Untranslated Region Mutation Research Articles

A comparison of complete untranslated regions of measles virus genomes derived from wild-type viruses and SSPE brain tissues

We compared complete untranslated regions (UTRs) of two subacute sclerosing panencephalitis (SSPE) measles virus (MV) strains and two wild-type (wt) MV strains, all belonging to the same genotype (D6). In comparison to wt MVs of the same genotype, base changes were identified in the two SSPE measles virus strains at 27 and 33 noncoding positions, respectively. Majority of these residues are unique for each of the SSPE virus sequences in comparison to all other reported measles virus strain sequences. The location of some of these changes indicates that they may modify cis-acting regulatory sequences including gene-end signal of the P gene, H/L gene junction and Kozak consensus element of the L gene. Further, within the long UTR between M and F genes, deletions and insertions were identified. Thus, our study could be significant for additional investigation using reverse genetics and recombinant viruses, of possible influence of mutations in UTRs on establishment and maintenance of chronic progressive CNS disease caused by MV persistence.

CYP2A13 genetic polymorphism in French Caucasian, Gabonese and Tunisian populations

Since human CYP2A13 is expressed in the respiratory tract and is involved in the activation of tobacco-specific nitrosamines, some of the previously reported sequence variations may contribute to inter-individual and inter-ethnic differences in the susceptibility of tobacco-related tumorigenesis. The aim was to compare the frequencies of the 578C > T (Arg101Stop), 3375C > T (Arg257Cys) and 7520C > G (3′-untranslated region) mutations in several populations. The frequencies of the 578C > T polymorphism were 3.8, 0 and 1.0% in French Caucasians, Gabonese and Tunisians, respectively. In the same populations, the frequencies of the 3375C > T mutation were 0, 15.3 and 4.2%, respectively, whereas the frequencies of the 7520C > G mutation were 1.0, 20.8 and 7.3%, respectively. Marked inter-ethnic variations in CYP2A13 were identified and confirmed. These findings provide data for further studies that associate CYP2A13 haplotypes with an incidence of smoking-related tumours in respect of ethnicity.

Complete genome sequencing of dengue virus type 1 isolated in Buenos Aires, Argentina

Dengue (DEN) constitutes a major viral arthropod-borne human illness. South America was last considered free of dengue two decades ago when a dramatic increase in the number of dengue fever and hemorrhagic dengue cases had been reported. Five viruses were isolated in Buenos Aires City from the 1999–2000 Paraguay outbreak. RT-PCRs obtained directly from plasma were cloned into pGemT vectors and sequences of the structural genes and NS1 were analyzed. Three viruses were full-length sequenced from RT-PCR obtained from cell-culture isolates. Excess of synonymous over non-synonymous mutations suggested that the structural proteins were under strong functional constraints while a weak purifying selection was operating in the whole polyprotein. Sequence diversity and selective pressures varied among patients but results were significantly above the procedure threshold. One sample showed small-plaque phenotype and impaired growth coupled to 3′untranslated region mutations. Phylogenetic analysis of full-length sequences split Buenos Aires isolates into two clusters within American DEN-1 genotype V: Clade I was phylogenetically linked to Brazilian samples and Clade II with samples from Paraguay and Northeastern Argentina. In Buenos Aires City, only dengue virus serotype 1 imported from Paraguay has been detected, though without evidence of local transmission.

The Neurofibromatosis Type 2 Gene Is Mutated in Perineurial Cell Tumors: A Molecular Genetic Study of Eight Cases

Perineurial cell tumors (PNTs) are rare neoplasms derived from or showing differentiation toward specialized lining cells of the nerve sheath, the perineurial cells. In this study, we have evaluated neurofibromatosis type 2 (NF2) gene alterations in eight PNTs using archival formaldehyde-fixed, paraffin-embedded tissue. Two conventional soft-tissue PNTs from the upper back and chest wall, one retiform soft tissue variant from the scapular region, and five sclerosing PNTs from the fingers and palm were studied. All cases showed histological features of PNTs, and the neoplastic cells were positive for epithelial membrane antigen and negative for S100 protein. The coding sequences (exons 1 to 15) of the NF2 gene were polymerase chain reaction (PCR) amplified and evaluated for mutations by direct sequencing of the PCR products. Five NF2 point mutations, two in the 5'-untranslated region (UTR) and three in exons 3, 6, and 8, were identified in four of eight cases (50%) studied. Exon mutations resulted in changes of predicted amino acids sequences: Asp-->Asn at codon 83, Glu-->Asp at codon 182, and Leu-->Val at codon 241. In two cases (one with a missense mutation in codon 241), the same point mutation in the 5'-UTR at the nucleotide position 8958 was identified. A loss of heterozygosity (LOH) study was performed in three cases. LOH at the NF2 locus was found in one case with a mutation in the 5'-UTR. However, in another case with exon 8 and 5'-UTR mutations, deletion of one allele of the NF2 gene was previously documented by fluorescence in situ hybridization. The coexistence of NF2 gene mutations and LOH at the NF2 locus indicates that the NF2 tumor suppressor gene is altered in PNTs by the two-hit mechanism.

Effects of 3' untranslated region mutations on plus-strand priming during moloney murine leukemia virus replication.

