Base Substitution Mutations Research Articles

The ChIP-seq-Defined Networks of Bcl-3 Gene Binding Support Its Required Role in Skeletal Muscle Atrophy

NF-kappaB transcriptional activation is required for skeletal muscle disuse atrophy. We are continuing to study how the activation of NF-kB regulates the genes that encode the protein products that cause atrophy. Using ChIP-sequencing we found that Bcl-3, an NF-kB transcriptional activator required for atrophy, binds to the promoters of a number of genes whose collective function describes two major aspects of muscle wasting. By means of bioinformatics analysis of ChIP-sequencing data we found Bcl-3 to be directing transcription networks of proteolysis and energy metabolism. The proteolytic arm of the Bcl-3 networks includes many E3 ligases associated with proteasomal protein degradation, including that of the N-end rule pathway. The metabolic arm appears to be involved in organizing the change from oxidative phosphorylation to glycolysis in atrophying muscle. For one gene, MuRF1, ChIP-sequencing data identified the location of Bcl-3 and p50 binding in the promoter region which directed the creation of deletant and base-substitution mutations of MuRF1 promoter constructs to determine the effect on gene transcription. The results provide the first direct confirmation that the NF-kB binding site is involved in the muscle unloading regulation of MuRF1. Finally, we have combined the ChIP-sequencing results with gene expression microarray data from unloaded muscle to map several direct targets of Bcl-3 that are transcription factors whose own targets describe a set of indirect targets for NF-kB in atrophy. ChIP-sequencing provides the first molecular explanation for the finding that Bcl3 knockout mice are resistant to disuse muscle atrophy. Mapping the transcriptional regulation of muscle atrophy requires an unbiased analysis of the whole genome, which we show is now possible with ChIP-sequencing.

Extraordinary genome stability in the ciliate Paramecium tetraurelia

Mutation plays a central role in all evolutionary processes and is also the basis of genetic disorders. Established base-substitution mutation rates in eukaryotes range between ∼5 × 10(-10) and 5 × 10(-8) per site per generation, but here we report a genome-wide estimate for Paramecium tetraurelia that is more than an order of magnitude lower than any previous eukaryotic estimate. Nevertheless, when the mutation rate per cell division is extrapolated to the length of the sexual cycle for this protist, the measure obtained is comparable to that for multicellular species with similar genome sizes. Because Paramecium has a transcriptionally silent germ-line nucleus, these results are consistent with the hypothesis that natural selection operates on the cumulative germ-line replication fidelity per episode of somatic gene expression, with the germ-line mutation rate per cell division evolving downward to the lower barrier imposed by random genetic drift. We observe ciliate-specific modifications of widely conserved amino acid sites in DNA polymerases as one potential explanation for unusually high levels of replication fidelity.

Sequence analysis for the complete proviral genome of avian leukosis virus subgroup J associated with haemangiomas, leiomyosarcomas and myelomas in layer flocks

Avian leukosis virus subgroup J (ALV-J) can cause a variety of neoplasms, including mainly myeloid leukosis (myelocytomatosis) and nephromas. Other tumours, such as histiocytic sarcoma (HS), haemangiosarcoma and mesothelioma, may also develop. In a previous article we described a case in which myeloid leukosis, haemangiomas and leiomyosarcomas appeared simultaneously in a commercial layer flock with infection by ALV-J. The present research was completed to understand the molecular characteristics of the ALV-J strain that induced clinical myeloid leukosis, haemangiomas and leiomyosarcomas. Two strains of ALV-J (SDAU1001 and SDAU1002) were isolated and identified, and their full-length sequences were analysed. The complete genome nucleotide sequences of these two isolates were different in length, 7652 nt and 7636 nt, respectively. They shared 98.9% identity with each other, and 93.4% to 97.8% nucleotide identity to the reference ALV-J isolates. A 19-nucleotide repeat sequence was identified in the primer binding site (PBS) leader region of isolate SDAU1001. A base substitution mutation (base 15 C-T) in this insertion was identified. However, the identical insertion at the same site was not found in SDAU1002. The gag and pol genes of the two viruses were more conserved than the env gene. One key deletion in the E element was a common feature of SDAU1001 and SDAU1002. SDAU1001 and SDAU1002, possibly recombinants of ALV-J and another avian retrovirus, may share the same ancestor. Co-infection by SDAU1001 and SDAU1002 isolates is a possible explanation why myeloid leukosis, haemangiomas, and leiomyosarcomas appeared simultaneously in the same commercial layer flock.

