The genetics of the repair of 5-azacytidine-mediated DNA damage in the fission yeastSchizosaccharomyces pombe

We have recently demonstrated thatSchizosaccharomyces pombe cells treated with the nucleoside analogue 5-azacytidine (5-azaC) require previously characterised G2 checkpoint mechanisms for survival. Here we present a survey of known DNA repair mutations which defines those genes required for survival in the presence of 5-azaC. Using a combination of single-mutant and epistasis analyses we find that the excision, mismatch and recombinational repair pathways are all required in some degree for the repair of 5-azaC-mediated DNA damage. There are distinct differences in the epistatic interactions of several of the repair mutations with respect to 5-azaC-mediated DNA damage relative to UV-mediated DNA damage.     

Ten novel mutations in the molybdenum cofactor genes MOCS1 and MOCS2 and in vitro characterization of a MOCS2 mutation that abolishes the binding ability of molybdopterin synthase

Molybdenum cofactor deficiency (MIM#252150) is a severe autosomal-recessive disorder with a devastating outcome. The cofactor is the product of a complex biosynthetic pathway involving four different genes (MOCS1, MOCS2, MOCS3 and GEPH). This disorder is caused almost exclusively by mutations in the MOCS1 or MOCS2 genes. Mutations affecting this biosynthetic pathway result in a lethal phenotype manifested by progressive neurological damage via the inactivation of the molybdenum cofactor-dependent enzyme, sulphite oxidase. Here we describe a total of ten novel disease-causing mutations in the MOCS1 and MOCS2 genes. Nine out of these ten mutations were classified as pathogenic in nature, since they create a stop codon, affect constitutive splice site positions, or change strictly conserved motifs. The tenth mutation abolishes the stop codon of the MOCS2B gene, thus elongating the corresponding protein. The mutation was expressed in vitro and was found to abolish the binding affinities of the large subunit of molybdopterin synthase (MOCS2B) for both precursor Z and the small subunit of molybdopterin synthase (MOCS2A).     

Helicase-inactivating mutations as a basis for dominant negative phenotypes

There is ample evidence from studies of both unicellular and multicellular organisms that helicase-inactivating mutations lead to cellular dysfunction and disease phenotypes. In this review, we will discuss the mechanisms underlying the basis for abnormal phenotypes linked to mutations in genes encoding DNA helicases. Recent evidence demonstrates that a clinically relevant patient missense mutation in Fanconi Anemia Complementation Group J exerts detrimental effects on the biochemical activities of the FANCJhelicase, and these molecular defects are responsible for aberrant genomic stability and a poor DNA damage response. The ability of FANCJ to use the energy from ATP hydrolysis to produce the force required to unwind duplex or G-quadruplex DNA structures or destabilize protein bound to DNA is required for its DNA repair functions in vivo. Strikingly, helicase-inactivating mutations can exert a spectrum of dominant negative phenotypes, indicating that expression of the mutant helicase protein potentially interferes with normal DNA metabolism and has an effect on basic cellular processes such as DNA replication, the DNA damage response, and protein trafficking. This review emphasizes that future studies of clinically relevant mutations in helicase genes will be important to understand the molecular pathologies of the associated diseases and their impact on heterozygote carriers.     

<Emphasis Type="Italic">RAD18</Emphasis> gene product of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> controls mutagenesis induced by hydrogen peroxide

Within eukaryotes, tolerance to DNA damage is determined primarily by the repair pathway controlled by the members of the RAD6 epistasis group. Genetic studies on a yeast Saccharomyces cerevisiae model showed that the initial stage of postreplication repair (PRR), i.e., initiation of replication through DNA damage, is controlled by Rad6–Rad18 ubiquitin-conjugating enzyme complex. Mutants of these genes are highly sensitive to various genotoxic agents and reduce the level of induced mutagenesis. In this case, the efficiency of mutagenesis suppression depends on the type of damage. In this study we showed that DNA damage induced by hydrogen peroxide at the same mutagen doses causes significantly more mutations and lethal events in the rad18 mutant cells compared to control wild-type cells.     

