Mutation Nt Research Articles

Evolution of the coronavirus spike protein in the full-length genome and defective viral genome under diverse selection pressures.

How coronaviruses evolve by altering the structures of their full-length genome and defective viral genome (DVG) under dynamic selection pressures has not been studied. In this study, we aimed to experimentally identify the dynamic evolutionary patterns of the S protein sequence in the full-length genome and DVG under diverse selection pressures, including persistence, innate immunity and antiviral drugs. The evolutionary features of the S protein sequence in the full-length genome and in the DVG under diverse selection pressures are as follows: (i) the number of nucleotide (nt) mutations does not necessarily increase with the number of selection pressures; (ii) certain types of selection pressure(s) can lead to specific nt mutations; (iii) the mutated nt sequence can be reverted to the wild-type nt sequence under the certain type of selection pressure(s); (iv) the DVG can also undergo mutations and evolve independently of the full-length genome; and (v) DVG species are regulated during evolution under diverse selection pressures. The various evolutionary patterns of the S protein sequence in the full-length genome and DVG identified in this study may contribute to coronaviral fitness under diverse selection pressures.

FOXO1 forkhead domain mutants in B-cell lymphoma lack transcriptional activity

Somatic point mutations of the FOXO1 transcription factor were reported in non-Hodgkin lymphoma including diffuse large B-cell lymphoma, follicular lymphoma and Burkitt lymphoma. These alterations were associated with a poor prognosis and resistance to therapy. Nearly all amino acid substitutions are localized in two major clusters, affecting either the N-terminal region (Nt mutations) or the forkhead DNA-binding domain (DBD mutations). While recent studies have focused on Nt mutations, we characterized FOXO1 DBD mutants. We analyzed their transcriptional activity, DNA binding, phosphorylation and protein–protein interaction. The majority of DBD mutants showed a decrease in activity and DNA binding, while preserving AKT phosphorylation and interaction with the cytoplasmic ATG7 protein. In addition, we investigated the importance of conserved residues of the α-helix 3 of the DBD. Amino acids I213, R214, H215 and L217 appeared to be crucial for FOXO1 activity. Our data underlined the key role of multiple amino-acid residues of the forkhead domain in FOXO1 transcriptional activity and revealed a new type of FOXO1 loss-of-function mutations in B-cell lymphoma.

Molecular epidemiology and phylodynamics of goose haemorrhagic polyomavirus

Goose haemorrhagic polyomavirus (GHPV, or Anser anser polyomavirus 1) is a small dsDNA virus of the Polyomaviridae family. The virus infects the internal organs causing haemorrhagic nephritis and enteritis of geese that may be fatal for goslings. In this study, GHPV positivity was examined in goose and duck samples collected in Hungary between 2005 and 2019. In this period, 384 of the investigated 1,111 specimens were diagnosed as GHPV-positive by PCR assay. Twenty-two GHPV genomes were sequenced and subjected to phylogenetic and evolutionary analysis. Based on the sequence data, the mean evolutionary rates were estimated 6.57×10-6 -5.82×10-5 s/s/y for both GHPV complete genomes and individual genes, with negative selection acting on each gene. When GHPV VP1 sequences originating from wild birds were also included in the analyses, the nt and aa mutations inflated the substitution rate to 1.54×10-4 s/s/y that may imply adaptation of the virus to novel host species. Our data suggested the co-circulation of various GHPV strains in Hungarian goose farms; the source of these may be persistently infected domesticated or migratory wild birds. Detection and characterization of GHPV in wild birds and domestic waterfowls may help to elaborate new strategies for more effective disease control and prevention.

