Large Chromosomal Rearrangements Research Articles

Rapid mapping of insertional mutations to probe cell wall regulation in Cryptococcus neoformans

Random insertional mutagenesis screens are important tools in microbial genetics studies. Investigators in fungal systems have used the plant pathogen Agrobacterium tumefaciens to create tagged, random mutations for genetic screens in their fungal species of interest through a unique process of trans-kingdom cellular transconjugation. However, identifying the locations of insertion has traditionally required tedious PCR-based methods, limiting the effective throughput of this system. We have developed an efficient genomic sequencing and analysis method (AIM-Seq) to facilitate identification of randomly generated genomic insertions in microorganisms. AIM-Seq combines batch sampling, whole genome sequencing, and a novel bioinformatics pipeline, AIM-HII, to rapidly identify sites of genomic insertion. We have specifically applied this technique to Agrobacterium-mediated transconjugation in the human fungal pathogen Cryptococcus neoformans. With this approach, we have screened a library of C. neoformans cell wall mutants, selecting twenty-seven mutants of interest for analysis by AIM-Seq. We identified thirty-five putative genomic insertions in known and previously unknown regulators of cell wall processes in this pathogenic fungus. We confirmed the relevance of a subset of these by creating independent mutant strains and analyzing resulting cell wall phenotypes. Through our sequence-based analysis of these mutations, we observed “typical” insertions of the Agrobacterium transfer DNA as well as atypical insertion events, including large deletions and chromosomal rearrangements. Initially applied to C. neoformans, this mutant analysis tool can be applied to a wide range of experimental systems and methods of mutagenesis, facilitating future microbial genetic screens.

Genetic mosaics and the germ line lineage.

Genetic mosaics provide information about cellular lineages that is otherwise difficult to obtain, especially in humans. De novo mutations act as cell markers, allowing the tracing of developmental trajectories of all descendants of the cell in which the new mutation arises. De novo mutations may arise at any time during development but are relatively rare. They have usually been observed through medical ascertainment, when the mutation causes unusual clinical signs or symptoms. Mutational events can include aneuploidies, large chromosomal rearrangements, copy number variants, or point mutations. In this review we focus primarily on the analysis of point mutations and their utility in addressing questions of germ line versus somatic lineages. Genetic mosaics demonstrate that the germ line and soma diverge early in development, since there are many examples of combined somatic and germ line mosaicism for de novo mutations. The occurrence of simultaneous mosaicism in both the germ line and soma also shows that the germ line is not strictly clonal but arises from at least two, and possibly multiple, cells in the embryo with different ancestries. Whole genome or exome DNA sequencing technologies promise to expand the range of studies of genetic mosaics, as de novo mutations can now be identified through sequencing alone in the absence of a medical ascertainment. These technologies have been used to study mutation patterns in nuclear families and in monozygotic twins, and in animal model developmental studies, but not yet for extensive cell lineage studies in humans.

Abstract LB-314: Whole exome sequencing analyses of gastric cancers reveal two distinct genomic alteration patterns with implications in drug sensitivity

