Analysis Of Other Mutations Research Articles

PB2193 GENE EXPRESSION PROFILE IN PRIMARY MYELOFIBROSIS: ROLE OF ASXL1

Background:Primary myelofibrosis (PMF) is a BCR‐ABL1‐negative myeloproliferative neoplasm characterized by aberrant differentiation of all myeloid lineages and profound disruption of the bone marrow niche. The presence of JAK2, CALR or MPL [R1] mutation supports the diagnosis. In approximately 10% of cases classified as PMF, the genetic cause remains unknown and they are known as triple negative. Recent prognostic scores like GIPSS or MIPSS recommend the analysis of other mutations in non‐driver genes, such as ASXL1, SRSF2, EZH2, IDH1/2 and U2AF1Q157, since they influence the risk stratification.Aims:To analyze the mRNA expression profiles of EZH2, ASXL1, RB1 and TET2 genes in a series of PMF patients with known mutational status of JAK2V617F, CALR and MPL MethodsOur series consisted of 21 PMF patients, 6 females and 15 males, with a mean age of 70 years (range 41–87), diagnosed and treated between 1996 and 2018 at the Hospital Universitario de Gran Canaria Dr. Negrín, Spain. Four patients of our series were triple negative, 13 JAK2V167mut, 3 CALRmut and 1 MPLmut. mRNA expression levels were determined by real time PCR in a LightCycler 480 Instrument II (Roche) using ABL as a control gene. Results were normalized with a cDNA pool from the peripheral blood of 10 healthy donors, which was introduced as an internal control in each experiment. SPPS V.23 software was used for statistical analyses.Results:We found that ASXL1 expression levels were significantly higher in patients with JAK2V617F mutated compared with JAK2V617F non‐mutated (mean 0.47 vs. 0.25, p = 0.037, Mann–Whitney U test). In the study of progression free survival, we detected a 36% decrease in the probability of progression to AML or death per unit of ASXL1 expression (p = 0.007, Cox regression test). No significant associations were found between expression levels of the other genes studied (RB1, TET2 and EZH2) and triple negative status.Summary/Conclusion:ASXL1 mutations are common in myeloid diseases and, in these cases, ASXL1 mutations are associated with worse overall survival and leukemia‐free survival. While the impact of ASXL1 mutations has been well‐studied, the impact of ASXL1 expression levels on patient outcome is less studied. The positive association between higher ASXL1 expression plus JAK2V617Fmut and better progression free survival is in agreement with the fact that the opposite, that is, inactivating ASXL1 mutations plus triple negative status, contribute to poor prognosis. These preliminary results should be confirmed in further studies with a larger series.

RET Proto-Oncogene Genotyping Using Unlabeled Probes, the Masking Technique, and Amplicon High-Resolution Melting Analysis

Single bp mutations in the RET proto-oncogene can cause multiple endocrine neoplasia type 2 syndromes. The conventional approach for genotyping RET mutations is sequencing the exons. A closed-tube RET genotyping assay using a saturating DNA dye, unlabeled probes, and amplicon high-resolution melting analysis was developed. The method required two sequential polymerase chain reaction stages, a primary and secondary assay. The primary assay analyzed RET exons 10, 11, 13, 14, and 16 with a total of seven reactions using eight unlabeled probes. The primary assay genotyped wild-type exons, a common exon 13 polymorphism, and an exon 16 mutation, whereas other RET sequence variation was detected. The primary unlabeled probe data limited the possible genotypes for the detected RET sequence variation, which permitted genotyping in a secondary assay with only two to five reactions. Six probes were designed with the masking technique and masked selected sequence variations to allow unambiguous analysis of other mutations elsewhere under the probe. After this two-stage RET genotyping assay, less than 0.2% of exons tested would require sequencing for genotype. A blinded study generated from five wild type and 29 available RET sequence variation samples was 100% concordant with sequencing. Amplicon high-resolution melting analysis with unlabeled probes and the masking technique is a fast, accurate method for genotyping the >50 RET sequence variations.