Characterization of significant molecular determinants of virulence of Enterovirus 71 sub-genotype B4 in Rhabdomyosarcoma cells

One of the leading causes of the hand, foot and mouth disease (HFMD) is Enterovirus 71 (EV-A71), displaying symptoms such as fever and ulcers in children but some strains can produce cardiopulmonary oedema which leads to death. There is no FDA-approved vaccine for prevention of severe HFMD. The molecular determinants of virulence for EV-A71 are unclear. It could be a single or a combination of amino acids that determines virulence in different EV-A71 genotype/sub-genotypes. Several EV-A71 strains bearing single nucleotide (nt) mutations were constructed and the contribution of each mutation to virulence was evaluated. The nt(s) that contributed to significant reduction in virulence in vitro were selected and each mutation was introduced separately into the genome to construct the multiply mutated EV-A71 strain (MMS) which carried six substitutions of nt(s) at the 5′-NTR (U700C), VP1-145 (E to G), VP1-98E, VP1-244K and G64R in the vaccine seed strain that had a partial deletion within the 5′-NTR region (nt. 475–485) of Δ11bp. In comparison to the wild type strain, the MMS showed low virulence as it produced very low RNA copy number, plaque count, VP1 and had 105-fold higher TCID50, indicative of a promising LAV candidate that should be further evaluated in vivo.

Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations.

Diversity generation by random mutagenesis is often the first key step in directed evolution experiments and screening of 1,000-2,000 clones is in most directed evolution campaigns sufficient to identify improved variants. For experimentalists important questions such as how many positions are mutated in the targeted gene and what amino acid substitutions can be expected after screening of 1,000-2,000 clones are surprisingly not answered by a statistical analysis of mutant libraries. Therefore three random mutagenesis experiments (epPCR with a low- and a high-mutation frequency and a transversion-enriched sequence saturation mutagenesis method named SeSaM-Tv P/P) were performed on the lipase BSLA and in total 3,000 mutations were analyzed to determine the diversity in random mutagenesis libraries employed in directed evolution experiments. The active fraction of the population ranged from 15% (epPCR-high), to 52% (SeSaM-Tv P/P), and 55% (epPCR-low) which correlates well with the average number of amino acid substitutions per protein (4.1, 1.6 and 1.1). In the epPCR libraries transitions were the predominant mutations (>72%), and >82% of all mutations occurred at A- or T-nts. Consecutive nucleotide (nt) mutations were obtained only with a low fraction (2.8%) under highly error-prone conditions. SeSaM-Tv P/P was enriched in transversions (43%; >1.7-fold more than epPCR libraries), and consecutive nt mutations (30.5%; 11-fold more than epPCR-high). A high fraction of wild-type BSLA protein (33%) was found in the epPCR-low mutant library compared to 2% in epPCR-high and 13% in SeSaM-Tv P/P. An average of 1.8-1.9 amino acid substitutions per residue was obtained with epPCR-low and -high compared to 2.1 via SeSaM-Tv P/P. The chemical composition of the amino acid substitutions differed, however, significantly from the two epPCR methods to SeSaM-Tv P/P.

Reemergence of Recombinant Vaccine–derived Polioviruses in Healthy Children, Madagascar

Reemergence of Recombinant Vaccine–derived Polioviruses in Healthy Children, Madagascar

Detection and significance of gene variation in hepatitis B virus S region

To explore the variations of gene S in hepatitis B viruses of hepatitis B patients and provide experimental evidences for the mutation analysis of viral gene. The virus DNA load in hepatitis B patient donors was detected by real-time polymerase chain reaction (PCR) and gene sequence analysis. And a comparison was made with standard strain by the software DNAstar. (1) Gene S was successfully amplified and sequenced in 15 hepatitis B patients. Three samples had I→T mutation at residue 126 in HBsAg "a" antigenic determinant. (2) Sixteen hepatitis B patients had 67 nucleotide mutations, including 14 residues in PreS1 and 6 residues in PreS2. Mutations nt 3036 T→C, nt 3039 T→G, nt 3066 C→T and L88V existed in PreS1 gene in all samples. HBV genome is susceptible to nucleotide mutations. Some residues have geographically restricted mutations in gene S region. And understanding the significance of these mutations may help clarify the pathogenesis of hepatitis B and provide new experimental evidence for its gene diagnosis and prevention.

