Point Mutations In Genome Research Articles

Genomic Selective Constraints in Murid Noncoding DNA

Recent work has suggested that there are many more selectively constrained, functional noncoding than coding sites in mammalian genomes. However, little is known about how selective constraint varies amongst different classes of noncoding DNA. We estimated the magnitude of selective constraint on a large dataset of mouse-rat gene orthologs and their surrounding noncoding DNA. Our analysis indicates that there are more than three times as many selectively constrained, nonrepetitive sites within noncoding DNA as in coding DNA in murids. The majority of these constrained noncoding sites appear to be located within intergenic regions, at distances greater than 5 kilobases from known genes. Our study also shows that in murids, intron length and mean intronic selective constraint are negatively correlated with intron ordinal number. Our results therefore suggest that functional intronic sites tend to accumulate toward the 5′ end of murid genes. Our analysis also reveals that mean number of selectively constrained noncoding sites varies substantially with the function of the adjacent gene. We find that, among others, developmental and neuronal genes are associated with the greatest numbers of putatively functional noncoding sites compared with genes involved in electron transport and a variety of metabolic processes. Combining our estimates of the total number of constrained coding and noncoding bases we calculate that over twice as many deleterious mutations have occurred in intergenic regions as in known genic sequence and that the total genomic deleterious point mutation rate is 0.91 per diploid genome, per generation. This estimated rate is over twice as large as a previous estimate in murids.

A Novel 5′ ATRX Point Mutation with Splicing Consequences in Chronic Myelomonocytic Leukemia with Immune Dysregulation and Acquired Alpha Thalassemia.

BACKGROUND: Acquired hemoglobin disorders including α thalassemia (hemoglobin H (HbH) disease) can complicate neoplastic myeloid disorders, especially myelodysplastic syndrome (MDS). Acquired α thalassemia in MDS has recently been linked to mutations in the trans-acting ATRX at Xq13, a gene previously associated with inherited ATR-X (α thalassemia-mental retardation-X linked) syndrome. In the 3 instances where acquired ATRX point mutations predicted to affect splicing have been encountered, mRNA has been unavailable for assessment or unexpressed; in the 3 cases where ATRX splicing changes were described in MDS, the responsible genomic DNA mutation could not be located. Here we report a novel genomic point mutation in an ATRX splice donor motif and define the mutation's splicing and expression consequences.METHODS AND RESULTS: A 59 year old anemic Swiss man with chronic myelomonocytic leukemia (CMML) and multiple autoimmune complications including granulomatous uveitis, diabetes insipidus, and pleuropericarditis, developed anemia (Hb 10 g/dL) with MCH of 16.7 pg and MCV of 54 fL. We detected 50% HbH-containing cells by supravital staining and 7.5% HbH by weak cation chromatography, confirmed as HbH by isoelectric focusing. Peripheral blood granulocytes, mononuclear cells and bone marrow mononuclear cells were isolated using dual density Ficoll-Hypaque, and DNA and RNA extracted for mutation screening. ATRX coding region (35 exons) and splice site DNA from mononuclear cells was amplified and subjected to denaturing high performance liquid chromatography (DHPLC). A heteroduplex peak was encountered in the amplicon centered on exon 4. Sequencing demonstrated thymidine to cytidine point mutation with high proportional clonality in the canonical GT splice donor motif 3′ to ATRX exon 4 (IVS 4+2 T>C), also detectable in patient granulocytes. Electrophoresis of raw PCR product from the 5′ end of ATRX cDNA demonstrated multiple abnormal bands. Subcloning and sequencing of cDNA demonstrated 2 splicing consequences of this mutation: exon 4 skipping (i.e., exon 3 was spliced directly to exon 5), and a second isoform with intact exons 3, 4, and 5 but retaining 43 base pairs of intron 4 (including the T>C point mutation in the splice donor site), activating a previously unrecognized cryptic splice site. Since exon 4 is 53 base pairs in length, both aberrant splicing consequences result in a frameshift and generation of a premature stop codon in the 5′ end of exon 5. Exon 6 is normally alternatively spliced, and this variation was retained, so there were two subforms of each aberrant spliceoform. Expression of ATRX mRNA in the patient's monocytes, measured by RT-PCR probing the 3′ helicase domain, was 11.1% of healthy controls; in the granulocytes, expression was 15.3% of normal.CONCLUSION: Pre-mRNA splicing abnormalities such as that described here are increasingly recognized in association with human disease and particularly neoplasia, and may represent a potential therapeutic target. Point mutations in general are sought in myeloid disorders as they can provide proof of clonality in ambiguous cases and allow tracking of clonality with therapy and disease evolution. Germline 5′ ATRX splicing abnormalities are only rarely associated with inherited ATR-X syndrome, presumably because of the devastating consequences on gene expression and fetal development, but may be more common in acquired thalassemia. In future analyses of acquired HbH disease, when seeking ATRX mutations, splicing abnormalities should be routinely considered.