Abstract Gastric cancer is the fourth most common cancer diagnosed and the second most frequent cause of cancer-related death worldwide. Multiple factors can contribute to the development of gastric cancer, including H. pylori infection, dietary behaviour and life style, possibly resulting in distinct cancer subtypes with different drug sensitivity profiles. In the present study we searched for gastric cancer mutation patterns in the dataset of the “The Cancer Genome Atlas” (TCGA) and in our collection of patient derived xenografts (PDX). In a second part, we evaluated gene alteration patterns for their implications for drug sensitivity. In both TCGA and our PDX datasets, Whole Exome Sequencing analyses revealed two subsets of gastric tumors characterized by specific mutation signatures, with different types and numbers of genomic alterations. The first subset (60% and 75% of samples) contained lower levels of mutations and was characterized by increased numbers of large chromosomal rearrangements resulting in gene loss or amplifications. The second subset of tumors (25%-40% of samples) revealed higher levels of mutations that were predominantly nucleic acid substitutions and small indels linked to mismatch repair genes including MLH1 or MSH3 and to high microsatellite instability. In both subsets, the mutation spectrum was dominated by C>T transitions with an increase of small indels in the subset of highly-mutated tumors. At the gene level, the genes which were mutated in our gastric PDX collection overlapped to great extent with the mutations found in TCGA tumors, especially regarding the most frequently mutated genes. In the first subset, high levels of gene amplifications and deletions were found, including growth factor receptor amplifications in EGFR and HER2. Furthermore, the mutation frequency in genes associated with drug resistance such as KRAS was decreased. The tumors with growth factor receptor amplification responded consistently to therapies such as Cetuximab or Trastuzumab. In contrast, an increased frequency of mutations in oncogenes and tumor suppressors, including KRAS (n=5/10), PIK3CA (n=5/10) and PTEN (n=7/10), was found in the second subset. The mutational profile of these tumors suggest the use of compounds targeting downstream molecules, such as PIK3CA, or targeting effectors of DNA repair, such as PARP, for anti-cancer therapy. Of note, no association was found between the mutation groups and sensitivity to chemotherapeutic agents such as 5FU, Cisplatin or Paclitaxel. In conclusion, we identified two subsets of gastric tumors both in the TCGA dataset and in our collection of PDX models, characterized by distinct genomic alteration profiles suggesting different therapeutic approaches. Currently, we are assessing drug sensitivity profiles within the two subsets in our PDX models. Citation Format: Anne-Lise Peille, Swee-Seong Wong, Florian Kiefer, Bruno Zeitouni, Armin Maier, Frederic Foucault, Tim Kees, Vincent Vuaroqueaux, Amit Aggarwal, Christoph Reinhard, Heinz Herbert Fiebig. Whole exome sequencing analyses of gastric cancers reveal two distinct genomic alteration patterns with implications in drug sensitivity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-314. doi:10.1158/1538-7445.AM2014-LB-314

Large chromosomal rearrangements during a long-term evolution experiment with Escherichia coli.

ABSTRACTLarge-scale rearrangements may be important in evolution because they can alter chromosome organization and gene expression in ways not possible through point mutations. In a long-term evolution experiment, twelve Escherichia coli populations have been propagated in a glucose-limited environment for over 25 years. We used whole-genome mapping (optical mapping) combined with genome sequencing and PCR analysis to identify the large-scale chromosomal rearrangements in clones from each population after 40,000 generations. A total of 110 rearrangement events were detected, including 82 deletions, 19 inversions, and 9 duplications, with lineages having between 5 and 20 events. In three populations, successive rearrangements impacted particular regions. In five populations, rearrangements affected over a third of the chromosome. Most rearrangements involved recombination between insertion sequence (IS) elements, illustrating their importance in mediating genome plasticity. Two lines of evidence suggest that at least some of these rearrangements conferred higher fitness. First, parallel changes were observed across the independent populations, with ~65% of the rearrangements affecting the same loci in at least two populations. For example, the ribose-utilization operon and the manB-cpsG region were deleted in 12 and 10 populations, respectively, suggesting positive selection, and this inference was previously confirmed for the former case. Second, optical maps from clones sampled over time from one population showed that most rearrangements occurred early in the experiment, when fitness was increasing most rapidly. However, some rearrangements likely occur at high frequency and may have simply hitchhiked to fixation. In any case, large-scale rearrangements clearly influenced genomic evolution in these populations.

A model for genome size evolution.

We present a model for genome size evolution that takes into account both local mutations such as small insertions and small deletions, and large chromosomal rearrangements such as duplications and large deletions. We introduce the possibility of undergoing several mutations within one generation. The model, albeit minimalist, reveals a non-trivial spontaneous dynamics of genome size: in the absence of selection, an arbitrary large part of genomes remains beneath a finite size, even for a duplication rate 2.6-fold higher than the rate of large deletions, and even if there is also a systematic bias toward small insertions compared to small deletions. Specifically, we show that the condition of existence of an asymptotic stationary distribution for genome size non-trivially depends on the rates and mean sizes of the different mutation types. We also give upper bounds for the median and other quantiles of the genome size distribution, and argue that these bounds cannot be overcome by selection. Taken together, our results show that the spontaneous dynamics of genome size naturally prevents it from growing infinitely, even in cases where intuition would suggest an infinite growth. Using quantitative numerical examples, we show that, in practice, a shrinkage bias appears very quickly in genomes undergoing mutation accumulation, even though DNA gains and losses appear to be perfectly symmetrical at first sight. We discuss this spontaneous dynamics in the light of the other evolutionary forces proposed in the literature and argue that it provides them a stability-related size limit below which they can act.