Analysis of human disease genes in the context of gene essentiality

The characteristics of human disease genes were investigated through a comparative analysis with mouse mutant phenotype data. Mouse orthologs with mutations that resulted in discernible phenotypes were separated from mutations with no phenotypic defect, listing ‘phenotype’ and ‘no phenotype’ genes. First, we showed that phenotype genes are more likely to be disease genes compared to no phenotype genes. Phenotype genes were further divided into ‘embryonic lethal’, ‘postnatal lethal’, and ‘non-lethal phenotype’ groups. Interestingly, embryonic lethal genes, the most essential genes in mouse, were less likely to be disease genes than postnatal lethal genes. These findings indicate that some extremely essential genes are less likely to be disease genes, although human disease genes tend to display characteristics of essential genes. We also showed that, in lethal groups, non-disease genes tend to evolve slower than disease genes indicating a strong purifying selection on non-disease genes in this group. In addition, phenotype and no phenotype groups showed differing types of disease mutations. Disease genes in the no phenotype group displayed a higher frequency of regulatory mutations while those in the phenotype group had more frequent coding mutations, indicating that the types of disease mutations vary depending on gene essentiality. Furthermore, missense disease mutations in no phenotype genes were found to be more radical amino acid substitutions than those in phenotype genes.     

Two new genes involved in signalling ambient pH in Aspergillus nidulans

Two new genes, palH and palI, where mutations mimic the effects of acidic growth pH have been identified in Aspergillus nidulans. A palH mutation is phenotypically indistinguishable from mutations in the palA, palB, palC, and palF genes, whereas palI mutations differ only in that they allow some growth at pH 8. Mutations in palA, B, C, F, and H are epistatic to a palI mutation and the significance of this epistasis is discussed. Additionally, palE and palB mutations have been shown to be allelic. Thus, a total of six genes where mutations mimic acidic growth conditions has been identified.     

Characterization of the first dominant dwarf maize mutant carrying a single amino acid insertion in the VHYNP domain of the <Emphasis Type="Italic">dwarf8</Emphasis> gene

The “green revolution” involving mainly wheat and rice was based on the use by breeders of semidominant mutations involved in the signal transduction pathway of Gibberellin (GA). In particular, mutations in the Reduced height (Rht) gene of wheat have been used to reduce plant height and consequently to avoid storm damage and lodging. These genes have been cloned and they encode for DELLA proteins which contain an N-terminal DELLA and a VHYNP domain essential for GA-dependent degradation of these proteins. In maize several mutations have been isolated which affect gibberellin biosynthesis and perception and in particular, mutations in Dwarf8 (D8) gene cause a severe dwarfing phenotype. D8 gene has been identified as an orthologue of Rht (Reduced height), Slr1(Slender rice 1) and Gibberellic Acid Insensitive (GAI) genes, this latter is a negative regulator of GA response in Arabidopsis. In this work, for the first time, we isolated and characterized a single amino acid insertion in the VHYNP domain of D8 maize gene causing the appearance of a dominant dwarf mutation. This spontaneous mutation, named D8-1023, showed a phenotype which is less severe in comparison with the other D8 mutants previously isolated which have modifications in the DELLA domain. This mutant appears to be an useful tool either to study the mechanism of GA-modulated growth in plants or to lower the height of maize tropical germplasm for breeding purposes.     

Hereditary breast cancer; Genetic penetrance and current status with BRCA

The most important cause of developing hereditary breast cancer is germline mutations occurring in breast cancer (BCs) susceptibility genes, for example, BRCA1, BRCA2, TP53, CHEK2, PTEN, ATM, and PPM1D. Many BC susceptibility genes can be grouped into two classes, high- and low-penetrance genes, each of which interact with multiple genes and environmental factors. However, the penetrance of genes can also be represented by a spectrum, which ranges between high and low. Two of the most common susceptibility genes are BRCA1 and BRCA2, which perform vital cellular functions for repair of homologous DNA. Loss of heterozygosity accompanied by hereditary mutations in BRCA1 or BRCA2 increases chromosomal instability and the likelihood of cancer, as well as playing a key role in stimulating malignant transformation. With regard to pathological features, familial breast cancers caused by BRCA1 mutations usually differ from those caused by BRCA2 mutations and nonfamilial BCs. It is essential to acquire an understanding of these pathological features along with the genetic history of the patient to offer an individualized treatment. Germline mutations in BRCA1 and BRCA2 genes are the main genetic and inherited factors for breast and ovarian cancer. In fact, these mutations are very important in developing early onset and increasing the risk of familial breast and ovarian cancer and responsible for 90% of hereditary BC cases. Therefore, according to the conducted studies, screening of BRCA1 and BRCA2 genes is recommended as an important marker for early detection of all patients with breast or ovarian cancer risk with family history of the disease. In this review, we summarize the role of hereditary genes, mainly BRCA1 and BRCA2, in BC.     