Genomic Selective Constraints in Murids

Recent work has suggested that there are many more selectively constrained, functional noncoding than coding sites in mammalian genomes. However, little is known about how selective constraint varies amongst different classes of noncoding DNA. We estimated the magnitude of selective constraint on a large dataset of mouse-rat gene orthologs and their surrounding noncoding DNA. Our analysis indicates that there are more than three times as many selectively constrained, nonrepetitive sites within noncoding DNA as in coding DNA in murids. The majority of these constrained noncoding sites appear to be located within intergenic regions, at distances greater than 5 kilobases from known genes. Our study also shows that in murids, intron length and mean intronic selective constraint are negatively correlated with intron ordinal number. Our results therefore suggest that functional intronic sites tend to accumulate toward the 59 end of murid genes. Our analysis also reveals that mean number of selectively constrained noncoding sites varies substantially with the function of the adjacent gene. We find that, among others, developmental and neuronal genes are associated with the greatest numbers of putatively functional noncoding sites compared with genes involved in electron transport and a variety of metabolic processes. Combining our estimates of the total number of constrained coding and noncoding bases we calculate that over twice as many deleterious mutations have occurred in intergenic regions as in known genic sequence and that the total genomic deleterious point mutation rate is 0.91 per diploid genome, per generation. This estimated rate is over twice as large as a previous estimate in murids.

Functional constraints and frequency of deleterious mutations in noncoding DNA of rodents.

Selection against deleterious mutations imposes a mutation load on populations because individuals die or fail to reproduce. In vertebrates, estimates of genomic rates of deleterious mutations in protein-coding genes imply the existence of a substantial mutation load, but many functionally important regions of the genome are thought to reside in noncoding DNA, and the contribution of noncoding DNA to the mutation load has been unresolved. Here, we infer the frequency of deleterious mutations in noncoding DNA of rodents by comparing rates of substitution at noncoding nucleotides with rates of substitution at the fastest evolving intronic sites of adjacent genes sampled from the whole genome sequences of mouse and rat. We show that the major elements of selectively constrained noncoding DNA are within 2,500 bp upstream and downstream of coding sequences and in first introns. Our estimate of the genomic deleterious point mutation rate for noncoding DNA (0.22 per diploid per generation) is similar to that for coding DNA. Mammalian populations therefore experience a substantial genetic load associated with selection against deleterious mutations in noncoding DNA. Deleterious mutations in noncoding DNA have predominantly quantitative effects and could be an important source of the burden of complex genetic disease variation in human populations.

Evolving beyond point mutations

Genomic structure and content are remarkably labile over evolutionary time. Yet we know little about the relative importance of genomic changes versus point mutations in adaptive evolution. Using microarray technology, a new paper [ 1 Riehle M.M. et al. Genetic architecture of thermal adaptation in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 525-530 Crossref PubMed Scopus (198) Google Scholar ] suggests that large-scale duplications and deletions might play a more common role in adaptation than was previously thought.

Molecular analysis of 5-azacytidine-induced variants in mammalian cells.