Polynucleotide Phosphorylase Plays an Important Role in the Generation of Spontaneous Mutations in Escherichia coli

Polynucleotide phosphorylase (PNP) plays a central role in RNA degradation, generating a pool of ribonucleoside diphosphates (rNDPs) that can be converted to deoxyribonucleoside diphosphates (dNDPs) by ribonucleotide reductase. We report here that spontaneous mutations resulting from replication errors, which are normally repaired by the mismatch repair (MMR) system, are sharply reduced in a PNP-deficient Escherichia coli strain. This is true for base substitution mutations that occur in the rpoB gene leading to Rif(r) and the gyrB gene leading to Nal(r) and for base substitution and frameshift mutations that occur in the lacZ gene. These results suggest that the increase in the rNDP pools generated by polynucleotide phosphorylase (PNP) degradation of RNA is responsible for the spontaneous mutations observed in an MMR-deficient background. The PNP-derived pool also appears responsible for the observed mutations in the mutT mutator background and those that occur after treatment with 5-bromodeoxyuridine, as these mutations are also drastically reduced in a PNP-deficient strain. However, mutation frequencies are not reduced in a mutY mutator background or after treatment with 2-aminopurine. These results highlight the central role in mutagenesis played by the rNDP pools (and the subsequent dNTP pools) derived from RNA degradation.

P2.06 Exploiting The Mutanome for Tumor Vaccination

ABSTRACT We demonstrate the first preclinical proof-of-concept of a mutation-based individualized cancer vaccine. Multiple genetic events and subsequent clonal evolution drive carcinogenesis, making disease elimination with single targeted drugs difficult. The multiplicity of gene mutations derived from clonal heterogeneity therefore represents an ideal setting for multi-epitope tumor vaccination. Here we used NGS exome resequencing to identify 941 non-synonymous somatic point mutations in B16F10 murine melanoma cells, with 589 of those mutations in expressed genes. Potential driver mutations occurred in classical tumor suppressor genes and genes involved in proto-oncogenic signaling pathways that control cell proliferation, adhesion, migration and apoptosis. The immunogenicity and specificity of 50 validated mutations was determined by immunizing mice with long peptides encoding the mutated epitopes. One-third of these peptides were found to be immunogenic, with 60 % in this group eliciting immune responses directed preferentially against the mutated sequence as compared to the wild type sequence. In tumor transplant models, peptide immunization conferred in vivo tumor control in protective and therapeutic settings, thereby qualifying mutated epitopes that include single amino acid substitutions as effective vaccines. Not only do our findings provide the first comprehensive picture of the mutanome of B16F10 melanoma, the most widely used cell line in immunotherapy studies, they also they offer insight into the extent of the immunogenicity of non-synonymous base substitution mutations. Lastly, the findings demonstrate the use of deep sequencing to identify immunogenic mutations and paves the way for individualized immunotherapy of cancer patients.