Complete Genome Sequence of an Overattenuated Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

JXA1-P170 is an overattenuated highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) that has been passaged in vitro 170 times. Vaccination with JXA1-P170 cannot protect pigs against JXA1 challenge. Compared with the parental virus JXA1, JXA1-P170 contains 1 nucleotide (nt) deletion and 113 nt mutations leading to 59 amino acid substitutions. Here we announce the first complete genome sequence of the overattenuated HP-PRRSV.

Multiple genomic sequences of hepatitis delta virus are associated with cDNA promoter activity and RNA double rolling-circle replication

To understand how DNA-dependent RNA polymerase II (pol II) recognizes hepatitis delta virus (HDV) RNA as a template, it is first necessary to identify the HDV sequence that acts as a promoter of pol II-initiated RNA synthesis. Therefore, we isolated the pol II-response element from HDV cDNA and examined the regulation by hepatitis delta antigens (HDAgs). Two HDV cDNA fragments containing bidirectional promoter activity were identified. One was located at nt 1582-1683 (transcription-promoter region 1, TR-P1) and the other at nt 1223-1363 (transcription-internal region 5, TR-I5). The promoter activities of these two regions were enhanced by HDAgs to differing degrees. Next, the role of these sequences in an HDV cDNA-free RNA replication system was characterized by site-directed mutagenesis. Our data showed that: (i) the AUG codon at the HDAg ORF of HDV RNA (nt 1599-1601) that mutates to UAG (amber stop codon) results in loss of dimeric but not monomeric HDV RNA synthesis. (ii) A 5 nt mutation of TR-P1 (P1-m5, nt 1670-1674) abolishes RNA replication completely. Two-nucleotide-mutated RNA (P1-m2, nt 1662-1663) is able to synthesize short RNAs but not monomeric HDV RNA. (iii) A mutation in 5 nt at the TR-I5 region (I5-m5, nt 1351-1355) also abolishes HDV replication. Mutants with 2 nt mutations (I5-m2, nt 1351-1352) or 3 nt mutations (I5-m3, nt 1353-1355) inhibit HDV dimeric but not monomeric RNA synthesis. Furthermore, large HDAg is expressed in cells transfected with I5-m3 and I5-m2 RNAs and that demonstrate the RNA-editing event in the monomeric HDV RNA. These results provide further understanding of the double rolling-circle mechanism in HDV RNA replication.

Evidence that Cymbidium mosaic virus (CymMV) isolates divide into two subgroups based on nucleotide diversity of coat protein and replicase genes

The genetic diversity of Cymbidium mosaic virus (CymMV, family Flexiviridae) was assessed by analysing the nucleotide sequences of coat protein (CP) and partial RNA-dependent RNA polymerase (RdRp) genes. Thirty CymMV sequences from vanilla isolates, obtained in this work by direct sequencing of RT-PCR products, were compared to the sequences from ornamental orchid isolates available in GenBank. The CymMV population exhibited overall low genetic diversity (pi=0.054 and pi=0.053 for CP and RdRp genes, respectively). Phylogenetic analyses of the 85 CP and 37 RdRp sequences revealed the segregation of the isolates into two congruent monophyletic clusters; however these two subgroups did not cluster in amino sequence analysis because most of the nt mutations were synonymous. Nevertheless, the subgrouping was confirmed by highly significant Kst tests for the CP and RdRp genes. Analysis of population genetic parameters and distribution of synonymous and nonsynonymous mutations revealed that both genes were under negative selection with no recombination events. These results suggested that the CymMV isolates found in cultivated orchids worldwide have a dual origin and are expanding as if following bottlenecks.

The Presence of FYAnull Alele of Duffy Blood Group System in Blood Donors and Individuals from a Malarial Endemic Region of Brazil.