Rastreamento da fibrose cística usando-se a análise combinada do teste de IRT neonatal e o estudo molecular da mutação deltaF508

A total of 117 newborn screening cards were anonymously selected for cystic fibrosis (CF) screening, searching for the DF508 mutation using polymerase chain reaction (PCR) followed by polyacrylamide gel electrophoresis (Page) and by quantification of Immunoreactive trypsin (IRT, Delfia). In 116 newborns an IRT concentration lower than 140ng/ml was evidenced. One of these was a DF508 heterozygote with an IRT concentration of 4,44ng/ml. One newborn was a non-DF508 homozygote with an IRT concentration of 410,7ng/ml. The IRT concentration average was significantly different if the newborn with the abnormal IRT was included or excluded from the population sample (n = 117, means = 8.207 ± 38.101; n = 116, means = 4.737 ± 6.597, respectively). Another sample of 8 newborns previously screened by IRT test and with elevated IRT levels was analyzed for DF508 mutation. The DF508 mutation was found in one or both chromosomes in five newborns, corresponding to 62.25% of the sample. Results obtained with two-tier IRT/DNA analysis showed that the approach only will be effective if: a) factors leading to false positives and false negatives are excluded; and b) molecular analysis of other mutations are included among those children with undefined results.

Dihydropyrimidine Dehydrogenase Enzyme Deficiency: Clinical and Genetic Assessment of Prevalence in Turkish Cancer Patients

Background: Fluorouracil has been reported to induce severe side-effects in particular subjects who have deficiency in dehydropyrimidine dehyrogenase activity, the major enzyme in the catabolism of fluorouracil.Patients and methods: In this study, we aimed to analyze the heterozygote and homozygote frequencies of dehydropyrimidine dehyrogenase gene mutation in 200 patients receiving fluorouracil based chemotherapy together with the assessment of the toxicity profile of these chemotherapy regimens.Results: According to the results of clinical toxicity assessments, grade 3–4 hematologic toxicity was noted in 12% of the patients. Grade 3 gastrointestinal toxicity was present in 5% of the subjects with no grade 4 side effects. The heterozygote (2q(1−q)) and homozygote (q2) frequencies of dehydropyrimidine dehyrogenase gene mutation were calculated as 1.5% (3/200) and 0.000055% (1/18,043) in the analyzed samples.Conclusion: In this report, for the first time we documented the frequency of dehydropyrimidine dehydrogenase gene mutation in Turkish cancer patients. The determination of enzyme activity in suspected individuals and analysis of other mutations on a population basis would be the next steps for our country.

Molecular analysis of wild-type and mutant alleles at the Opaque-2 regulatory locus of maize reveals different mutations and types of O2 products.

The expression of the various members of the zein multigene family in maize endosperm is controlled by different regulatory loci. One of these loci, Opaque-2, coding for a bZIP transcriptional factor, controls the expression of a subset of zein genes. Analysis of genomic DNA from plants carrying wild-type (O2) or mutant o2 alleles shows specific DNA restriction patterns that correlate with transcript types and their various gene products. Northern and western analyses show the presence in different wild types of a 1.7 kb transcript coding for different sizes of normal O2 proteins that migrate as doublets in the 68-72 kDa range. Among the various o2 mutants analysed we showed the occurrence of various null-transcript alleles, the presence of alleles with a normal size transcript which, however, produce a different-sized o2 protein, and a mutant producing both a normal size transcript and a longer transcript, but generating only a single o2 product migrating around 40 kDa. Analysis of other mutations (o7, fl2) known to affect zein polypeptide synthesis shows no interference of these mutations in the expression of the O2 gene products. The overall results indicate the occurrence of micro heterogeneity in the O2 wild-type genes and a broad spectrum of o2 mutations, both producing different sizes of O2 or o2 proteins. A nomenclature of the O2 and o2 genes based on the RFLP, transcripts and products of the various alleles is presented.

Genetic analysis of the dominant white-spotting (W) region on mouse chromosome 5: identification of cloned DNA markers near W.