2.3 Exome sequencing improves genetic diagnosis of structural fetal abnormalities revealed by ultrasound

BackgroundThe genetic aetiology of non-aneuploid fetal structural abnormalities is typically investigated by karyotyping and array-based detection of microscopically detectable rearrangements, and submicroscopic copy number variants (CNVs), which collectively yield a...

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see Frequency of Prader–Willi syndrome in births conceived via assisted reproductive technology Assisted reproductive technology (ART), which enables couples having difficulty conceiving to bear children, is not without risk. The technology has been associated with an increased frequency of certain imprinting disorders. Gold et al. studied a cohort of children with Prader–Willi syndrome (PWS) and found that the use of ART by their parents did not seem to be higher than that for parents of other children. In an analysis of surveys completed by members of the Prader–Willi Syndrome Association, along with medical records, the research team determined that 1.1% of individuals with PWS were conceived using ART, compared with 1% of the general population, an insignificant difference. However, there was a significant difference in the cause of PWS among ART users. In this group, more cases were caused by maternal uniparental disomy (UPD) and imprinting defects than in the general population. The study is the first to report cases of an imprinting-center defect in PWS patients conceived via ART. The investigators suggest that the increase may be explained by the fact that the parents of those in the ART-conceived group were significantly older (mean age 36.3 and 39.7 years for mothers and fathers, respectively) than those of the naturally conceived group (31.5 and 31.6 years, respectively), and maternal UPD is associated with maternal age at conception. The age of parents using reproductive technologies must be taken into account when evaluating the risks associated with the technology. —Karyn Hede, News Editor see Next-generation carrier screening Next-generation DNA sequencing is poised to supplant traditional genotyping methods for many genetic screening applications. But before it can do so, the technique must jump the hurdles of speed, accuracy, and cost per test. Researchers from the clinical diagnostic company Good Start Genetics, based in Cambridge, Massachusetts, report that they have developed a proprietary method that can provide cost-efficient genotyping that is as accurate as traditional methods while providing more detailed information. Noting that accuracy and reproducibility have been an issue with previously described next-gen methods, the authors explain that the new method uses commercial sequencing technology combined with probes designed to capture regions of interest in 15 well-known recessive Mendelian disorders. The sample set of 194 yielded a single-nucleotide-variant false-positive rate of 1.1 per million base pairs and one false negative. The authors do not provide precise measurements of insertion/deletion detection accuracy but report a sensitivity of 95.3% in the reference set. The company has developed an automated workflow that processes the steps from isolating DNA from blood or cell lines to delivery of a clinical report within 6 days. Of course, next-gen methods are not yet appropriate for some types of mutations, including certain large deletions and chromosomal rearrangements, but for routine testing of common Mendelian disease traits, they are rapidly gaining ground. —Karyn Hede, News Editor

Exome sequencing improves genetic diagnosis of structural fetal abnormalities revealed by ultrasound