Azidothymidine and other chain terminators are mutagenic for template-switch-generated genetic mutations

The accumulation of mutations causes cell lethality and can lead to carcinogenesis. An important class of mutations, which are associated with mutational hotspots in many organisms, are those that arise by nascent strand misalignment and template-switching at the site of short repetitive sequences in DNA. Mutagens that strongly and specifically affect this class, which is mechanistically distinct from other mutations that arise from polymerase errors or by DNA template damage, are unknown. Using Escherichia coli and assays for specific mutational events, this study defines such a mutagen, 3'-azidothymidine [zidovudine (AZT)], used widely in the treatment and prevention of HIV/AIDS. At sublethal doses, AZT has no significant effect on frame shifts and most base-substitution mutations. AT-to-CG transversions and deletions at microhomologies were enhanced modestly by AZT. AZT strongly stimulated the "template-switch" class of mutations that arise in imperfect inverted repeat sequences by DNA-strand misalignments during replication, presumably through its action as a chain terminator during DNA replication. Chain-terminating 2'-3'-didehydro 3'-deoxythymidine [stavudine (D4T)] and 2'-3'-dideoxyinosine [didanosine (ddI)] likewise stimulated template-switch mutagenesis. These agents define a specific class of mutagen that promotes template-switching and acts by stalling replication rather than by direct nucleotide base damage.

Exploiting the Mutanome for Tumor Vaccination

Multiple genetic events and subsequent clonal evolution drive carcinogenesis, making disease elimination with single-targeted drugs difficult. The multiplicity of gene mutations derived from clonal heterogeneity therefore represents an ideal setting for multiepitope tumor vaccination. Here, we used next generation sequencing exome resequencing to identify 962 nonsynonymous somatic point mutations in B16F10 murine melanoma cells, with 563 of those mutations in expressed genes. Potential driver mutations occurred in classical tumor suppressor genes and genes involved in proto-oncogenic signaling pathways that control cell proliferation, adhesion, migration, and apoptosis. Aim1 and Trrap mutations known to be altered in human melanoma were included among those found. The immunogenicity and specificity of 50 validated mutations was determined by immunizing mice with long peptides encoding the mutated epitopes. One-third of these peptides were found to be immunogenic, with 60% in this group eliciting immune responses directed preferentially against the mutated sequence as compared with the wild-type sequence. In tumor transplant models, peptide immunization conferred in vivo tumor control in protective and therapeutic settings, thereby qualifying mutated epitopes that include single amino acid substitutions as effective vaccines. Together, our findings provide a comprehensive picture of the mutanome of B16F10 melanoma which is used widely in immunotherapy studies. In addition, they offer insight into the extent of the immunogenicity of nonsynonymous base substitution mutations. Lastly, they argue that the use of deep sequencing to systematically analyze immunogenicity mutations may pave the way for individualized immunotherapy of cancer patients.

Amber mutants of bacteriophage T4D: their isolation and genetic characterization.

We have isolated a large number of mutants of bacteriophage T4D that are unable to form plaques on strain B of Escherichia coli, but are able to grow (nearly) normally on some other strains of E. coli, in particular strain CR63. These mutants, designated amber (am), have been characterized by complementation tests, by genetic crosses, and by their response to chemical mutagens. It is concluded that a particular subclass of base substitution mutations may give rise to amber mutants and that such mutants occur in many genes, which are widely distributed over the T4 genome.

Delivery Method, Target Gene Structure, and Growth Properties of Target Cells Impact Mutagenic Responses to Reactive Nitrogen and Oxygen Species