We have assigned several mouse cDNA and genomic clones to the W region of mouse chromosome 5, established their position with respect to various marker loci in the region, and provided molecular verification that the W19H mutation is a deletion. Meiotic recombination analysis of an interspecific mouse backcross indicated the following gene order and distances [in centimorgans (cM)]: centromere-Emv-1-(13 cM)-D4S76-(17 cM)-D5SC25-(5 cM)-alpha-casein-(1 cM)-beta- casein-(6 cM)-alpha-fetoprotein-(18 cM)-beta-glucuronidase. D5SC25, an anonymous locus defined by a mouse brain cDNA, maps near the map position of W and within the breakpoints of the presumed genetic deletion that causes the W19H phenotype. Southern analysis of DNAs of W19H/+ interspecific F1 hybrid mice and somatic cell hybrid lines carrying the W19H deletion chromosome showed the deletion of D5SC25. In fact, analysis of other mutations at or near the W locus, which had been transferred from the strain of origin for many backcross generations, revealed the retention of donor restriction fragment length polymorphisms at the D5SC25 locus. Such evidence confirms close linkage between D5SC25 and W (within 1 cM) and indicates that the D5SC25 cDNA clone could serve as a starting point in a chromosome "walk" to W and other closely linked loci that affect development.

Mutations in the pho80 gene confer permeability to 5'-mononucleotides in Saccharomyces cerevisiae.

Yeast mutants permeable to dTMP (tup) were selected and two new complementation groups (tup5 and tup7) were identified. Assay of the levels of both acid and alkaline phosphatase in cells grown under either repressing (5 mM PO4(-3) or derepressing (0.03 mM PO4(-3) conditions indicated that, in general, tup mutations cause cells to be defective in their regulation of phosphatase synthesis. In addition, three of the tup mutations (tup1, tup4 and tup7) displayed markedly elevated rates of inorganic phosphate transport. The tup7 locus was found to be tightly centromere-linked on the right arm of chromosome XV, and was shown to be allelic with the pho80 regulatory locus on the basis of both genetic and biochemical criteria. Analysis of other mutations known to affect phosphatase levels (pho) indicated that some also conferred permeability to dTMP. Possible allelic relationships between tup genes and certain of these pho mutations are discussed. Regardless of the culture conditions, wild-type strains were not permeable to dTMP; in contrast, it was found in the course of this work that normal yeast cells were permeable to dUMP and that dUMP permeability was regulated by the concentration of inorganic phosphate present in the medium used to grow the cells. Thus, permeability to 5'-mononucleotides appears to be under coordinate control with phosphatase synthesis.

Genetics of Ustilago violacea. X. The Pseudohyphal Mutation and Tetrad Analysis

The pseudohyphal mutation is responsible for elongate cells in chains and dull colonies with a ropy surface. A cross between pink pseudohyphal (y+ ph) and yellow sporidial (y ph+) strains was analyzed by random sporidial analysis, teliospore colony (intact tetrad) analysis, and teliospore colony-sporidia sampling. Both types of teliospore colony analysis gave parental ditype, nonparental ditype, and tetratype tetrads. The y and ph loci independently segregate. Because the y locus is centromere linked, tetratype tetrads result from recombination between the ph locus and its centromere (ca. 4 cM). Tetratype tetrads provide sporidia for the tetrad analysis of other mutations in parental strains.

Physiological Genetics of the Mouse

Publisher Summary This chapter discusses the physiological genetics of mouse. It reviews the effect of mutations in different organ systems of the mouse that include skeleton, integument, nervous system, pigment-forming system, blood, and developmental systems of the embryo. Mutations is discussed where an effect on enzyme systems can be demonstrated, such as those affecting pigmentation. Hemoglobin synthesis is shown to be disturbed in different hematopoietic tissues and in different phases of development by the action of mutational changes with far-reaching effects. One syndrome of abnormalities is discussed that is shown to have arisen as a result of the mutational effect on a hormone. The developmental analysis of some abnormalities of the nervous system demonstrates how the original effect of a mutation may show no direct relation to the eventual symptoms. The analysis of other mutations reveals that their effects are due to the changed rates of growth and differentiation of certain tissues.