The genetic etiology of non-aneuploid fetal structural abnormalities is typically investigated by karyotyping and array-based detection of microscopically detectable rearrangements, and submicroscopic copy-number variants (CNVs), which collectively yield a pathogenic finding in up to 10% of cases. We propose that exome sequencing may substantially increase the identification of underlying etiologies. We performed exome sequencing on a cohort of 30 non-aneuploid fetuses and neonates (along with their parents) with diverse structural abnormalities first identified by prenatal ultrasound. We identified candidate pathogenic variants with a range of inheritance models, and evaluated these in the context of detailed phenotypic information. We identified 35 de novo single-nucleotide variants (SNVs), small indels, deletions or duplications, of which three (accounting for 10% of the cohort) are highly likely to be causative. These are de novo missense variants in FGFR3 and COL2A1, and a de novo 16.8 kb deletion that includes most of OFD1. In five further cases (17%) we identified de novo or inherited recessive or X-linked variants in plausible candidate genes, which require additional validation to determine pathogenicity. Our diagnostic yield of 10% is comparable to, and supplementary to, the diagnostic yield of existing microarray testing for large chromosomal rearrangements and targeted CNV detection. The de novo nature of these events could enable couples to be counseled as to their low recurrence risk. This study outlines the way for a substantial improvement in the diagnostic yield of prenatal genetic abnormalities through the application of next-generation sequencing.

Mutational landscape of yeast mutator strains

The acquisition of mutations is relevant to every aspect of genetics, including cancer and evolution of species on Darwinian selection. Genome variations arise from rare stochastic imperfections of cellular metabolism and deficiencies in maintenance genes. Here, we established the genome-wide spectrum of mutations that accumulate in a WT and in nine Saccharomyces cerevisiae mutator strains deficient for distinct genome maintenance processes: pol32Δ and rad27Δ (replication), msh2Δ (mismatch repair), tsa1Δ (oxidative stress), mre11Δ (recombination), mec1Δ tel1Δ (DNA damage/S-phase checkpoints), pif1Δ (maintenance of mitochondrial genome and telomere length), cac1Δ cac3Δ (nucleosome deposition), and clb5Δ (cell cycle progression). This study reveals the diversity, complexity, and ultimate unique nature of each mutational spectrum, composed of punctual mutations, chromosomal structural variations, and/or aneuploidies. The mutations produced in clb5Δ/CCNB1, mec1Δ/ATR, tel1Δ/ATM, and rad27Δ/FEN1 strains extensively reshape the genome, following a trajectory dependent on previous events. It comprises the transmission of unstable genomes that lead to colony mosaicisms. This comprehensive analytical approach of mutator defects provides a model to understand how genome variations might accumulate during clonal evolution of somatic cell populations, including tumor cells.

Copy‐number variation in the pathogenesis of autism spectrum disorder

Autism spectrum disorder is a neurodevelopmental disorder present in 1% of the population, characterized by impairments in reciprocal social interaction, communication deficits and restricted patterns of behavior. Approximately 10% of the autism spectrum disorder population is thought to have large chromosomal rearrangements. Copy-number variations (CNV) alter the genome structure either by duplication or deletion of a chromosomal region. The association between CNV and autism susceptibility has become more apparent through the use of methods based on comparative genomic hybridization in screening CNV. The nature of the high CNV rate in the human genome is partly explained by non-allelic homologous recombination between flanking repeated sequences derived from multiple copies of transposons or mobile genetic elements. There are hotspots for CNV in the human genome, such as 16p11.2 and 22q11.2. Genes involved in CNV are supposed to have copy-number dose-dependent effects on the behavior of affected individuals. Animal models give insight into the possible interactions between core genetic loci and additional factors contributing to the phenotypes of each individual. If affected genes code for cellular signaling molecules, reducing the dosage in the intracellular signaling pathway may result in the malfunction of the nervous system. The genetic background of autism spectrum disorder is highly heterogenic and most common or rare CNV do not lead to autism spectrum disorders in the majority of cases, but may occasionally increase the risk of developing an autism spectrum disorder.

Interclonal Variations in the Molecular Karyotype of Trypanosoma cruzi: Chromosome Rearrangements in a Single Cell-Derived Clone of the G Strain

Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure.

