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Maple syrup urine disease (branched-chain oxoaciduria)
Published in William L. Nyhan, Georg F. Hoffmann, Aida I. Al-Aqeel, Bruce A. Barshop, Atlas of Inherited Metabolic Diseases, 2020
William L. Nyhan, Georg F. Hoffmann, Aida I. Al-Aqeel, Bruce A. Barshop
MSUD is transmitted as an autosomal recessive trait. This is true of each of the variants. Classic MSUD has been seen throughout the world and in all ethnic groups. It is common among the Mennonites of Pennsylvania in whom the incidence is one in 400 [20]. In the German screening program, an incidence of one in 165,000 was encountered [40]. Heterozygote detection by enzymatic assay may not be reliable. Once a mutation is identified in a proband, molecular techniques may be used to establish carrier status. The activity of the enzyme can be measured in cultured amniotic fluid cells, and the disease has been diagnosed prenatally in a substantial number of patients. Mutations can readily be tested for in prenatal diagnosis, especially with allele-specific oligonucleotide probes [12].
Atherosclerosis and Coronary Heart Disease
Published in Victor A. Bernstam, Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
The analysis of apo E alleles at the DNA level allowed the demonstration of structural alterations affecting apo E isoform receptor-binding capacities. The routine detection of apo E alleles is possible using allele-specific oligonucleotides (ASOs) in hybridization and PCR amplification. The discrimination of ε3-ε4 and ε2-ε3 alleles by the ASO hybridization approach is based on the difference in thermal stability between the hybrids formed by fully matched 19- or 20-mer oligonucleotides and duplexes with a minor G-T mismatch or duplexes with a more destabilizing mismatch. PCR amplification produces much stronger signals that are less affected by nonhomologous hybridization. Combining PCR with Southern blotting allowed the identification of apo E isoforms and could be modified to recognize any mutation in the gene of interest.
Detection Techniques for Single Nucleotide Polymorphisms
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
W. Mathias Howell, Johan Stenberg, Chatarina Larsson, Mats Nilsson, Ulf Landegren
The SNPlex assay102 recently released by Applied Biosystems is a multiplexed ligation-based assay performed on fragmented genomic DNA. The allele discrimination procedure basically involves differential ligation of one locus-specific oligonucleotide with a pair of allele-specific oligonucleotides. Each allele-specific oligonucleotide encodes a unique tag termed a ZipCode™. Following a clean-up step, successfully ligated products are amplified by PCR and immobilized to a streptavidin-coated plate via a biotin label on one of the PCR primers. A second clean-up step serves to remove excess PCR reagents and the non-biotinylated strand of the PCR product. A set of Zip-Chute™ detection probes is added to the plate and allowed to hybridize.
Mutation patterns of epidermal growth factor receptor gene in non-small cell lung cancer among Egyptian patients
Published in Egyptian Journal of Basic and Applied Sciences, 2022
Wafaa H. Elmetnawy, Mona Qenawi, Salwa Sabet, Heba Bassiony
Extracted DNA was amplified by Multiplex PCR amplification of specific DNA sequences using biotinylated primers. Then, the amplification products were analyzed by gel electrophoresis. The amplified products were hybridized to a test strip containing allele-specific oligonucleotide probes immobilized as an array of parallel lines (Precise selective hybridization of specific sequences onto Strip Assay). Malignant tumor and control samples were tested for specific EGFR mutations using ASO strip assay that targets three mutations affecting codon 719 in exon 18 (G719A, G719C, and G719S), grouped deletions within exon 19, insertions within exon 20, and the individual mutations T790M, L858R, L861Q, and S768I. The 30 investigated mutations in the four exons of EGFR gene are listed in Table 1.
Rare Pathogenic β0-Thalassemia Mutation, Codon 7 (GAG>TAG) (HBB: c.22G>T). Report of the First Two Cases in Albanian Immigrants of Northern Greece
Published in Hemoglobin, 2022
Evangelia Zarkada, Eleni Yfanti, Aikaterini Teli, Angeliki Balassopoulou, Klio Sinopoulou, Stamatia Theodoridou
The denaturing gradient gel electrophoresis (DGGE) showed that the woman was a carrier of the IVS-I-6 (T>C) mutation on the β gene. Her 37-year-old husband, Albanian from Elbasan, was also screened and the results showed a Hb level of 13.0 g/dL, RBC count of 6.14 × 1012/L, MCV 64.4 fL, MCH 21.1 pg and ferritin level of 64.0 ng/mL; HPLC analysis showed a Hb A2 level of 5.5% and Hb F of 7.4% with negative sickle test. His red cell morphology included target cells, hypochromasia, microcytosis, anisocytosis, and basophilic punctuation. Moreover, the man showed an unknown electrophoretic pattern on DGGE. None of the known mutations corresponded to this pattern and sequencing revealed a point mutation at codon 7 (G>T), where guanine was replaced by thymine and thus the codon corresponding to glutamic acid (GAG) was replaced by the codon leading to termination of translation (TAG) and non-production of the β chain. The presence of the mutation was confirmed by hybridization allele-specific oligonucleotide (ASO). This rare codon 7 mutation was reported for the first time worldwide [1,14] and these are the hematological results in this Albanian male. The α-globin genotype was found to be normal. As the first couple came late in the pregnancy, fetal blood testing was taken for PND and biosynthesis of fetal blood and analysis of the DNA. The fetus proved to be a carrier of the new codon 7 mutation with a biosynthesis β to α ratio of 0.030, characteristic of a β0-thal mutation (Table 1).
Volanesorsen for treatment of familial chylomicronemia syndrome
Published in Expert Review of Cardiovascular Therapy, 2021
Julieta Lazarte, Robert A. Hegele
Two of the main implementation platforms to downregulate mRNA are as follows: 1) short interfering RNAs (siRNAs); and 2) allele-specific oligonucleotides (ASOs), which are both discussed at length in excellent comprehensive reviews that the reader is encouraged to seek out [49,50]. Briefly, siRNAs act within the cytoplasm, while ASOs act within the nucleus. siRNAs are double stranded DNAs that are delivered as duplexes, are taken up by the RNA-induced silencing complex (called Argonaute or AGO), and they guide AGO to the target complementary mRNA, which can then be cleaved or repressed. In contrast, ASOs are delivered as a single strand of either RNA or DNA and once inside the nucleus are able to find their target sequence alone. After binding to the target mRNA, ASOs can sterically block protein-building interactions with ribosomes, or they can recruit degrading factors (e.g. RNase H1, an endogenous ribonuclease expressed widely in mammalian cells) or they can modulate splicing to yield an ineffective transcript [50–52]. Furthermore, both siRNAs and ASOs can be chemically coupled with certain sugars that direct them through specific receptors on hepatocytes, such as the asialoglycoprotein receptor, which increases the efficiency of delivery of the therapy.