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PubMed2000

Molecular epidemiology of HIV-1 in Switzerland: evidence for a silent mutation in the C2V3 region distinguishing intravenous drug users from homosexual men. Swiss HIV Cohort Study.

OBJECTIVES: To study the molecular epidemiology of HIV-1 strains found in Switzerland and to determine possible genetic linkages among strains sorted by risk group or geographic region. DESIGN: A cross-sectional, clinic-based survey of HIV-1 molecular sequences and linked patient history from Swiss people. METHODS: Specimens were collected from 215 HIV-1-infected people in HIV outpatient clinics of four tertiary referral centers (Lausanne, St. Gallen, Zurich, and Basel) between May and August 1996, mainly from homosexual men, injecting drug users (IDU), and heterosexually infected people. In addition, specimens collected between 1991 and 1995 in the HIV outpatient clinic at University of Geneva were included into this survey. These specimens were collected primarily for an ongoing, prospective cohort (Swiss HIV Cohort Study). Direct C2V3C3 sequences of the env gene were determined from 158 samples of peripheral blood mononuclear cells. Genetic data were analyzed with the available patient history on each specimen. RESULTS: As found in other previous studies in Europe, primarily subtype B viruses were identified, whereas seven (4%) of 158 were non-subtype B: one subtype D, four subtype A, and two subtype E. Five of seven non-B subtypes occurred in immigrants from African or Asian countries and all seven were found exclusively in individuals who had been infected by heterosexual contact. No significant clustering of strains within different study sites or risk groups was found. A silent mutation (LAI env 834) occurred significantly more often in IDU than in homosexual men (p<.001). CONCLUSIONS: Although the lack of significant clustering of strains by risk group or geographic region may result from early introduction of subtype B viruses in Switzerland, the strong association of a silent mutation with IDU suggests that, early in the epidemic, there was a unique founder virus among IDUs. The HIV epidemic in Switzerland is still predominantly caused by subtype B viruses.

Amino Acid Sequence↗
PubMed2000

Progressive supranuclear palsy pathology caused by a novel silent mutation in exon 10 of the tau gene: expansion of the disease phenotype caused by tau gene mutations.

Genetic mutations in the tau gene on chromosome 17 are known to cause frontotemporal dementias. We have identified a novel silent mutation (S305S) in the tau gene in a subject without significant atrophy or cellular degeneration of the frontal and temporal cortices. Rather the cellular pathology was characteristic of progressive supranuclear palsy, with neurofibrillary tangles concentrating within the subcortical regions of the basal ganglia. Two affected family members presented with symptoms of dementia and later developed neurological deficits including abnormality of vertical gaze and extrapyramidal signs. The third presented with dystonia of the left arm and dysarthria, and later developed a supranuclear gaze palsy and falls. The mutation is located in exon 10 of the tau gene and forms part of a stem-loop structure at the 5' splice donor site. Although the mutation does not give rise to an amino acid change in the tau protein, functional exon-trapping experiments show that it results in a significant 4.8-fold increase in the splicing of exon 10, resulting in the presence of tau containing four microtubule-binding repeats. This study provides direct molecular evidence for a functional mutation that causes progressive supranuclear palsy pathology and demonstrates that mutations in the tau gene are pleiotropic.

Aged↗
PubMed2001

A silent mutation induces exon skipping in the phenylalanine hydroxylase gene in phenylketonuria.

An A-->T substitution in cDNA nucleotide 1197 (c.1197A/T) of the human phenylalanine hydroxylase (PAH) gene has been regarded as a silent mutation, because both the wild-type (GUA) and the mutant (GUU) alleles encode a valine residue at codon 399 (V399 V). The nucleotide c.1197 is located at the 3'-end of exon 11at position -3 of the exon-intron junction. To explore whether the substitution exerts any effects on the processing of the PAH mRNA, illegitimate PAH transcripts from lymphoblast cultures of a phenylketonuria (PKU) patient heterozygous for c.1197A/T were analyzed by the polymerase chain reaction following reverse-transcription (RT-PCR). mRNAs with an exon 11 deletion were revealed. Furthermore, by using an R408 W mutation in the paternal allele as a marker, sequence analysis of the RT-PCR products indicates that virtually all PAH transcripts from the maternal allele with the c. 1197A/T substitution do not contain exon 11. To address whether this substitution is the main determinant for exon skipping, PAH minigenes with or without the substitution were constructed and transfected to a human hepatoma cell line. Analysis of the transcription products by S1 nuclease mapping clearly indicated that such exon 11 skipping was directly associated with the c.1197A/T substitution. Thus, this study demonstrates that the c.1197A/T substitution in the PAH gene is not just a neutral polymorphism but a mutation that induces post-transcriptional skipping of exon 11 leading to a PKU phenotype.