Exome profiling of primary, metastatic and recurrent ovarian carcinomas in a BRCA1-positive patient

BackgroundOvarian carcinoma is a common, and often deadly, gynecological cancer. Mutations in BRCA1 and BRCA2 genes are present in at least a fifth of patients. Uncovering other genes that become mutated subsequent to BRCA1/BRCA2 inactivation during cancer development will be helpful for more effective treatments.MethodsWe performed exome sequencing on the blood, primary tumor, omental metastasis and recurrence following therapy with carboplatin and paclitaxel, from a patient carrying a BRCA1 S1841R mutation.ResultsWe observed loss of heterozygosity in the BRCA1 mutation in the primary and subsequent tumors, and somatic mutations in the TP53 and NF1 genes were identified, suggesting their role along with BRCA1 driving the tumor development. Notably, we show that exome sequencing is effective in detecting large chromosomal rearrangements such as deletions and amplifications in cancer. We found that a large deletion was present in the three tumors in the regions containing BRCA1, TP53, and NF1 mutations, and an amplification in the regions containing MYC. We did not observe the emergence of any new mutations among tumors from diagnosis to relapse after chemotherapy, suggesting that mutations already present in the primary tumor contributed to metastases and chemotherapy resistance.ConclusionsOur findings suggest that exome sequencing of matched samples from one patient is a powerful method of detecting somatic mutations and prioritizing their potential role in the development of the disease.

1866 – Novel candidate genomic loci for mental retardation

Introduction Genetic disorders underlie a significant number of cases of mental retardation (MR). However, the traditionally used routine karyotyping is not able to detect small but often clinically relevant aberrations. High-resolution genome-wide microarray technologies may help to increase the detectability of MR etiology. Objectives To improve MR diagnostics. Aims To identify the novel CNV regions and susceptibility genes in MR patients. Methods Large chromosomal rearrangements, biochemical defects, and known monogenic syndromes accompanied by MR were ruled out for 71 patients. Further the genome-wide analysis using CGH Microarray Kits 4×44K and 8×60K (Agilent Technologies, USA) was performed. Results Eight novel CNVs in 9 patients not described in healthy individuals and not associated with any disease were indentified implicating regions at 11q22.3, 5q33.1, 3p26.3, 15q11.1-q11.2, 15q22.2, 1q25.1-q25.2, 7q21.3, 2q12.3. They include candidate genes DDX10 (putative RNA helicase), IL17B (cytokine, primarily localized to neuronal cell bodies), CNTN6 (may play a role in the formation of axon connections), TLN2 (plays a role in cell adhesion and recycling of synaptic vesicles), TNR (an extracellular matrix protein, expressed primarily in the central nervous system), ASTN1 (a neuronal adhesion molecule), PON1 (detoxifies organophosphate neurotoxicants), and SLC5A7 (choline transporter in the brain and periphery). Conclusions Previously unknown CNVs, containing genes responsible for proper central nervous system functioning, were detected in 13% of patients. The novel CNVs and genes identified by aCGH may help discovering new etiological mechanisms of MR. This study was supported by European Community's Seventh Framework Programme, CHERISH (project 223692) and by Federal Program (grant 8727).

Karyotype polymorphism and chromosomal rearrangement in populations of the phytopathogenic fungus, Ascochyta rabiei

The fungus Ascochyta rabiei is the causal agent of Ascochyta blight of chickpea and the most serious threat to chickpea production. Little is currently known about the genome size or organization of A. rabiei. Given recent genome sequencing efforts, characterization of the genome at a population scale will provide a framework for genome interpretation and direction of future resequencing efforts. Electrophoretic karyotype profiles of 112 isolates from 21 countries revealed 12–16 chromosomes between 0.9 Mb and 4.6 Mb with an estimated genome size of 23 Mb–34 Mb. Three general karyotype profiles A, B, and C were defined by the arrangement of the largest chromosomes. Approximately one-third of isolates (group A) possessed a chromosome larger than 4.0 Mb that was absent from group B and C isolates. The ribosomal RNA gene (rDNA) cluster was assigned to the largest chromosome in all except four isolates (group C) whose rDNA cluster was located on the second largest chromosome (3.2 Mb). Analysis of progeny from an in vitro sexual cross between two group B isolates revealed one of 16 progeny with an rDNA-encoding chromosome larger than 4.0 Mb similar to group A isolates, even though a chromosome of this size was not present in either parent. No expansion of the rDNA cluster was detected in the progeny, indicating the increase in chromosome size was not due to an expansion in number of rDNA repeats. The karyotype of A. rabiei is relatively conserved when compared with published examples of asexual ascomycetes, but labile with the potential for large scale chromosomal rearrangements during meiosis. The results of this study will allow for the targeted sequencing of specific isolates to determine the molecular mechanisms of karyotype variation within this species.