5-azacytidine induces 6-thioguanine resistance in AS52 cells. To characterize these resistant clones, we isolated 148 of them from 50 independently treated flasks. Less than nine (6%) of the 148 variants were spontaneous. PCR amplification of the DNA primers flanking the gpt gene produced no product in 15 clones (10%). Of the 133 remaining clones, 52 showed sequence alterations in the gpt structural gene. Of these 52, 34 (65%) were GC-->CG transversions. Only seven were located in CpG sequences. Thus, methyltransferase complexes are not major contributors to 5-azacytidine-induced point mutations in AS52 cells. The remaining 81 clones had no sequence alterations within the coding region of the gpt gene. Southern blot analysis of a sample of these variants (37/81) indicated that the 6-thioguanine-resistant phenotype was not due to local rearrangements or deletions (resolution 50 bp). Sequence analysis of the early promoter region of another sample of these variants (24/81) indicated that lesions in the promoter could not be responsible for the 6-thioguanine resistance observed. Thus, a majority of these variants were formed via a mechanism other than small genomic rearrangements, point mutations or deletions of the gpt structural gene or its promoter. Neither the mechanisms leading to these variants nor the biological and morphological consequences of these variants are known.

Integrins and E-Cadherin Expression – A Way for the Better Understanding of the Biological Behaviour of Colorectal Cancer

Colorectal carcinoma ranks second in frequency among all malignant tumors in the developed industrial countries. Determination of tumor cell differentiation together with pathological and clinical staging are currently the most important prognostic markers and thus decisive for the proper planning of therapy. The expression of adhesion molecules is subject to a number of genomic DNA alterations, e.g., point mutations and deletions. The resulting changes in the expression and function of adhesion molecules, (e.g., integrins and E-cadherin), lead to functional impairments in the cell-cell and cell-extracellular matrix interactions, which in turn can cause changes in cell differentiation and transformation. Our paper is intended as an overview and critical discussion of the significance of integrins and E-cadherin in tumor progression and the metastatic spread of colorectal carcinoma.

Accumulation of deletions and point mutations in mitochondrial genome in degenerative diseases.

Accumulation of various mutations in the mitochondrial genome is proposed as an important contributor to aging and degenerative diseases. Extensive fragmentation of mtDNA was detected in association with increased 8-hydroxydeoxyguanosine content in the heart mitochondrial DNA (mtDNA) from a patient with premature aging and mitochondrial cardiomyopathy, who carried a mutation within the mitochondrial tRNA(Asp) gene. This result suggests that damage to mtDNA by hydroxyl radical and accumulation of deleted mtDNA can be accelerated by a specific mitochondrial genotype. Similarly, extensive fragmentation of mtDNA was also detected in cultured cells exposed to a high oxygen concentration atmosphere, implying that mtDNA is vulnerable to reactive oxygen species. To clarify the role of point mutations accumulated in mtDNA, we examined the sequence heterogeneity of mtDNA in the skeletal muscle of a MELAS patient who carried a mutation within the mitochondrial tRNA(leu)(UUR) gene. The analysis revealed that the frequency of mutant clones in the MELAS muscle was significantly higher than those in an age-matched control muscle and a control placenta. Some of these nucleotide substitutions were missense and nonsense mutations, which potentially have deleterious effects on the mitochondrial function. The frequency of nucleotide substitutions in the striatum of three patients with Parkinson's disease was also significantly higher than that in control tissues. We also observed increased protein modification by 4-hydroxy-2-nonenal, a lipid peroxidation by-product, in Parkinson's disease. These results suggests that a vicious cycle contributes to the progression of degenerative process. In this cycle, first a primary mitochondrial mutation(s) induces a mitochondrial respiratory defect, which increases the leakage of reactive oxygen species (ROS) from the respiratory chain. Then the ROS would trigger accumulation of secondary mtDNA mutations in postmitotic cells, leading to further aggravation of mitochondrial respiratory defects and increased production of ROS and lipid peroxides from mitochondria, and thus resulting in degeneration of cellular components.