Base Sequence↗
PubMed1998

alpha-Thalassemia Subtyping and the Detection of Silent Mutations by High-Resolution Fragment Analysis and DNA Sequencing.

Background: Sets of polymerase chain reaction (PCR) primers were used that make it possible to study subtypes of the most prevalent mutations that cause alpha-thalassemia (-alpha3.7 deletions). These primers (Dev and C3, Dev and C9c) can be used in PCR to amplify regions that are characteristic of the three - alpha3.7 deletion subtypes (alpha3.7i, alpha3.7ii, alpha3.7iii). These subtypes are important because the type of alpha-chain deletion can affect the level of alpha-globin production. Methods and Results: The PCR products were screened using automated high-resolution electrophoresis on a DNA sequencer. Subtypes were then identified by automated DNA sequencing. During the screening for subtypes with high-resolution fragment analysis, new fragments were detected that were slightly different from the expected size. DNA sequencing of these fragments demonstrated the presence of three mutations that had not been described or fully characterized before. Conclusions: High-resolution fragment analysis is a convenient way to detect alpha-thalassemia subtypes, and DNA sequencing of silent mutations can lead to a better understanding of the cause of these mutations.

Journal Article↗
PubMed1993

A silent mutation in HLA-DPB1*0101 and its evolutionary implications.

A novel HLA-DPB1 allele (DPB1*/A6) has been identified in West Africa, and the occurrence of the recently reported allele DPB1*4001 (DPB1*BRI-5) in Negroid populations has been confirmed DPB1*WA6 differs from DPB1*0101 by a silent nucleotide exchange at codon position 43 only. It constitutes, thereby, a link to DPB1*1101, *1501, and *2601 and suggests and evolutionary relationship with these alleles. Carrying the same silent mutation, DPB1*1101, *1501, and *2601 may, by recombinations, have evolved from DPB1*WA6. An apparently older evolutionary branch arose directly from DPB1*0101 and comprises the closely related alleles DPB1*2701 and DPB1*4001 reported only from individuals of African descent.

Alleles↗
PubMed2002

Association of HSP70 with endonucleases allows the expression of otherwise silent mutations.

A subpopulation of the 70 kDa heat shock protein (HSP70) found within the mitochondria of Saccharomyces cerevisiae functions as a stable binding partner of the endonuclease SceI. We have previously found that the SceI endonuclease monomer recognizes and cleaves a unique, 26 bp sequence in vitro. Dimerization with HSP70 changes the specificity of SceI, allowing it to cleave at multiple sequences. This study shows that SuvI, an ortholog of SceI isolated from a different yeast strain, contains two amino acid substitutions, yet it shows the same uni-site specificity in its monomeric form. Binding of HSP70 to the SuvI monomer confers multi-site specificity that is different from that exhibited by the HSP70/SceI heterodimer. Mutation of single residues of SceI to the corresponding residue in SuvI provides enzymes with specificities intermediate between SceI and SuvI when complexed with HSP70. These results suggest that HSP70 interaction with certain endonucleases allows the expression of otherwise silent mutations in them, causing a change in enzyme cleavage specificity.

Amino Acid Sequence↗
PubMed1994

Aberrant splicing of exon 6 in the pyruvate dehydrogenase-E1 alpha mRNA linked to a silent mutation in a large family with Leigh's encephalomyelopathy.

Pyruvate dehydrogenase (PDH)-E1 alpha deficiency has recently been studied at the molecular-genetic level. The gene is situated on the X chromosome. We report on an unusual mutation in a familial E1 alpha deficiency. In fibroblasts, PDH deficiency was diagnosed in a young infant presenting with Leigh's encephalomyelopathy and in a maternal nephew with episodes of "malaises." In the two affected children as well as their mothers we found a silent mutation in exon 6 of the PDH-E1 alpha and an aberrant splicing of exon 6 in some of the cDNA clones. This case emphasizes the need for both genomic and cDNA analysis in cases where a PDH-E1 alpha deficiency is strongly suspected.