TMEM127Screening in a Large Cohort of Patients with Pheochromocytoma and/or Paraganglioma

TMEM127 is a novel pheochromocytoma (PCC) susceptibility gene. Our aim was to clearly determine the indications for TMEM127 genetic testing in patients with PCC and/or paraganglioma (PGL). Germline DNA from 642 unrelated patients who did not carry mutations in major PCC susceptibility genes was analyzed. Five hundred fifty-nine patients were affected by PCC, 72 by PGL (22 with head and neck and 50 with thoracic or abdominal location), and 11 by both PCC and PGL. Analysis of the TMEM127 gene was performed by direct sequencing and quantitative multiplex PCR of short fluorescent fragments. In our cohort six mutations (0.9%) were identified. Three of them (p.Ala47Asp, p.Gln64HisfsX18, p.Tyr164X) were found in patients exhibiting clinical criteria for a hereditary disease (young age at diagnosis, bilateral PCC, or family history). The three others (p.Gln157X, p.Val68SerfsX13, p.Val90Met) were detected in patients with an apparently sporadic presentation. No mutation was found among patients with PGL, and no large chromosomal rearrangement spanning the TMEM127 gene was detected. Our results combined with the two previous studies suggest that direct sequencing of TMEM127 should be considered, after a negative screening of VHL, RET, SDHB, and SDHD genes, in patients with PCC.

Identification of new genetic alterations in MLH1, MSH2 and MSH6 using IHC and HRM analysis in Lynch syndrome-suspected patients

Mutations in DNA MMR genes, mainly MSH2 and MLH1, are the most frequent cause of HNPCC, an autosomal dominant predisposition to colorectal cancer and other malignancies. In our study we tested 46 unrelated patients with suspected HNPCC, who met Bethesda criteria. Tumors from probands (when available) were tested by immunohistochemistry for deficiencies in MLH1, PMS2, MSH2 and MSH6. DNA samples were analyzed using high-resolution melting (HRM). PCR amplicons were designed to scan complete MLH1, MSH2 and MSH6 coding sequences using HRM method. In the first stage of screening HRM analysis was performed by scanning 14 amplicons selected as potentially harboring most frequent mutations in Polish population (Kurzawski et al., 2005). Sequencing was used to confirm and characterize affected exons identified in HRM. Three novel deleterious mutations were found in MSH2 gene, one of them being splice acceptor site mutation in exon 5, and two of them being nonsense mutations in exons 6 and 8. Tumours from patients bearing these mutations were lacking MSH2 protein. We found also missense mutation in exon 8 of MLH1, which has not been previously reported in Polish population. Tumour from this patient exhibited weak expression of MLH1 and PMS2 proteins. In 42 patients, only unspecified variants and polymorphisms have been found so far (analysis is still in progress). In our opinion, HRM is a rapid, inexpensive and high-throughput method to prescreen for point mutation and small deletions in MMR genes. Technical aspects concerning analysis of one-replicate versus 2-replicates data will be discussed. It must be noted that HRM cannot be used alone, MLPA and array-CGH are still required for detection of large deletions and chromosome rearrangements.

Whole-Genome Sequencing and Phenotypic Analysis of Bacillus subtilis Mutants following Evolution under Conditions of Relaxed Selection for Sporulation