Dysregulated production of nitric oxide (NO•) and reactive oxygen species (ROS) by inflammatory cells in vivo may contribute to mutagenesis and carcinogenesis. Here, we compare cytotoxicity and mutagenicity induced by NO• and ROS in TK6 and AS52 cells, delivered by two methods: a well-characterized delivery system and a novel adaptation of a system for coculture. When exposed to preformed NO•, a cumulative dose of 620 μM min reduced the viability of TK6 cells at 24 h to 36% and increased mutation frequencies in the HPRT and TK1 genes to 7.7 × 10⁻⁶ (p < 0.05) and 24.8 × 10⁻⁶ (p < 0.01), 2.7- and 3.7-fold higher than background, respectively. In AS52 cells, cumulative doses of 1700 and 3700 μM min reduced viability to 49 and 22%, respectively, and increased the mutation frequency 10.2- and 14.6-fold higher than the argon control (132 × 10⁻⁶ and 190 × 10⁻⁶, respectively). These data show that TK6 cells were more sensitive than AS52 cells to killing by NO•. However, the two cell lines were very similar in relative susceptibility to mutagenesis; on the basis of fold increases in MF, average relative sensitivity values [(MF(exp)/MF(control))/cumulative NO• dose] were 5.16 × 10⁻³ and 4.97 × 10⁻³ μM⁻¹ min⁻¹ for TK6 cells and AS52 cells, respectively. When AS52 cells were exposed to reactive species generated by activated macrophages in the coculture system, cell killing was greatly reduced by the addition of NMA to the culture medium and was completely abrogated by combined additions of NMA and the superoxide scavenger Tiron, indicating the relative importance of NO• to loss of viability. Exposure in the coculture system for 48 h increased mutation frequency in the gpt gene by more than 9-fold, and NMA plus Tiron again completely prevented the response. Molecular analysis of gpt mutants induced by preformed NO• or by activated macrophages revealed that both doubled the frequency of gene inactivation (40% in induced vs 20% in spontaneous mutants). Sequencing showed that base-substitution mutations dominated the spectra, with transversions (30-40%) outnumbering transitions (10-20%). Virtually all mutations took place at guanine sites in the gene. G:C to T:A transversions accounted for about 30% of both spontaneous and induced mutations; G:C to A:T transitions amounted to 10-20% of mutants; insertions, small deletions, and multiple mutations were present at frequencies of 0-10%. Taken together, these results indicate that cell type and proximity to generator cells are critical determinants of cytotoxic and genotoxic responses induced by NO• and reactive species produced by activated macrophages.

Genome Sequencing and Analysis of the Tasmanian Devil and Its Transmissible Cancer

SummaryThe Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations.PaperClip

A System for the Rapid Determination of the Mutation Spectrum in Escherichia coli

The first step toward elucidating the mutagenic effects of chemicals and pathways is to determine the specificity of the mutations generated spontaneously or in response to treatment with mutagens. We constructed a set of plasmid-encoded probes for the specific detection of each type of base substitution mutation. Using these probes, we were able to quickly determine both the mutation rate and the specificity of the mutations caused by different types of mutagens and mutagenic conditions. We also developed a PCR-based method to rapidly and robustly determine the mutation spectrum in response to various mutagenic samples in parallel. This system allows one to not only analyze the mutation specificity of various chemicals, but also to search for novel genetic elements that promote the specific mutation events.

Variation in Base-Substitution Mutation in Experimental and Natural Lineages of Caenorhabditis Nematodes

Variation among lineages in the mutation process has the potential to impact diverse biological processes ranging from susceptibilities to genetic disease to the mode and tempo of molecular evolution. The combination of high-throughput DNA sequencing (HTS) with mutation-accumulation (MA) experiments has provided a powerful approach to genome-wide mutation analysis, though insights into mutational variation have been limited by the vast evolutionary distances among the few species analyzed. We performed a HTS analysis of MA lines derived from four Caenorhabditis nematode natural genotypes: C. elegans N2 and PB306 and C. briggsae HK104 and PB800. Total mutation rates did not differ among the four sets of MA lines. A mutational bias toward G:C→A:T transitions and G:C→T:A transversions was observed in all four sets of MA lines. Chromosome-specific rates were mostly stable, though there was some evidence for a slightly elevated X chromosome mutation rate in PB306. Rates were homogeneous among functional coding sequence types and across autosomal cores, arms, and tips. Mutation spectra were similar among the four MA line sets but differed significantly when compared with patterns of natural base-substitution polymorphism for 13/14 comparisons performed. Our findings show that base-substitution mutation processes in these closely related animal lineages are mostly stable but differ from natural polymorphism patterns in these two species.

