infertile couples.
Basic genetics and patterns of inheritance
Published in Hung N. Winn, Frank A. Chervenak, Roberto Romero, Clinical Maternal-Fetal Medicine Online, 2021
Various banding techniques are used to visualize the chromosomes. The most frequently used method is Giemsa banding or G-banding, which results in a specific pattern of dark and light bands on each chromosome. The older method, quinacrine banding, or Q-banding, produces the same dark and light patterns, but requires the use of a fluorescence microscope and is not used routinely. Reverse banding or R-banding, results in the opposite of the dark and light pattern seen with G-banding; this may be used to better see the ends of the chromosomes. C-banding stains the constitutive heterochromatin, which is near the centromeres and NOR stain visualizes the nucleolar organizing regions of the satellites and stalks of acrocentric chromosomes. For routine karyotype analysis, G-banding is typically used by most laboratories. Routine karyotyping cannot detect gains or losses of cytogenetic material smaller than about 4Mb in size and therefore can miss significant abnormalities.
Genetics
Published in Cathy Laver-Bradbury, Margaret J.J. Thompson, Christopher Gale, Christine M. Hooper, Child and Adolescent Mental Health, 2021
There are a number of different laboratory methods for identifying genetic variation. Karyotyping is used for the identification of chromosomal abnormalities such as trisomies (e.g. Down’s syndrome). Large copy number variants can be identified using fluorescent in-situ hybridisation (FISH), in which specially designed probes can be designed to target specific known DNA sequences. However, this approach requires variation in specific chromosomal regions to be suspected.
Prenatal chromosomal microarray analysis in foetuses with isolated absent or hypoplastic nasal bone
Published in Annals of Medicine, 2022
Xiaomei Shi, Jian Lu, Ling Li, Ran Wei, Jing Wu
Cell-free DNA or so-called non-invasive prenatal testing (NIPT) is now widely used in clinical practice as a prenatal screening method for common aneuploidies. It also has the potential to detect foetal CNVs, but with false-positive and false-negative results. Recent studies have showed that the accuracy of NIPT for CNVs is still unsatisfactory and needs to be improved [20,21]. CMA is still the most effective method for CNVs detection. On the other hand, conventional karyotyping can identify the majority of foetal chromosomal abnormalities, at a resolution of greater than 10 Mb. In this study, the deletion or duplication sizes of the seven cases were ranged from 713 kb to 4.9 Mb, and all of them may not be diagnosed by the G-banding karyotyping. Finally, from a patient's point of view, they want to exclude as many abnormalities as possible to ensure that the foetus is healthy. Therefore, for prenatal genetic counselling, counsellors should discuss the possibility of pathogenic CNVs to parents when foetuses with isolated absent or hypoplastic NB. CMA should be recommended when isolated absent or hypoplastic NB is suspected antenatally.
Adult acute lymphoblastic leukemia in a resource-constrained setting: outcomes after expansion of genetic evaluation
Published in Hematology, 2022
Wellington F. Silva, Mariane T. Amano, Luiza L. Perruso, Maria Gabriella Cordeiro, Renata Kiyomi Kishimoto, Aline de Medeiros Leal, Luciana Nardinelli, Israel Bendit, Elvira DRP Velloso, Eduardo M. Rego, Vanderson Rocha
All diagnostic samples were processed by the Laboratory of Tumor Biology and Cytogenetics from Hospital das Clínicas (HCFMUSP). Bone marrow (BM) or peripheral blood enriched with blasts (in ‘dry tap’ cases) were analyzed. All patients were screened for BCR-ABL1 rearrangement through FISH or RT–PCR during the corticosteroid pre-phase. In addition, all samples from patients with B-lineage ALL were evaluated for TCF3-PBX1, KMT2A-AFF4, and ETV6-RUNX1 (patients < 25 years old) fusions through RT–PCR[15]. Conventional karyotyping was attempted in all samples by standardized techniques [16]. In CD10-negative cases, dual-color break-apart FISH probe for 11q23 gene (KMT2A) was carried out. Patients who did not have any of these mentioned fusions detected were eligible to retrospectively screen for CRLF2 rearrangement (CRLF2-r, dual-color break-apart FISH probe, Cytocell). Fusions involving ABL1 and PDGFRB genes were also investigated by FISH (dual-color break-apart probes, Metasystems and Cytocell, respectively). Deletions encompassing IKAROS (IKZF1 gene) could be inferred from finding expression of IKZF1 dominant-negative isoforms (ik6 and ik10, resulting from exons 4–7 and 2–7 deletions, respectively) as previously reported [17]. All primers followed internal validation and manufacturer’s recommendations (Invitrogen). Part of genetic experiments was performed in remnant cytogenetic pellets or RNA stored at the diagnostic laboratory. Genetic evaluation is summarized in a flowchart available in supplementary appendix.
Clarifying the Blurry Boundaries between Research and Clinical Care
Published in The American Journal of Bioethics, 2022
Good ethics begins with good facts, and methodology matters. We are told that the researchers diagnosed Klinefelter Syndrome by genomic sequencing in a non-CLIA lab. CLIA regulations were designed to reduce failures of labeling and specimen identification (CDC 2022). Returning research results from a non-CLIA lab raises issues of sample labeling accuracy. Also significant is how the diagnosis was made. We do not know if the researchers conducted formal karyotyping after identifying Klinefelter Syndrome with sequencing. The majority of karyotypes detected in Klinefelter Syndrome individuals are 47,XXY while others are non-mosaic (e.g., 48,XXXY) or mosaic aneuploidy (i.e., 46,XY/47,XXY). Karyotyping is necessary to determine whether the participant is a mosaic (which often has milder symptoms) (Matsumoto and Anawalt 2019). This may tip the scales of risks and benefits in deciding whether the finding is “urgently important.”