BACKGROUND: Malaria is a virulent disease caused by the Plasmodium parasite. Innate resistance to malaria infections in humans is conferred by various blood group polymorphisms. The Duffy blood group system consists of Fya and Fyb antigens which are encoded by codominant alleles FYA and FYB. Four phenotypes are defined: Fy(a+b+), Fy(a+b−), Fy(a−b+) and Fy(a−b−). Erythrocytes of Duffy-negative individuals are resistant to invasion by P. vivax. In Blacks the Fy(a−b−) phenotype is associated with a single point mutation (-33T-C) in the GATA-1 binding motif for the erythroid promoter of FYB.STUDY DESIGN AND METHODS: We investigated the phenotypes and the genotypes of Duffy blood group system of 250 individuals living in a malarial endemic region (MER) in the state of Amazon (Brazil), and of 199 blood donors (BD) from a non-endemic region. The phenotyping was done by agglutination gel tests (DiaMed-Latino América) using anti-Fya and anti-Fyb reagents. The molecular analysis for FYA, FYB, FYBES (GATA box mutation nt -33T-C), and FYBWeak (mutations 265 C-T, and 298 G-A) alleles, were performed by PCR-RFLP. The PCR products were digested by Ban I for FYA and FYB identifications; by Sty I for GATA box mutation; Acy I and Mwo I for 265 C-T and 298 G-A mutations, respectively. Some samples that showed discrepancy between the phenotype and genotype results were examined by sequence analysis using the ABI PrismâBig Dyeä Terminator Cycle Sequencing Ready Reaction Kit” (Perkin Elmer), and the interpretation by the software ABI PRISMä 377 DNA Sequencer”, 3.3 version (Perkin Elmer).RESULTS: We found that 34/250 (13.6%) of 250 persons living in the MER and 37/199 (18.6%) of BD had phenotype and genotype discrepant results [Fy(a+b−) FYA/FYB]. In addition, we found that 16/34 (47%) of people living in the MER, and 4/37 (10.8%) of BD did not present the -33T-C mutation, the 265 C-T, or the 298 G-A mutations. The sequence analysis of 2 samples from persons from MER indicated the presence of -33T-C mutation in the FYA allele in one individual (1 FYA/FYB and W/M; FYA/FYB and M/M). Additionally, we detected that 18/34 (53%) of people living in the MER, and 33/37 (89.2%) of BD presented the -33T-C mutation. The sequence analysis of 5 samples indicated the presence of -33T-C mutation in the FYA allele in 4 cases [2 persons from MER and 2 from BD (FYA/FYB e M/M)].CONCLUSION: Recently the mutation responsible for erythrocyte Duffy antigen-negativity [Fy(a−b−)] was demonstrated in FYA allele in a malarial endemic region of Papua New Guinea. The present data demonstrated the presence of the FYAnull allele not only in persons living in a malarial endemic region but also in Brazilian blood donors from non-endemic areas. In contrast with that which happens with the FYB allele, our results indicated that the presence of the -33T-C mutation in the FYA allele does not abolish the expression of the Fya antigen in the erythrocyte.

Genotypic and phenotypic heterogeneity of African Americans with primary iron overload