DNA Unwinding by ASCC3 Helicase Is Coupled to ALKBH3-Dependent DNA Alkylation Repair and Cancer Cell Proliferation

Demethylation by the AlkB dioxygenases represents an important mechanism for repair of N-alkylated nucleotides. However, little is known about their functions in mammalian cells. We report the purification of the ALKBH3 complex and demonstrate its association with the activating signal cointegrator complex (ASCC). ALKBH3 is overexpressed in various cancers, and both ALKBH3 and ASCC are important for alkylation damage resistance in these tumor cell lines. ASCC3, the largest subunit of ASCC, encodes a 3'-5' DNA helicase, whose activity is crucial for the generation of single-stranded DNA upon which ALKBH3 preferentially functions for dealkylation. In cell lines that are dependent on ALKBH3 and ASCC3 for alkylation damage resistance, loss of ALKBH3 or ASCC3 leads to increased 3-methylcytosine and reduced cell proliferation, which correlates with pH2A.X and 53BP1 foci formation. Our data provide a molecular mechanism by which ALKBH3 collaborates with ASCC to maintain genomic integrity in a cell-type specific manner.

Structure of the 2-aminopurine-cytosine base pair formed in the polymerase active site of the RB69 Y567A-DNA polymerase.

The adenine base analogue 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the polymerase active site of the RB69 Y567A-DNA polymerase. A modified wobble 2AP-C base pair was detected with one H-bond between N1 of 2AP and a proton from the C4 amino group of cytosine and an apparent bifurcated H-bond between a proton on the 2-amino group of 2-aminopurine and the ring N3 and O2 atoms of cytosine. Interestingly, a primer-terminal region rich in AT base pairs, compared to GC base pairs, facilitated dCTP binding opposite template 2AP. We propose that the increased flexibility of the nucleotide binding pocket formed in the Y567A-DNA polymerase and increased "breathing" at the primer-terminal junction of A+T-rich DNA facilitate dCTP binding opposite template 2AP. Thus, interactions between DNA polymerase residues with a dynamic primer-terminal junction play a role in determining base selectivity within the polymerase active site of RB69 DNA polymerase.

Hypoxia induces CD133 expression in human lung cancer cells by up-regulation of OCT3/4 and SOX2

CD133 has been recognized as a specific cell surface marker for cancer stem cells in various tumors, although its biological functions and transcriptional regulation remain unclear. We found that the CD133 expression level was up-regulated in the lung cancer cell lines N417, H358, and A549, when these cell lines were cultured under hypoxic conditions. Among the five promoters (P1-P5) of human CD133 gene loci, P1 promoter was most strongly associated with hypoxia-induced promoter activity of CD133 gene expression. The P1 promoter possesses several cis-regulatory elements, including RUNT, GATA, ETS, OCT, SRY, and CREB-binding sites. A series of deletion and base substitution mutants of the P1 promoter revealed that OCT- and SRY-binding sites are important for hypoxia-induced promoter activity. The chromatin immunoprecipitation assay further confirmed the direct binding of Octamer biding trans-cription factor 3/4 (OCT4) and/or SRY-box containing gene2 (SOX2) to the P1 promoter region of CD133 gene loci. In addition, the enhancement of both OCT4 and SOX2 expression by the α subunit of hypoxia-inducible factors (HIF1α and HIF2α) was required for hypoxia-induced CD133 expression. Knockdown of OCT4 or SOX2 expression in N417 cells with stabilized HIF1α and/or HIF2α abolished CD133P1 activity, while ectopic OCT4 or SOX2 expression triggers CD133P1 activity in the absence of HIF1α or HIF2α. Thus, in the hypoxic conditions, OCT4 and SOX2, both of which are induced by HIF1α/HIF2α. promote CD133 expression in the lung cancer cells via their direct interaction with the P1 promoter.