Sequencing <Emphasis Type="Italic">EVC</Emphasis> and <Emphasis Type="Italic">EVC2</Emphasis> identifies mutations in two-thirds of Ellis–van Creveld syndrome patients

Ellis–van Creveld syndrome (EvC) is caused by mutations in EVC and EVC2, genes in a divergent orientation separated by only 2.6 kb. We systematically sought mutations in both genes in a panel of 65 affected individuals to assess the proportion of cases resulting from mutations in each gene. We PCR amplified and sequenced the coding exons of both genes. We investigated mutations that could affect splicing by in vitro splicing assays and cDNA analysis. We have identified EVC mutations in 20 cases (31%); in all of these we have detected the mutation on each allele. We have identified EVC2 mutations in 25 cases (38%); in 22 of these we have isolated a mutation on each allele. The majority of the mutations introduce a premature termination codon. We sequenced the region between the two genes in 10 of the 20 cases in which we had not identified a mutation in either gene, revealing only one SNP that was not a common polymorphism. As we have not identified mutations in either gene in 20 cases (31%) it is possible that there is further genetic heterogeneity. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure

Background  

Glaucoma is a common disease but its molecular etiology is poorly understood. It involves retinal ganglion cell death and optic nerve damage that is often associated with elevated intraocular pressure. Identifying genes that modify glaucoma associated phenotypes is likely to provide insights to mechanisms of glaucoma. We previously reported glaucoma in DBA/2J mice caused by recessive alleles at two loci, isa and ipd, that cause iris stromal atrophy and iris pigment dispersion, respectively. A approach for identifying modifier genes is to study the effects of specific mutations in different mouse strains. When the phenotypic effect of a mutation is modified upon its introduction into a new strain, crosses between the parental strains can be used to identify modifier genes. The purpose of this study was to determine if the effects of the DBA/2J derived isa and ipd loci are modified in strain AKXD-28/Ty.     

Novel genes influencing the expression of the yellow locus and mdg4 (gypsy) in Drosophila melanogaster

Summary We used a system with a mobilized Stalker transposable element, sometimes in combination with P-M hybrid dysgenesis, in the search for new mutations interfering with the y ² mutation induced by mdg4 (gypsy) insertion into the yellow locus. A novel gene, modifier of mdg4, was detected in chromosome 3. The mutation mod(mdg4) either enhanced or suppressed phenotypic changes in different mutations induced by mdg4 insertions. Thus, mod(mdg4) seems to be involved in the control of mdg4 expression. Six other loci designated as enhancers of yellow were also detected. The e(y) ⁿ (with n from 1–6) mutations enhanced the expression of several y mutations induced by different insertions into the yellow locus. The major change is a damage of bristle and hair pigmentation which is not suppressed by su(Hw) mutations. On the other hand, e(y) ⁿ alleles do not interact with mdg4 induced mutations in other loci. All e(y) ⁿ genes are located in different regions of the X chromosome. One may speculate that e(y) ⁿ genes are involved in trans-regulation of the yellow locus and possibly of some other loci.     

Comprehensive landscape and interference of clonal haematopoiesis mutations for liquid biopsy: A Chinese pan-cancer cohort