Primary iron overload may be relatively common in African Americans, but its cause is incompletely understood. Thus, we evaluated genotype and phenotype characteristics of unselected African American index patients with primary iron overload who reside in central Alabama. All had hepatic iron concentration ≥30 μmol/g dry wt or ≥2.0 g of iron mobilized by phlebotomy to achieve iron depletion. Genotype analyses were performed in African American control subjects from the same region. There were 23 patients (19 men, 4 women); mean age at diagnosis was 52 ± 12 years (1 SD) (range 32–69 years). Nine (39.1%) reported that they consumed ≥45 g of ethanol daily; five had chronic hepatitis C. Eight had some form of hemoglobinopathy or thalassemia. Mean serum transferrin saturation was 56 ± 28% (range 15–100%). The geometric mean serum ferritin at diagnosis was 1076 ng/mL [95% confidence interval 297–3473 ng/mL]. Increased stainable liver iron was observed in hepatocytes only in 4 patients, in macrophages only in 8 patients, and in hepatocytes and macrophages in 8 patients. The mean quantity of iron mobilized by phlebotomy (corrected for iron absorbed during treatment) was 5.3 ± 2.0 g (range 4.0–8.4 g). Iron removed by phlebotomy was greater in patients with hemoglobinopathy or thalassemia than in those without these forms of anemia (6.6 ± 1.3 g vs 3.9 ± 1.6 g, respectively; P = 0.0144). Daily consumption of ≥45 g of ethanol or chronic hepatitis C was not associated with an increased or decreased amount of phlebotomy-mobilized iron, on the average. The percentage of index patients positive for HFE C282Y was greater than that of controls ( P = 0.0058). The respective percentages of phenotype positivity for HFE H63D, D6S105(8), and HLA-A*03 were similar in patients and controls. HFE S65C, I105T, and G93R were not detected in index or control subjects. Two of 13 patients were heterozygous for the ferroportin allele nt 744 G→T (Q248H), although the phenotype frequency of this allele was similar in patients and 39 controls. Synonymous ferroportin alleles were also detected in some patients. The ceruloplasmin mutation nt 1099C→T (exon 6; Arg367Cys) was detected in 1 of 2 patients tested. Abnormal alleles of β-2 microglobulin, Nramp2, TFR2, hepcidin, or IRP2 alleles were not detected in either of the 2 patients so tested. We conclude that primary iron overload in African Americans is not the result of the mutation of a single gene. HFE C282Y, ferroportin 744 G→T, and common forms of heritable anemia appear to account for increased iron absorption or retention in some patients.

Systematic analysis of the ABO gene diversity within exons 6 and 7 by PCR screening reveals new ABO alleles.

Mutations critical for ABO blood group phenotypes have predominantly been found in exons 6 and 7 of the ABO gene, both of which encode the catalytic domain of ABO glycosyltransferase. To design rapid and reliable ABO genotyping assays, a profound knowledge of the prevalent alleles is required and a reliable sequence database needs to be established. A PCR screening system was established consisting of 102 different PCRs, each specific for a single nucleotide (nt) variation. The primer mixes were developed to walk from the 5' to the 3' end of exons 6 and 7 of the ABO gene to screen for nt mutations at 50 known polymorphic sites. A total of 109 unrelated individuals with common and rare ABO characteristics were screened. All blood samples in which the PCR results were inconclusive or inconsistent with the ABO phenotypes were subjected to sequence analysis of exons 6 and 7. The results of PCR screening were conclusive and consistent with the ABO phenotypes in 90 cases. In the remaining 19 cases, PCR screening revealed unusual allele combinations or amplification results that were incompatible with known ABO allele combinations or subgroups predicted by serologic analysis. In these 19 cases, sequencing revealed new ABO alleles (one ABO*Ael allele, one ABO*B(A) allele and two ABO*O alleles) in two individuals with common and seven individuals with variant ABO phenotypes. This PCR screening strategy is an effective tool for obtaining deeper insight into the ABO gene diversity and diversification and may be useful to increase the quality of the ABO sequence database.