Impact of a stress-inducible switch to mutagenic repair of DNA breaks on mutation in Escherichia coli

Basic ideas about the constancy and randomness of mutagenesis that drives evolution were challenged by the discovery of mutation pathways activated by stress responses. These pathways could promote evolution specifically when cells are maladapted to their environment (i.e., are stressed). However, the clearest example--a general stress-response-controlled switch to error-prone DNA break (double-strand break, DSB) repair--was suggested to be peculiar to an Escherichia coli F' conjugative plasmid, not generally significant, and to occur by an alternative stress-independent mechanism. Moreover, mechanisms of spontaneous mutation in E. coli remain obscure. First, we demonstrate that this same mechanism occurs in chromosomes of starving F(-) E. coli. I-SceI endonuclease-induced chromosomal DSBs increase mutation 50-fold, dependent upon general/starvation- and DNA-damage-stress responses, DinB error-prone DNA polymerase, and DSB-repair proteins. Second, DSB repair is also mutagenic if the RpoS general-stress-response activator is expressed in unstressed cells, illustrating a stress-response-controlled switch to mutagenic repair. Third, DSB survival is not improved by RpoS or DinB, indicating that mutagenesis is not an inescapable byproduct of repair. Importantly, fourth, fully half of spontaneous frame-shift and base-substitution mutation during starvation also requires the same stress-response, DSB-repair, and DinB proteins. These data indicate that DSB-repair-dependent stress-induced mutation, driven by spontaneous DNA breaks, is a pathway that cells usually use and a major source of spontaneous mutation. These data also rule out major alternative models for the mechanism. Mechanisms that couple mutagenesis to stress responses can allow cells to evolve rapidly and responsively to their environment.

The Riboflavin analog Roseoflavin targets an FMN-riboswitch and blocks Listeria monocytogenes growth, but also stimulates virulence gene-expression and infection

During recent years, riboswitches have emerged as potential targets for novel antibacterial substances. In this study, we investigated how one flavin analog, roseoflavin, affected the gene-expression, growth and infectivity of the human bacterial pathogen Listeria monocytogenes to determine the potential of this analog to function as an antibacterial substance. The results indicate that roseoflavin has a profound inhibiting effect on the growth of L. monocytogenes at very low concentrations. Also, expression of the gene located downstream of the FMN riboswitch, a riboflavin transporter, was blocked by the addition of roseoflavin. Base-substitution mutations in the FMN riboswitch allowed the bacteria to grow in the presence of roseoflavin, showing that roseoflavin targeted the FMN riboswitch directly. Surprisingly, we found that roseoflavin stimulated L. monocytogenes virulence gene expression and infection abilities in a mechanism independent of the FMN riboswitch. Our results suggest that roseoflavin can block growth but also enhance Listeria virulence.

Random Mutagenesis of the Proton-coupled Folate Transporter (SLC46A1), Clustering of Mutations, and the Bases for Associated Losses of Function

Loss-of-function mutations in the proton-coupled folate transporter (PCFT, SLC46A1) result in the autosomal recessive disorder, hereditary folate malabsorption (HFM). Identification and characterization of HFM mutations provide a wealth of information on the structure-function relationship of this transporter. In the current study, PCR-based random mutagenesis was employed to generate unbiased loss-of-function mutations of PCFT, simulating the spectrum of alterations that might occur in the human disorder. A total of 26 mutations were generated and 4 were identical to HFM mutations. Eleven were base deletion or insertion mutations that led to a frameshift and, along with similar HFM mutations, are predominantly localized to two narrow regions of the pcft gene at the 5'-end. Base substitution mutations identified in the current study and HFM patients were largely distributed across the pcft gene. Elimination of the ATG initiation codon by a one-base substitution (G > A) did not result in a complete lack of translation at the same codon consistent with rare non-ATG translation initiation. Among six missense mutants evaluated, three mutant PCFTs were not detected at the plasma membrane, one mutation resulted in decreased binding to folate substrate, and one had a reduced rate of conformational change associated with substrate translocation. The remaining PCFT mutant had defects in both processes. These results broaden understanding of the regions of the pcft gene prone to base insertion and deletion and inform further approaches to the analysis of the structure-function of PCFT.

Mucopolysaccharidosis type I: molecular characteristics of two novel alpha-L-iduronidase mutations in Tunisian patients.