The Pathogenesis of Graves’ Disease

We have hypothesized over many years that Graves' disease (GD) and the other autoimmune thyroid diseases (AITD) are each due to antigen-specific defects in suppressor (regulatory) T lymphocyte function. There have been several reports dealing with the role of regulatory T lymphocyte subsets, ie., that will prevent autoimmune disease in these and other organ-specific autoimmune diseases. In AITD, suppressor T cells have been shown to be less well activated by relevant antigen, but are normally activated by irrelevant antigen; suppressor T cells from normal persons react equally well to both. In GD, these cells have been shown to be inadequately activated by TSH receptor antigen, but are normally activated by irrelevant autoantigen. This reduction is partial only, and insufficient itself to precipitate the autoimmune disease; further insults from the environment are necessary to further reduce generalized regulatory cell activity, adding to the genetically induced specific regulatory cell dysfunction, which appears in turn to be due to a specific defect in the presentation of a specific antigen. This, in turn, may relate to abnormalities of the genes responsible for antigen presentation. The end result is activation of appropriate helper and effector T cells, the stimulation by these of appropriate B lymphocytes, and the concurrent production of cytokines. These events lead to functional changes within the target cell which itself will express Class II antigens, heat shock proteins, and intercellular adhesion molecules, all of which amplify the immune response. Moreover, the activation of helper T lymphocytes by specific antigen depends on the availability of normal amounts of antigen being presented to them by antigen-presenting cells. Thus, there is no need to invoke any primary abnormality or infection of the thyroid cell, or any cross-reacting antigen of microorganismic origin to initiate this process. What is required is an abnormality of antigen-presentation such that regulatory cells are not properly activated, plus some additive environmental disturbance acting on the immune system. GD specifically results from the production by B lymphocytes of an antibody directed against the TSH receptor which stimulates the thyrocyte in a manner similar to TSH, but for a much longer interval. There are also antibodies to the thyrotrophin (TSH) receptor which block the action of TSH. Thyroid stimulating antibody is typical of GD and is detectable in about 95% of cases, but is also seen in destructive thyroiditis transiently. It tends to decline with antithyoid drug therapy, and rises further (for several months) after 131 I treatment. It may slowly decline after subtotal thyroidectomy. It also declines in the third trimester of pregnancy but sometimes is sufficiently high to cause foetal and neonatal passive transfer GD. It tends to rebound in the mother after delivery and may result in postpartum GD. The blocking antibody may cause atrophic thyroiditis and hypothyroidism. Antimicrosomal antibody has now been shown to be antithyroperoxidase. It correlates moderately well with thyroid dysfunction in Hashimoto's thyroiditis (HT) and GD, while antithyroglobulin is of much less value. Graves' ophthalmopathy is still not well understood, and its precise relationship to Graves' hyperthyroidism has yet to be worked out. However the retroorbital fibroblast is now emerging as the most likely target cell, with retroorbital muscle involvement possibly secondary. A recent observation of a genomic point mutation on the TSH receptor on fibroblasts from patients with Graves' ophthalmopathy but not normal persons raises interesting possibilities.

Point mutations of mitochondrial genome in Parkinson's disease.

Oxidative stress and subsequent energy crisis have been proposed as the cause of nigral neuronal cell death in Parkinson's disease. We have reported defects in the mitochondrial respiratory chain and increased amount of deleted mitochondrial genome in the nigrostriatal system of patients with Parkinson's disease. Deletion in mitochondrial DNA could be ascribed to somatically acquired premature aging leading to cell death. To elucidate the contribution of maternally transmitted point mutations in mitochondrial DNA to the premature DNA damages, we employed a direct sequencing system and analyzed the total nucleotide sequences of mitochondrial DNA in the brains of five patients with idiopathic Parkinson's disease. There were no predominant point mutations among the patients in contrast to some neuromuscular diseases. However, each patient had several point mutations that would result in a significant change in the gene products. Some of these mutations may be involved either in the increased production of oxygen radicals from the mitochondrial respiratory chain or in the increased susceptibility of the respiratory chain components to oxidative damage. We propose that some of these mutations can be regarded as one of the risk factors accelerating degeneration of nigrostriatal pathway in Parkinson's disease.

A genomic point mutation in the extracellular domain of the thyrotropin receptor in patients with Graves' ophthalmopathy

A genomic point mutation in the extracellular domain of the thyrotropin receptor in patients with Graves' ophthalmopathy

A genomic point mutation in the extracellular domain of the thyrotropin receptor in patients with Graves' ophthalmopathy.