Genetic and Clinical Description of Hemochromatosis Probands and Heterozygotes: Evidence That Multiple Genes Linked to the Major Histocompatibility Complex Are Responsible for Hemochromatosis

ABSTRACT: We evaluated Alabama hemochromatosis probands ( n= 74) and normal control subjects ( n= 142) for expression of the hemochromatosis-associated mutations nt 845G→A (845A; Cys282Tyr) and nt 187C→G (His63Asp) in a gene linked to the major histocompatibility complex (MHC). We also tabulated parameters of iron metabolism and iron overload in probands and in obligate heterozygote family members of homozygous Cys282Tyr probands. Among probands, 59.4% were Cys282Tyr homozygotes and 20.3% were heterozygotes; 20.3% did not express this mutation. In normal control subjects, 14.7% were heterozygous for the Cys282Tyr mutation; one normal control subject was homozygous for the Cys282Tyr mutation. None (0 of 44) of our Cys282Tyr-homozygous hemochromatosis probands had the His63Asp mutation. Of the Cys282Tyr-heterozygous and -negative probands, the His63Asp mutation occurred in 26.7% (4/15) and 53.3% (8/15), respectively. In normal control subjects, 23.2% were heterozygous for the His63Asp mutation; 2.8% were homozygous. Induction phlebotomy requirements and other manifestations of iron overload were significantly greater in Cys282Tyr homozygotes than among other probands. Cys282Tyr-heterozygous probands had significantly higher values of serum iron parameters than did obligate Cys282Tyr heterozygotes whose values were, on the average, normal. Co-expression of HLA-A3, HLA-B7, and D6S105(8) was significantly more frequent in all subgroups of probands stratified by Cys282Tyr expression than in normal control subjects. These results demonstrate that the severity of iron overload in hemochromatosis is affected significantly by genetic factors. Further, our findings support the hypothesis that one or more MHC-linked genes other than that corresponding to the Cys282Tyr and His63Asp mutations contributes to increased iron absorption and iron overload in hemochromatosis probands.

Prevalence of factor V Leiden mutation in various populations.

Resistance to activated protein C (APC) is the most common inherited risk factor for venous thrombosis. Most cases of APC resistance are caused by the point mutation nt 1691 G-A in factor V gene, referred to as factor V Leiden mutation. As initially shown in a Dutch population, this mutation has a carrier rate of 2.9%, the most frequent genetic disposition for thrombophilia and deep venous thrombosis. By large-scale epidemiological studies we have determined the prevalence of factor V Leiden mutation in populations from Poland (200), Argentina (215), Venezuela (126), Costa Rica (196), and India (150). The prevalences have been estimated for Poland (Warsaw) 5.0%, Argentina (Buenos Aires) 5.1%, Venezuela (Valencia) 1.6%, Costa Rica (San José) 2.0%, and India (Punjab) 1.3%. Based on worldwide distribution, it can be hypothesized that the factor V Leiden mutation has originated and accumulated in central European Caucasians and spread over the world by migration.

Molecular characterization of glucose-6-phosphate dehydrogenase deficiency in Brazil.

Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) variants was carried out in 150 unrelated G6PD deficient blood donors from the region of Campinas, Brazil. By allele specific oligomer hybridization or digestion of exon 4 of the G6PD gene with the restriction endonuclease NlaII, we detected the 202 G-->A mutation in 146 individuals. This mutation was associated with the 376 G-->A substitution and only one haplotype was observed in these individuals. Digestion of exon 6 with the restriction enzyme MboII showed the presence of the Mediterranean variant in three individuals. Haplotype analysis showed, in all three samples, a T at nt 1311 and the C at nt 13 in intron 11, suggesting a European origin of this variant. By SSCP analysis and direct sequencing we detected the mutation nt 1003 G-->A (335 Ala-->Thr) in one blood donor. This mutation was previously described in a boy of Indian ancestry and the variant was denominated G6PD Chatam. The case described here has no Indian ancestry; thus, we presume that the mutations have arisen independently, although we do not know the haplotype of the Indian patient. The haplotype of our case was the most common observed in our population (PvuII, BspHI+, PstI+, 1311C, NlaIII-). Thus, our data indicate that G6PD A- with the 202 G-->A mutation is the most frequent G6PD deficiency in the population of southeastern Brazil. The remaining variants had a Mediterranean origin. These results are in agreement with the origin of the Brazilian population.