BackgroundMucopolysaccharidosis type I (MPS I) is an autosomal storage disease resulting from defective activity of the enzyme α-L-iduronidase (IDUA). This glycosidase is involved in the degradation of heparan sulfate and dermatan sulfate. MPS I has severe and milder phenotypic subtypes.Aim of study: This study was carried out on six newly collected MPS I patients recruited from many regions of Tunisia.Patients and methods: Mutational analysis of the IDUA gene in unrelated MPS I families was performed by sequencing the exons and intron-exon junctions of IDUA gene.ResultsTwo novel IDUA mutations, p.L530fs (1587_1588 insGC) in exon 11 and p.F177S in exon 5 and two previously reported mutations p.P533R and p.Y581X were detected. The patient in family 1 who has the Hurler phenotype was homozygous for the previously described nonsense mutation p.Y581X.The patient in family 2 who also has the Hurler phenotype was homozygous for the novel missense mutation p.F177S. The three patients in families 3, 5 and 6 were homozygous for the p.P533R mutation. The patient in family 4 was homozygous for the novel small insertion 1587_1588 insGC. In addition, eighteen known and one unknown IDUA polymorphisms were identified.ConclusionThe identification of these mutations should facilitate prenatal diagnosis and counseling for MPS I in Tunisia.BackgroundMucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder caused by the deficient activity of the enzyme of α-L-iduronidase (IDUA, EC 3.2.1.76). This glycosidase is involved in the degradation of heparan sulfate and dermatan sulfate. The clinical phenotype of MPS I ranges from the very severe in Hurler syndrome (MPS IH) to the relatively benign in Scheie syndrome (MPS IS), with an intermediate phenotype designated Hurler/Scheie (MPS IH/S) [1]. Isolation of complementary and genomic DNAs encoding human α -L- iduronidase [2,3] have enable the identification of mutations underlying the enzyme defect and resulting in MPS I clinical phenotype. More than 100 mutations have been reported in patients with the MPS I subtypes (Human Gene Mutation Database; http://www.hgmd.org). High prevalence of the common mutations p.W402X and p.Q70X has been described; both of them in the severe clinical forms [4,5]. A high prevalence of common mutation p.P533R has also been described in MPS I patients with various phenotypes [5,6]. In addition, rare mutations including single base substitution, deletion, insertion and splicing site mutation have been identified [7], indicating a high degree of allelic heterogeneity in IDUA gene.Here, we described two novel IDUA mutations in MPS I Tunisian patients. These lesions were homoallelic in all the patients of the six families investigated as consanguineous marriages are still frequent in Tunisia [8].

Genome-wide survey of post-meiotic segregation during yeast recombination

BackgroundWhen mismatches in heteroduplex DNA formed during meiotic recombination are left unrepaired, post-meiotic segregation of the two mismatched alleles occurs during the ensuing round of mitosis. This gives rise to somatic mosaicism in multicellular organisms and leads to unexpected allelic combinations among progeny. Despite its implications for inheritance, post-meiotic segregation has been studied at only a few loci.ResultsBy genotyping tens of thousands of genetic markers in yeast segregants and their clonal progeny, we analyzed post-meiotic segregation at a genome-wide scale. We show that post-meiotic segregation occurs in close to 10% of recombination events. Although the overall number of markers affected in a single meiosis is small, the rate of post-meiotic segregation is more than five orders of magnitude larger than the base substitution mutation rate. Post-meiotic segregation took place with equal relative frequency in crossovers and non-crossovers, and usually at the edges of gene conversion tracts. Furthermore, post-meiotic segregation tended to occur in markers that are isolated from other heterozygosities and preferentially at polymorphism types that are relatively uncommon in the yeast species.ConclusionsOverall, our survey reveals the genome-wide characteristics of post-meiotic segregation. The results show that post-meiotic segregation is widespread in meiotic recombination and could be a significant determinant of allelic inheritance and allele frequencies at the population level.