Orbital and pretibial fibroblasts are targets of autoimmune attack in Graves' ophthalmopathy (GO) and pretibial dermopathy (PTD). The fibroblast autoantigen involved in these peripheral manifestations of Graves' disease and the reason for the association of GO and PTD with hyperthyroidism are unknown. RNA encoding the full-length extracellular domain of the TSH receptor has been demonstrated in orbital and dermal fibroblasts from patients with GO and normal subjects, suggesting a possible antigenic link between fibroblasts and thyrocytes. RNA was isolated from cultured orbital, pretibial, and abdominal fibroblasts obtained from patients with severe GO (n = 22) and normal subjects (n = 5). RNA was reverse transcribed, and the resulting cDNA was amplified by the polymerase chain reaction, using primers spanning overlapping regions of the entire extracellular domain of the TSH receptor. Nucleotide sequence analysis showed an A for C substitution in the first position of codon 52 in 2 of the patients, both of whom had GO, PTD, and acropachy. Genomic DNA isolated from the 2 affected patients, and not from an additional 12 normal subjects, revealed the codon 52 mutation by direct sequencing and AciI restriction enzyme digestions. In conclusion, we have demonstrated the presence of a genomic point mutation, leading to a threonine for proline amino acid shift in the predicted peptide, in the extracellular domain of the TSH receptor in two patients with severe GO, PTD, acropachy, and high thyroid-stimulating immunoglobulin levels. RNA encoding this mutant product was demonstrated in the fibroblasts of these patients. We suggest that the TSH receptor may be an important fibroblast autoantigen in GO and PTD, and that this mutant form of the receptor may have unique immunogenic properties.

Molecular mechanisms of the formation of DNA double-strand breaks and induction of genomic rearrangements

The probability that damage occurs in closely opposed sites on complementary DNA strands increases when DNA is heavily modified with mutagenic agents. Enzymatic excision of the opposite lesions produces DNA double-strand breaks which give rise to genomic rearrangements (deletions, insertions, etc.). Plasmid systems were developed for studying chemical lesions leading to double-strand breaks and the fate of broken plasmid molecules within bacterial cells. Deletions result from the base-pairing of fortuitously located direct repeats flanking the DNA broken ends; as a consequence, the latter are joined, while the DNA fragment between the direct repeats is deleted. Genomic rearrangements arise during the repair of the DNA double-strand breaks, and both events are due to similar repair enzymes which maintain the integrity of the DNA primary structure when conditions are not stressful. A number of genomic rearrangements and point mutations seem to be predetermined by the DNA primary structure.

Mitochondrial mutagenesis in yeast: mutagenic specificity of EMS and the effects of RAD9 and REV3 gene products

EMS is capable of inducing point mutations in mitochondrial genomes of yeast. It induces efficiently the mitochondrial suppressor mutation of the mitochondrial ochre mutation oxi 1-V25. The base changes leading to the suppression effect have not been identified. AT → GC base substitutions in mitochondrial genomes are inefficiently induced by EMS. The RAD9 and REV3 gene products participate in EMS mutagenesis in nuclear, as well as mitochondrial genomes of yeast.

Genomic alterations associated with persistent infections by equine infectious anaemia virus, a retrovirus.

The unique periodic nature of equine infectious anaemia (EIA) is believed to result from the ability of the infecting virus. EIAV, to undergo relatively rapid antigenic variations which circumvent host immune responses resulting in distinct virus populations in sequential clinical episodes in the persistently infected horse. This model was examined by oligonucleotide mapping comparisons of the RNA genomes of selected isolates of EIAV. Variations in oligonucleotide maps could be reproducibly demonstrated (i) after adaptation of the laboratory strain of EIAV to replication in a pony, (ii) after serial passage of virus between two ponies, and (iii) after a prolonged persistent infection in a single pony. In the latter case, the two isolates examined were recovered from different clinical episodes and were shown to be antigenic variants. In contrast, no variations in RNA structure could be detected in oligonucleotide maps of virus isolated after prolonged passage in tissue culture. Thus, these results support our concept that EIAV is a highly mutable virus, which may given rise to antigenic variants in the presence of immune pressures. The degree of variation observed between oligonucleotide maps is similar to that observed previously between variants of visna virus. These similarities between EIAV an visna suggest that genomic point mutations producing antigenic variants may be a more important mechanism of retrovirus persistence than was previously recognized.