Tumour-derived DNA found in the plasma of cancer patients provides the probability to detect somatic mutations from circulating cell-free DNA (cfDNA) in plasma samples. However, clonal hematopoiesis (CH) mutations affect the accuracy of liquid biopsy for cancer diagnosis and treatment. Here, we integrated landscape of CH mutations in 11,725 pan-cancer patients of Chinese and explored effects of CH on liquid biopsies in real-world. We first identified 5933 CHs based on panel sequencing of matched DNA of white blood cell and cfDNA on 301 genes for 5100 patients, in which CH number of patients had positive correlation with their diagnosis age. We observed that canonical genes related to CH, including DNMT3A, TET2, ASXL1, TP53, ATM, CHEK2 and SF3B1, were dominant in the Chinese cohort and 13.29% of CH mutations only appeared in the Chinese cohort compared with the Western cohort. Analysis of CH gene distribution bias indicated that CH tended to appear in genes with functions of tyrosine kinase regulation, PI3K-Akt signalling and TP53 activity, suggesting unfavourable effects of CH mutations in cancer patients. We further confirmed effect of driver genes carried by CH on somatic mutations in liquid biopsy of cancer patients. Forty-eight actionable somatic mutations in 17 driver genes were considered CH genes in 92 patients (1.80%) of the Chinese cohort, implying potential impacts of CH on clinical decision-making. Taken together, this study exhibits strong evidence that gene mutations from CH interfere accuracy of liquid biopsies using cfDNA in cancer diagnosis and treatment in real-world.     

Mutational accessibility of essential genes on chromosome I(left) in Caenorhabditis elegans

We have analyzed a region of approximately 5.4 million base pairs for mutations, which under standard laboratory conditions result in developmental arrest, sterility, or maternal-effect lethality in Caenorhabditis elegans. Lethal mutations were isolated, maintained, and genetically manipulated as homozygotes using sDp2– a duplication of the left half of chromosome I. All of the lethals and rearrangements used in this analysis were balanced by sDp2. Relatively low doses of mutagen, (approximately 15 mM ethylmethane sulfate; EMS), were used so as to limit the occurrence of second-site mutations, thus increasing the probability of recovering single nucleotide substitutions. Treatment of over 32,400 marked chromosomes resulted in 486 analyzed mutations. In this paper, we add 133 previously unidentified let genes, isolated in the EMS screens, and one let gene identified by a γ-ray induced mutation, to our collection of 103 essential genes. We also recovered lethal alleles of genes for which visible mutants already existed. In total, eight deficiencies and alleles of 237 essential genes were identified. Eighty-nine of the previously unidentified let genes are represented by more than one lethal allele. Statistical analysis indicates a minimum estimate of 400 essential genes in the region of chromosome I balanced by sDp2. This region occupies approximately half of chromosome I, and contains over 1135 protein-coding genes predicted from the genomic sequence data. Thus, approximately one-third of the predicted genes are estimated to be essential. Of these approximately 60% are represented by lethal alleles. Less than 2% of the lethal-bearing strains recovered in our analysis, including the eight genetically definable deficiencies, carried more than one lethal mutation. Several screens were used to recover mutations for this analysis. Because all the mutations were isolated using the same balancer, under similar screening conditions, it was possible to compare intervals within the sDp2 region with each other. The fraction of essential genes that present relatively large targets for EMS was highest within the central cluster (dpy-5 to unc-13). Received: 12 July 1999 / Accepted: 6 December 1999     

Mutant copies of mitochondrial DNA in tissues and plasma of mice subjected to X-ray irradiation

Impairments of mitochondrial genome are associated with a wide spectrum of degenerative diseases, development of tumors, aging, and cell death. We studied the content of mitochondrial DNA (mtDNA) with mutations and the total content of mutations in the brain and the spleen of mice subjected to X-ray irradiation at a dose of 1–5 Gy at 8–28 days after treatment. In these mice, we studied the number of mutant copies of extracellular mtDNA (ec-mtDNA) and its total content in blood plasma. We estimated mutations in control and irradiated mice using cleavage of heteroduplexes prepared by hybridization of PCR amplicons of mtDNA (D-loop region) mediated by CEL-I endonuclease, an enzyme that specifically cleaves unpaired bases. Changes in the total number of mtDNA copies relative to nuclear DNA were assessed by real time PCR using the ND-4 and GAPDH genes, respectively. We found that the number of mutant mtDNA copies was significantly increased in the brain and the spleen of irradiated mice and reached the maximum level at the eighth day after treatment; it then decreased by the 28th day after treatment. In nuclear genes similar to mutagenesis, mutagenesis of mtDNA in the brain and spleen tissues linearly depended on irradiation dose. In contrast to mutant nuclear genes, most mutant mtDNA copies were eliminated in the brain and spleen tissues, whereas the total content of mtDNA did not change within 28 days after irradiation. Our data show that, during this period, a high level of ec-mtDNA with mutations was observed in DNA circulating in blood plasma with the maximum level found at the 14th day. We suppose that mutant mtDNA copies are eliminated from cells of animals subjected to irradiation during the posttreatment period. Higher content of ec-mtDNA in blood plasma can be considered as a potential marker of radiation damage to the body.     