Base Sequence↗
PubMed2006

Silent mutations at the 5'-end of the cDNA of actinoporins from the sea anemone Stichodactyla helianthus allow their heterologous overproduction in Escherichia coli.

Wild-type actinoporins StnI and StnII from the sea anemone Stichodactyla helianthus, as well as their NH(2)-terminal six-His tagged versions, have been overproduced in Escherichia coli. Overproduction of both wild-type proteins was only possible after introducing silent mutations within the 5'-end of their original cDNA sequences. These mutations would prevent the formation of RNA secondary structures blocking the ribosome-binding site and the initiation codon. The four recombinant proteins were purified to homogeneity in milligrams amount and characterized from spectroscopic and functional points of view. All the isolated proteins behaved as the corresponding natural ones although the six-His tagged variants exhibited a decreased lytic activity. The strategy described will be useful to allow the production of mutant variants of these proteins and probably of other actinoporins.

Animals↗
PubMed2002

Silent mutations affect in vivo protein folding in Escherichia coli.

As an approach to investigate the molecular mechanism of in vivo protein folding and the role of translation kinetics on specific folding pathways, we made codon substitutions in the EgFABP1 (Echinococcus granulosus fatty acid binding protein1) gene that replaced five minor codons with their synonymous major ones. The altered region corresponds to a turn between two short alpha helices. One of the silent mutations of EgFABP1 markedly decreased the solubility of the protein when expressed in Escherichia coli. Expression of this protein also caused strong activation of a reporter gene designed to detect misfolded proteins, suggesting that the turn region seems to have special translation kinetic requirements that ensure proper folding of the protein. Our results highlight the importance of codon usage in the in vivo protein folding.

Amino Acid Substitution↗
PubMed1999

Use of silent mutations in cDNA encoding human glutathione transferase M2-2 for optimized expression in Escherichia coli.

Heterologous expression of human glutathione transferase M2-2 (GST M2-2) using Escherichia coli was improved 140-fold by mutating the cDNA expressing the enzyme. Expression of GST M2-2 from this cDNA clone, pKHXhGM2, generated approximately 190 mg protein per liter of bacterial culture, corresponding to approximately 12% of the total amount of soluble protein. The high-level-expressing cDNA was generated by oligonucleotide-directed mutagenesis introducing alternative silent mutations into the third nucleotide of codons 2, 4-7, and 10-14 in the 5' end of the cDNA coding region. The choice of alternative codons was restricted to those naturally occurring in highly biased genes in E. coli. Furthermore, the wild-type TAG stop codon at the 3' end was replaced with the two stop codons TAA and TGA in tandem to increase translation termination efficiency. The resulting partially randomized cDNA library was assayed for high-level expression using immunoscreening. Sequence similarities between the constructed high-level-expressing GST M2-2 cDNA and a similarly designed cDNA encoding the closely related human GST M1-1 suggest that the codons in the region immediately following the start codon are influential in achieving high-level expression. Pyrimidines seem to be more favorable than purines in the third position of codons in optimizing the expression of these enzymes in E. coli.

Amino Acid Sequence↗
PubMed1996

Optimized heterologous expression of the polymorphic human glutathione transferase M1-1 based on silent mutations in the corresponding cDNA.

An expression clone for large-scale production of the polymorphic human glutathione transferase (GST) M1-1 has been developed. Heterologous expression in Escherichia coli afforded a yield of 100 mg of GST M1-1 per 3 liters of culture medium, corresponding to an approximately 10-fold increased yield compared to the parental expression construct. Overproduction of the enzyme was dependent on the codon usage in the 5' region of the DNA sequence encoding glutathione transferase M1-1. High-level expression clones were generated by a combination of random silent mutations in selected wobble positions in the coding sequence and immunoselection of clones from the library of random mutants. The strategy used is generally applicable for the production of recombinant proteins provided that a suitable selection procedure is available for identifying the desired mutants.

Amino Acid Sequence↗
PubMed1988

An extensive search for RFLP in the human glucose-6-phosphate dehydrogenase locus has revealed a silent mutation in the coding sequence.