A conserved purine-rich motif located near the 3' end of retroviral genomes is involved in the initiation of plus-strand DNA synthesis. We mutated sequences both within and flanking the Moloney murine leukemia virus polypurine tract (PPT) and determined the effects of these alterations on viral DNA synthesis and replication. Our results demonstrated that both changes in highly conserved PPT positions and a mutation that left only the cleavage-proximal half of the PPT intact led to delayed replication and reduced the colony-forming titer of replication defective retroviral vectors. A mutation that altered the cleavage proximal half of the PPT and certain 3' untranslated region mutations upstream of the PPT were incompatible with or severely impaired viral replication. To distinguish defects in plus-strand priming from other replication defects and to assess the relative use of mutant and wild-type PPTs, we examined plus-strand priming from an ectopic, secondary PPT inserted in U3. The results demonstrated that the analyzed mutations within the PPT primarily affected plus-strand priming whereas mutations upstream of the PPT appeared to affect both plus-strand priming and other stages of viral replication.

Double Fluorescent-amplification Refractory Mutation Detection (dF-ARMS) of the Factor V Leiden and Prothrombin Mutations

Simultaneous fluorescent [F] detection of the factor V Leiden (G1691A) and the prothrombin 3'-untranslated region (G20210A) mutations were performed in a single tube polymerase chain reaction (PCR). Amplification refractory mutation detection system (ARMS) formed the basis of this assay design. Fluorescent-labelled primers incorporated into amplicons during the reaction facilitated detection directly by GeneScan analysis without further manipulation. To test the efficacy of this double [F]-ARMS (dF-ARMS) method, 48 patients with unexplained thrombotic tendencies were investigated for their factor V Leiden and prothrombin genotypes. These results corresponded exactly with data achieved using the more conventional methods of restriction fragment length polymorphism (RFLP)-PCR and direct DNA sequencing. Three out of the 48 patients in this group were found to be compound heterozygotes.

Mechanisms of Increased NGF Production in Vascular Smooth Muscle of the Spontaneously Hypertensive Rat

The spontaneously hypertensive rat (SHR) was developed as a genetic model of essential hypertension.In vivoandin vitroevidence demonstrates that vascular smooth muscle cells (VSMCs) from the SHR produce more nerve growth factor (NGF) than the normotensive Wistar-Kyoto (WKY) control strain. This increased NGF production is accompanied by excessive innervation of target tissues in the SHR. In the present study, a sensitive, competitive, quantitative, reverse-transcriptase polymerase chain reaction (C Q RT-PCR) assay is characterized and used to analyze levels of NGF mRNA in cultured VSMCs derived from the SHR and WKY strains as well as bladder tissue. Differences in NGF secretion rates between SHR and WKY VSMCs were partially due to an increased stability of NGF mRNA in SHR VSMCs. Following treatment with platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β1) to elevate NGF production, the half-life of the NGF mRNA was 104.5 ± 18.0 min in SHR VSMCs, compared to only 36.5 ± 11.6 min in WKY VSMCs. Sequence analysis of the 3′ untranslated region (UTR) revealed no strain differences incis-acting sequences potentially involved in determining mRNA stability. Thus, it seems unlikely to be a 3′UTR mutation that prolongs mRNA lifetime. Rather, differential regulation of an RNA-binding protein may play a role in the abnormal NGF mRNA stability in SHR VSMCs. SHR VSMCs also demonstrate an increased translational efficiency of NGF protein; more NGF protein is synthesized per unit of NGF mRNA. The use of a C Q RT-PCR assay has allowed the determination that abnormal NGF mRNA stabilization as well as altered translational efficiency may contribute to excess NGF synthesis and progressive hypertension in the SHR.

The Prothrombin Gene 3′-Untranslated Region Mutation Is Frequently Associated With Factor V Leiden in Thrombophilic Patients and Shows Ethnic-Specific Variation in Allele Frequency

To the Editor: As data accumulate on the recently identified genetic variant in the 3′-untranslated region of the prothrombin (factor II) gene (G20210A), it is apparent that this mutation confers a moderate risk for venous thrombosis.[1-3][1] Data from several abstracts presented at the 1997 ISTH

Point mutations of the connexin32 (GJB1) gene in X-linked dominant Charcot-Marie-Tooth neuropathy.

Ten families with X-linked dominant CMT neuropathy (CMTX1) were screened for point mutations of the connexin32 (Cx32, GJB1) gene. Two families showed missense mutations, respectively an A-->G transition at amino acid 102 (glutamate to glycine) and a C-->T transition at amino acid 142 (arginine to tryptophan). Three families showed nonsense mutations, respectively a C-->T transition at amino acid 22 (arginine to stop) a G-->T transversion at amino acid 186 (glutamate to stop), and a T-->A transversion at amino acid 217 (cysteine to stop). Five CMTX1 neuropathy families showed no evidence of point mutations of the connexin32 coding sequence. These findings suggest that the CMTX1 neuropathy genotype is heterogeneous or the result of promoter mutations, 3'-untranslated region mutations or exon/intron splice site mutations. Four of the reported mutations created or destroyed restriction enzyme sites: a HaeIII restriction enzyme site was destroyed by the mutation at amino acid position 22, a HpaII site was eliminated at amino acid position 142, a Bfal restriction site was created by the mutation at amino acid 186 and a Ddel restriction site was created by the mutation at amino acid 217. These changes allowed us to test family members for the mutations and observe the segregation of the disease with the mutations.