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Preimplantation Genetic Testing for Aneuploidies: Where We Are and Where We're Going
Published in Darren K. Griffin, Gary L. Harton, Preimplantation Genetic Testing, 2020
Andrea Victor, Cagri Ogur, Alan Thornhill, Darren K. Griffin
During PGT-A, aside from the analysis of nuclear DNA content, cellular biopsies can be evaluated for mitochondrial DNA (mtDNA) load. mtDNA is a circular nucleic acid molecule present in each human cell as multiple copies. Two groups published independent studies in 2015 indicating that high mtDNA content per cell correlated with poor embryo viability [153,154]. They described a threshold of mtDNA copy number that if surpassed always led to failed implantation upon transfer. One of the groups published two additional studies supporting the findings, although noting that some clinics did not generate blastocysts with a greatly elevated mtDNA copy number [155,156]. Publications from other laboratories could not reproduce the results [157–160]. Noting that vastly different mtDNA quantitation methods were used between studies, it was suggested that technical variability could have caused the contradictory observations. Guidelines were hence proposed with the intent to standardize mtDNA copy number analysis during PGT-A [161]. Recently, a study adhering to those guidelines reported the implantation of blastocysts with a highly elevated mtDNA copy number, which led to subsequent healthy births [162]. The use of mtDNA copy number assessment as a routine PGT-A “add-on” to further rank euploid embryos has therefore largely been discredited. Whether it will find some use in particular settings remains to be determined.
Genetics
Published in Stephan Strobel, Lewis Spitz, Stephen D. Marks, Great Ormond Street Handbook of Paediatrics, 2019
Jane A. Hurst, Richard H. Scott
Since the 1980s, the mainstay of DNA sequencing has been Sanger sequencing, a technique which requires PCR amplification of the target gene in small fragments followed by sequencing of the amplified fragment. This is a highly accurate but labour intensive and therefore expensive technique. Sanger sequencing does not usually detect larger-scale copy number alterations. For many genes, comprehensive mutation testing therefore also requires the use of a copy number analysis technique such as multiplex ligation-dependent probe amplification (MLPA).
Radiogenomics
Published in Ruijiang Li, Lei Xing, Sandy Napel, Daniel L. Rubin, Radiomics and Radiogenomics, 2019
Besides gene expression data, also other types of molecular data have important applications in determining diagnosis or treatment of cancer patients. Examples are DNA sequencing, DNA copy number, DNA methylation analysis, and protein expression. DNA sequencing provides us with the full complement of mutations in the genome of a biological sample. For example, activation of oncogenes or inactivation of tumor suppressor genes are common drivers of many tumor types. Similarly, DNA copy number analysis allows to profile how many copies are present for each gene in the human genome. This analysis allows to identify if certain genes are amplified, e.g., oncogenes, or are deleted from the genome, e.g., tumor suppressor genes. These genes are often known as cancer driver genes and can drive tumor growth. Genome-wide DNA methylation measurements are possible thanks to microarray technology enabling reading out binary states in the genome to determine if they are methylated or not [36–40]. Finally, protein expression analysis is technologically more complicated, as profiling the human proteome is more complicated and expensive, but has recently seen an increase in use thanks to developments in mass spectrometry technology and large multi-cancer projects are being executed [41].
Profiling targetable immune checkpoints in osteosarcoma
Published in OncoImmunology, 2018
Troy A McEachron, Timothy J Triche, Laurie Sorenson, David M Parham, John D Carpten
To investigate if the targetable immune checkpoint genes PD-L1, PD-L2, B7-H3 and IDO1 are affected by the copy number alterations that typify osteosarcoma, genome-wide copy number analysis was performed on DNA from 195 pediatric osteosarcoma tumor specimens using SNP 6.0 arrays. This copy number data was combined with whole genome sequencing data from the TARGET osteosarcoma project (19 specimens) and internal whole genome data (1 specimen), totaling 215 specimens. Our meta-analysis revealed recurrent independent copy number gains at genomic loci encompassing PD-L1/PD-L2 (9p24.1), B7-H3 (15q24) and IDO1 (8p11.21) at frequencies of 8%-9% (Figure 1(a)). The nature of the PD-L1, PD-L2, and IDO1 copy number alterations ranged from low-level gains to high-level amplifications (Figures 1(b-e) and 2(a-c)). Although B7-H3 was gained in 8% of the specimens, we did not observe high level amplifications at this locus (Figures 1(d) and 2(b)). Furthermore, our data demonstrates a very strong correlation between samples with copy number gains of PD-L1 and PD-L2 (Figure 1(f)). With the exception of PD-L1 and PD-L2, simultaneous gains of more than one checkpoint gene in an individual specimen were rarely observed, suggesting that these are mutually exclusive and independent events (Figure 1(a)).
A novel deep intronic low penetrance RB1 variant in a retinoblastoma family
Published in Ophthalmic Genetics, 2018
Sameh E. Soliman, Hilary Racher, Melissa Lambourne, Donco Matevski, Heather MacDonald, Brenda Gallie
Complete retinoblastoma genetic testing was performed on DNA extracted from a peripheral blood sample taken from the affected child. Analysis included (i) sequencing of all 27 RB1 coding exons, flanking intronic regions, and promoter; (ii) copy number analysis using quantitative-multiplex PCR (5); (iii) allele-specific polymerase chain reaction (PCR) for 11 recurrent RB1 pathogenic variants, capable of detecting mosaicism at levels as low at 1% (6) and (iv) targeted sequencing analysis for four previously identified deep intronic pathogenic variants: c.608-3416A>G (7), c.861 + 828T>G (7), c.2490-1398A>G (8), and c.939 + 541A>G (Impact Genetics in-house data). No pathogenic variants were detected. Given the heritable presentation in this family, further molecular analysis was pursued to seek deep intronic variants that may disrupt splicing of the RB1 transcript.
Nance–Horan syndrome in females due to a balanced X;1 translocation that disrupts the NHS gene: Familial case report and review of the literature
Published in Ophthalmic Genetics, 2018
Laura Gómez-Laguna, Alejandro Martínez-Herrera, Alejandra del Pilar Reyes-de la Rosa, Constanza García-Delgado, Karem Nieto-Martínez, Fernando Fernández-Ramírez, Tania Yanet Valderrama-Atayupanqui, Ariadna Berenice Morales-Jiménez, Judith Villa-Morales, Susana Kofman, Alicia Cervantes, Verónica Fabiola Morán-Barroso
GTG banding karyotype analyses in peripheral blood lymphocytes of the patients identified the apparently balanced rearrangement between chromosomes X and 1: 46,X,t(X;1)(p22.13;q22) (Figure 2A). The subtelomeric regions of these chromosomes were analyzed in patient 2 by FISH with mixture number 1 from ToTelVysion™ multicolor DNA probes (Vysis Abbott Laboratories, Abbott Park, ILL, USA), the analysis confirmed the translocation (Figure 2B); therefore, her karyotype was as follows: 46,X,t(X;1)(p22.13;q22).ish t(X;1)(1QTEL10+,DXYS129-,DXZ1+;CEB108/T7+, 1QTEL10-,DXYS129+)[30]. Copy-number analysis was performed on peripheral blood DNA from patient 1 with the Cytoscan HD array kit (Affymetrix Inc., Santa Clara, CA, USA) as previously described(23). The array data indicated that the translocation was balanced (data not shown). Replicating R-banding chromosomal analysis in peripheral blood lymphocytes from Patient 2 was performed as previously described(24), and the results demonstrated that the normal X chromosome was late-replicating in 100 analyzed cells (Figure 2C).