The genetic polymorphism of an approximately 100-kb DNA region comprising and flanking the glucose-6-phosphate dehydrogenase (G6PD) gene on human chromosome Xq28 has been analyzed in detail. By using 14 unique sequence probes and 18 restriction enzymes, we have characterized 257 restriction fragments or 370 restriction sites. On testing 12-57 individual X chromosomes, all sites but one were nonpolymorphic. However, a PstI site that maps to exon 10 of the G6PD gene, which is still monomorphic in all British and Italian subjects tested, is polymorphic in west-African people. Specifically, it is absent from 22% of Nigerian X chromosomes. By sequence analysis we have shown that the absence of this PstI site results from a G----A replacement at position 1116, corresponding to the third base of a glutamine codon; no amino acid change is produced in the protein. Thus, a polymorphic silent mutation is demonstrated in a human gene.

DNA Restriction Enzymes↗
PubMed1993

Region-specific rates of molecular evolution: a fourfold reduction in the rate of accumulation of "silent" mutations in transcribed versus nontranscribed regions of homologous DNA fragments derived from two closely related mouse species.

We have sequenced homologous DNA fragments of 2.7 and 2.8 kbp derived from the closely related mouse species Mus musculus domesticus (M. domesticus) and Mus musculus musculus (M. musculus), respectively. These two species diverged approximately 1 million years ago. Each DNA fragment contains 1.35 kbp of the 3' end of the constitutively expressed 2.2-kbp aprt (adenine phosphoribosyltransferase) gene and a similarly sized nontranscribed region downstream of the aprt gene. The aprt gene region contains protein coding sequences (0.35 kbp), intronic sequences (0.75 kbp), and a 3' nontranslated sequence (0.25 kbp). Both the M. domesticus and M. musculus downstream regions share three partial copies of the B1 repetitive element with the M. musculus downstream region containing an additional complete copy of this element. A comparison of the 2.7- and 2.8-kbp DNA fragments revealed a total of 63 molecular alterations (i.e., mutations) that were approximately fourfold more abundant in the nontranscribed downstream region than in the transcribed aprt gene. Of the 11 mutations observed in the transcribed region, 7 were found in introns, 3 in the 3' untranslated sequence, and 1 was a synonymous change in an exon. A comparison of the human and M. domesticus aprt genes has previously revealed no homology in either the intronic or 3' nontranslated regions with the exception of a 26-bp sequence in intron 3 and sequences at the exon/intron boundaries necessary for correct mRNA splicing (Broderick et al., Proc. Natl. Acad. Sci. USA, 84:3349, 1987). Therefore, there does not appear to be selective pressure for sequences within these regions. We conclude that there is a lower rate of accumulation of "silent" mutations in the transcribed mouse aprt gene than in a contiguous nontranscribed downstream region. A possible molecular mechanism involving preferential DNA repair for the transcribed region is discussed.

Adenine Phosphoribosyltransferase↗
PubMed1990

The BALB/c secondary response to the Sb site of influenza virus hemagglutinin. Nonrandom silent mutation and unequal numbers of VH and Vk mutations.

We have determined the nucleotide sequences of the expressed VH and Vk genes from 13 secondary (2 degrees) hemagglutinin (HA) (Sb) specific hybridomas derived from a single mouse. These antibodies share an Id, H37-68 (68Id) that dominates the 2 degrees HA(Sb) response in this mouse, but is rare or absent from 2 degrees responses of other mice. We find that these antibodies derive from five clones. The H chains of these antibodies are encoded by a single VH gene joined to a variety of DH and JH genes. The length of complementarity-determining region (CDR) 3 and sequence of the D-J junction are restricted, suggesting selection on CDR3 of the H chain. The L chains are more diverse. In the presented examples, they are encoded by the Vk21C and Vk21E genes and a Vk9 gene, and are joined to Jk1, 2, or 4. Each antibody is extensively mutated. The nature and distribution of the mutations suggests that 68Id-producing cells have been selected by Ag, although there are differences regarding the domain (VH, Vk, or both) in which mutations were selected. The implications of these findings on the idiosyncratic nature of the 68Id antibody response to HA(Sb) are discussed. There are two unusual characteristics regarding somatic mutation in these hybridomas. Whereas the expressed VH and Vk21 genes appear to have accumulated mutations at a high rate (1 to 1.5 x 10(-3)/base pairs/division, the expressed Vk9 genes appear to have accumulated mutations at a 5 to 15-fold lower rate than the expressed VH genes in the same cells. There is also a surprisingly high number of parallel silent somatic mutations in the VH genes, of which all but one are clustered to a 28-bp region in framework region 2 and CDR 2-encoding segments. The probability that this could have occurred by a random mutational process is essentially zero.

