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Advances in Non-Invasive Diagnosis of Single-Gene Disorders and Fetal Exome Sequencing
Published in Carlos Simón, Carmen Rubio, Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
Liesbeth Vossaert, Roni Zemet, Ignatia B. Van den Veyver
Genome and exome sequencing are technologies that examine the genome at a nucleotide level. NGS is achieved by sequencing many distributed, overlapping sites throughout the genome in a massively parallel manner.56,57 Briefly, for NGS library preparation, genomic DNA is sheared into 50–400 nt fragments, ligated to adapters, and purified. For panel or exome sequencing, an enrichment step is included to capture regions of interest using “DNA baits.” Massively parallel sequencing of the NGS library then rapidly and accurately derives the nucleotide sequences of each fragment, which are then aligned to a reference genome to detect potential sequence variants56–58 (Figure 27.3). Two quality parameters for NGS accuracy are the sequencing depth, which refers to the number of overlapping reads for each base-pair, and the breadth of sequence coverage, which is the fraction of the reference sequence that is covered at sufficient depth. The American College of Medical Genetics and Genomics (ACMG) recommends an average depth of ≥ 100-fold with 90–95% of the sequence covered at least 10-fold for diagnostic exome sequencing (ES).59 With recent advances in NGS technology, clinical laboratories can offer increasingly better sequencing depth and shorter turnaround time (TAT) to results, which is crucial for fetal ES.60,61
Genetics
Published in Cathy Laver-Bradbury, Margaret J.J. Thompson, Christopher Gale, Christine M. Hooper, Child and Adolescent Mental Health, 2021
Exome sequencing studies have been used to identify individual genetic variants in the protein-coding regions of the genome (approximately 1%) in both case-control cohorts and in families (to identify de novo mutations). Whole-genome sequencing is more time-consuming and expensive than whole-exome sequencing since both the protein-coding and non-coding regions are sequenced. Work is currently underway through the Whole Genome Sequencing for Psychiatric Disorders Consortium to integrate sequencing data across disorders in large cohorts of patients and controls that should yield some interesting insights into biological mechanisms underlying cross-disorder risk (Sanders et al., 2017).
Lung Cancer
Published in Pat Price, Karol Sikora, Treatment of Cancer, 2020
The 2015 WHO classification encourages diagnostic precision, made possible with the greater availability of immunohistochemistry and molecular techniques, and is at the forefront of the international effort to reduce NSCLC not-otherwise-specified diagnosis rates. There are now several driver mutations that have been identified in NSCLC, and those in adenocarcinoma and squamous cell carcinoma are specifically depicted in Table 7.1. Some of the commonest molecular techniques used to identify these are summarized in Table 7.2. The table shows the applications of each technique for the different types of aberrations that may be encountered in lung cancer. Targeted exome sequencing on tissue or blood is being increasingly utilized to sequence a focused panel of genes as costs and turnaround times have reduced. Polymerase chain reaction (PCR) technology can also be used to target more specific areas of the genome, identifying single gene mutations and their RNA counterparts in tissue or in circulating tumor cells in blood. Fluorescent in-situ hybridization (FISH) makes use of microscopy to identify fusion and amplification products of fluorescently labeled genes, and although it has been transformational for identifying common fusion/amplifications such as anaplastic lymphoma kinase (ALK), it is also labor-intensive. Immunohistochemistry can be a very cheap, widely performed assay, which can be highly sensitive and specific for identifying protein products of fusions/amplifications such as ALK.
Concurrent PANK2 and OCA2 variants in a patient with retinal dystrophy, hypopigmented irides and neurodegeneration
Published in Ophthalmic Genetics, 2023
Eva Wai Nam Wong, Shirley S.W. Cheng, Tiffany T.Y. Woo, Robert F. Lam, Frank H.P. Lai
Genetic testing and counseling are increasingly recognized as an essential part of clinical management of inheritable ocular diseases. With the use of whole exome sequencing, there would be potential to reveal genetic information that may not pertain to the patient’s presenting condition or partially pertaining to the primary indication with additional disease phenotype (e.g. neurodegeneration) like our patient. Without appropriate genetic testing, the diagnosis of PKAN and OCA may not be apparent owing to the non-specific signs and symptoms in our case. Upon diagnosis, our patient was promptly referred to the neurologists for further workup and management of his neurodegenerative disorder. Although no active treatment could be offered to halt or reverse the disease processes, the patient and his family were informed of the disease course, mode of inheritance and the subsequent management required. Multidisciplinary care involving continuous support from ophthalmologists, neurologists, clinical geneticists, dermatologists, physiotherapists and occupational therapists is indicated for disease monitoring, symptomatic relief as well as to maximize daily functioning.
Genomic medicine in Africa: a need for molecular genetics and pharmacogenomics experts
Published in Current Medical Research and Opinion, 2023
Oluwafemi G. Oluwole, Marc Henry
Limited capacity is the forefront of the challenges facing the implementation of genomic medicine,5 because advanced genomic techniques are needed to implement genomic medicine. For example, it costs about $250 per sample for whole-exome sequencing of X50 coverage, and about $1800 for whole-genome sequencing. The issue regarding the storage systems and securing licenses for cloud computing also limit robust data analyses in genomic medicine. Beside, due to numerous identification of variants of unknown significance,6. more advanced knowledge and analyses are necessary to determine the relevance of these genetic variants to genomic medicine. The study aims to identify the gaps in knowledge and highlight the current state of genomic medicine in Africa to improve research interests in this area.
Identification of Missense Extracellular Matrix Gene Variants in a Large Glaucoma Pedigree and Investigation of the N700S Thrombospondin-1 Variant in Normal and Glaucomatous Trabecular Meshwork Cells
Published in Current Eye Research, 2022
Mary K. Wirtz, Renee Sykes, John Samples, Beth Edmunds, Dongseok Choi, Douglas R. Keene, Sara F. Tufa, Ying Ying Sun, Kate E. Keller
A standard salting-out procedure20 was used to isolate genomic DNA from blood samples from all family members. Whole genome sequencing (WGS) was performed commercially on DNA samples from five family members at Macrogen (Korea). WGS covers approximately 96% of the entire human genome (3 gigabases). Exons are not only covered more effectively than using an exome sequencing approach but variants falling outside of current exome capture designs (such as promoter or other regulatory variants) are not missed. Sequencing was performed on a HiSeq2000 (Illumina, San Diego, CA) to an average depth of 30x for the five samples. A total of 127,373 to 135,944 Mb was identified in the five individuals with 86.65% to 90.16% of ≥ Q30 bases. The mean quality score ranged from 34.45 to 35.53. VCF files were uploaded to the Golden Helix SNP & Variation Suite and the sequences were aligned with the GRCh37/hg19 assembly. The confidence filter retained variants with a call quality of at least 20 and read depth at least 30. Bioinformatics analyses were performed following Genome Analysis Toolkit (GATK) best practices.37,38 Filtration for nonsynonymous sequences was followed by in-silico prediction of functionality using PolyPhen, SIFT and LRT.39–42