Genetic analysis and complementation by germ-line transformation of lethal mutations in the unc-22 IV region of Caenorhabditis elegans

Summary The subject of this study is the organization of essential genes in the 2 map-unit unc-22 IV region of the Caenorhabditis elegans genome. With the goal of achieving mutational saturation of essential genes in this region, 6491 chromosomes mutagenized with ethyl methanesulfonate (EMS) were screened for the presence of lethal mutations in the unc-22 region. The genetic analysis of 21 lethal mutations in the unc-22 region resulted in the identification of 6 new essential genes, making a total of 36 characterized to date. A minimum of 49 essential genes are estimated to lie in this region. A set of seven formaldehyde-induced deficiencies of unc-22 and surrounding loci were isolated to facilitate the positioning of essential genes on the genetic and physical maps. In order to study essential genes at the molecular level, our approach was to rescue lethal mutations by the injection of genomic DNA in the form of cosmid clones into the germ-line of balanced heterozygotes carrying a lethal mutation. The cosmid clones containing let-56 and let-653 were identified by this method.     

Lethal mutations defining 112 complementation groups in a 4.5?Mb sequenced region of Caenorhabditis elegans chromosome III

The central gene cluster of chromosome III was one of the first regions to be sequenced by the Caenorhabditis elegans genome project. We have performed an essential gene analysis on the left part of this cluster, in the region around dpy-17III balanced by the duplication sDp3. We isolated 151 essential gene mutations and characterized them with regard to their arrest stages. To facilitate positioning of these mutations, we generated six new deficiencies that, together with preexisting chromosomal rearrangements, subdivide the region into 14 zones. The 151 mutations were mapped into these zones. They define 112 genes, of which 110 were previously unidentified. Thirteen of the zones have been anchored to the physical sequence by polymerase chain reaction deficiency mapping. Of the 112 essential genes mapped, 105 are within these 13 zones. They span 4.2 Mb of nucleotide sequence. From the nucleotide sequence data, 920 genes are predicted. From a Poisson distribution of our mutations, we predict that 234 of the genes will be essential genes. Thus, the 105 genes constitute 45% of the estimated number of essential genes in the physically defined zones and between 2 and 5% of all essential genes in C. elegans. Received: 23 April 1998 / Accepted: 18 August 1998     

Amplification of oncogenes in human cancer cells

Gene amplification refers to a genomic change that results in an increased dosage of the gene(s) affected. Amplification represents one of the major molecular pathways through which the oncogenic potential of proto-oncogenes is activated during tumorigenesis. The architecture of amplified genomic structures is simple in some tumor types, involving in the vast majority of cases only one gene, such as MYCN in neuroblastomas. On the other hand, it can be complex and discontinuous, involving several syntenic co-amplified genes, such as in the 11q13 amplification in breast cancer, although in many of these cases there may be a single target gene. The presence of different nonsyntenic amplified genes raises the possibility that cells of certain tumors are susceptible to independent amplification events. In general, the amplified genes do not undergo additional damage by mutations. The data indicate that it is the enhanced level of a wild-type protein that contributes to tumorigenesis. BioEssays 20 :473–479, 1998.© 1998 John Wiley & Sons, Inc.     

Zebrafish heart failure models: opportunities and challenges

Heart failure is a complex pathophysiological syndrome of pumping functional failure that results from injury, infection or toxin-induced damage on the myocardium, as well as genetic influence. Gene mutations associated with cardiomyopathies can lead to various pathologies of heart failure. In recent years, zebrafish, Danio rerio, has emerged as an excellent model to study human cardiovascular diseases such as congenital heart defects, cardiomyopathy, and preclinical development of drugs targeting these diseases. In this review, we will first summarize zebrafish genetic models of heart failure arose from cardiomyopathy, which is caused by mutations in sarcomere, calcium or mitochondrial-associated genes. Moreover, we outline zebrafish heart failure models triggered by chemical compounds. Elucidation of these models will improve the understanding of the mechanism of pathogenesis and provide potential targets for novel therapies.