Amino Acid Sequence↗
PubMed2004

Familial adenomatous polyposis: aberrant splicing due to missense or silent mutations in the APC gene.

Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, the relevance of rare exonic single-base substitutions at nucleotide positions close to splice sites that are predicted to result in missense or silent (SNP) variants or substitutions in introns at splice-site positions that are not highly conserved has not been systematically examined in FAP patients. In 34 index patients, we identified 26 different heterozygous single-base substitutions at or close to the splice sites. We characterized five exonic mutations in exon 4 (c.423G>T), exon 14 (c.1956C>T, c.1957A>G, and c.1957A>C), and exon 15 (c.1959G>A) by transcript analysis and by splice-prediction programs (BDGP, SpliceSiteFinder, and ESEfinder). The splicing patterns of these variants were compared to those of 16 different substitutions at highly or less-conserved intronic splice-site positions, and to normal controls. In addition, we analyzed cosegregation of the variants with affected family members and examined the genotype-phenotype correlation. We could demonstrate that the four unclear variants in exon 4 and 14 that are predicted to result in missense or silent mutations in fact lead to complete exon skipping due to aberrant splicing; one possible explanation for this observed effect might be the disruption of exonic splicing enhancer (ESE) motifs. In contrast, the substitution at the first position of exon 15 seems to actually be a silent variant. We present the first systematic evaluation of different single-base substitutions in APC at or close to splice sites at transcript level. We show that the consequence of exonic mutations cannot be evaluated only by the predicted change in amino acid sequence but rather by the change at DNA level. The functional analysis of variants with unknown pathogenic effect plays an important role in increasing the mutation detection rate and achieving validation of predictive testing.

Adenomatous Polyposis Coli↗
PubMed2005

Problems in using statistical analysis of replacement and silent mutations in antibody genes for determining antigen-driven affinity selection.

The analysis of molecular signatures of antigen-driven affinity selection of B cells is of immense use in studies on normal and abnormal B cell development. Most of the published literature compares the expected and observed frequencies of replacement (R) and silent (S) mutations in the complementarity-determining regions (CDRs) and the framework regions (FRs) of antibody genes to identify the signature of antigenic selection. The basic assumption of this statistical method is that antigenic selection creates a bias for R mutations in the CDRs and for S mutations in the FRs. However, it has been argued that the differences in intrinsic mutability among different regions of an antibody gene can generate a statistically significant bias even in the absence of any antigenic selection. We have modified the existing statistical method to include the effects of intrinsic mutability of different regions of an antibody gene. We used this method to analyse sequences of several B cell-derived monoclonals against T-dependent antigens, T-independent antigens, clones derived from lymphoma and amyloidogenic clones. Our sequence analysis indicates that even after correcting for the intrinsic mutability of antibody genes, statistical parameters fail to reflect the role of antigen-driven affinity selection in maturation of many clones. We suggest that, contrary to the basic assumption of such statistical methods, selection can act both for and against R mutations in the CDR as well as in the FR regions. In addition we have identified different methodological difficulties in the current uses of such statistical analysis of antibody genes.

Animals↗
PubMed2003

Splicing mosaic of the myophosphorylase gene due to a silent mutation in McArdle disease.

The authors report the molecular findings in a patient with McArdle disease who harbored a silent polymorphism (K608K) in the myophosphorylase gene. cDNA studies demonstrated that this polymorphism leads to a severe mosaic alteration in mRNA splicing, including exon skipping, activation of cryptic splice-sites, and exon-intron reorganizations. These findings suggest that, in patients with McArdle disease in whom no pathogenic mutation has been found, any a priori silent polymorphism should be re-evaluated as a putative splicing mutation.

Alternative Splicing↗