Little is known about how genetic variation at the nucleotide level contributes to competitive fitness within species. During a 6,000-generation study of Bacillus subtilis evolved under relaxed selection for sporulation, a new strain, designated WN716, emerged with significantly different colony and cell morphologies; loss of sporulation, competence, acetoin production, and motility; multiple auxotrophies; and increased competitive fitness (H. Maughan and W. L. Nicholson, Appl. Environ. Microbiol. 77:4105-4118, 2011). The genome of WN716 was analyzed by OpGen optical mapping, whole-genome 454 pyrosequencing, and the CLC Genomics Workbench. No large chromosomal rearrangements were found; however, 34 single-nucleotide polymorphisms (SNPs) and +1 frameshifts were identified in WN716 that resulted in amino acid changes in coding sequences of annotated genes, and 11 SNPs were located in intergenic regions. Several classes of genes were affected, including biosynthetic pathways, sporulation, competence, and DNA repair. In several cases, attempts were made to link observed phenotypes of WN716 with the discovered mutations, with various degrees of success. For example, a +1 frameshift was identified at codon 13 of sigW, the product of which (SigW) controls a regulon of genes involved in resistance to bacteriocins and membrane-damaging antibiotics. Consistent with this finding, WN716 exhibited sensitivity to fosfomycin and to a bacteriocin produced by B. subtilis subsp. spizizenii and exhibited downregulation of SigW-dependent genes on a transcriptional microarray, consistent with WN716 carrying a knockout of sigW. The results suggest that propagation of B. subtilis for less than 2,000 generations in a nutrient-rich environment where sporulation is suppressed led to rapid initiation of genomic erosion.

A novel syndrome of paediatric cataract, dysmorphism, ectodermal features, and developmental delay in Australian Aboriginal family maps to 1p35.3-p36.32.

BackgroundA novel phenotype consisting of cataract, mental retardation, erythematous skin rash and facial dysmorphism was recently described in an extended pedigree of Australian Aboriginal descent. Large scale chromosomal re-arrangements had previously been ruled out. We have conducted a genome-wide scan to map the linkage region in this family.MethodsGenome-wide linkage analysis using Single Nucleotide Polymorphism (SNP) markers on the Affymetrix 10K SNP array was conducted and analysed using MERLIN. Three positional candidate genes (ZBTB17, EPHA2 and EPHB2) were sequenced to screen for segregating mutations.ResultsUnder a fully penetrant, dominant model, the locus for this unique phenotype was mapped to chromosome 1p35.3-p36.32 with a maximum LOD score of 2.41. The critical region spans 48.7 cM between markers rs966321 and rs1441834 and encompasses 527 transcripts from 364 annotated genes. No coding mutations were identified in three positional candidate genes EPHA2, EPHB2 or ZBTB17. The region overlaps with a previously reported region for Volkmann cataract and the phenotype has similarity to that reported for 1p36 monosomy.ConclusionsThe gene for this syndrome is located in a 25.6 Mb region on 1p35.3-p36.32. The known cataract gene in this region (EPHA2) does not harbour mutations in this family, suggesting that at least one additional gene for cataract is present in this region.

Chromosomal Rearrangements Formed by rrn Recombination Do Not Improve Replichore Balance in Host-Specific Salmonella enterica Serovars

BackgroundMost of the ∼2,600 serovars of Salmonella enterica have a broad host range as well as a conserved gene order. In contrast, some Salmonella serovars are host-specific and frequently exhibit large chromosomal rearrangements from recombination between rrn operons. One hypothesis explaining these rearrangements suggests that replichore imbalance introduced from horizontal transfer of pathogenicity islands and prophages drives chromosomal rearrangements in an attempt to improve balance.Methodology/Principal FindingsThis hypothesis was directly tested by comparing the naturally-occurring chromosomal arrangement types to the theoretically possible arrangement types, and estimating their replichore balance using a calculator. In addition to previously characterized strains belonging to host-specific serovars, the arrangement types of 22 serovar Gallinarum strains was also determined. Only 48 out of 1,440 possible arrangement types were identified in 212 host-specific strains. While the replichores of most naturally-occurring arrangement types were well-balanced, most theoretical arrangement types had imbalanced replichores. Furthermore, the most common types of rearrangements did not change replichore balance.Conclusions/SignificanceThe results did not support the hypothesis that replichore imbalance causes these rearrangements, and suggest that the rearrangements could be explained by aspects of a host-specific lifestyle.

Population-based study of genetic variation in individuals with autism spectrum disorders from Croatia

BackgroundGenome-wide studies on autism spectrum disorders (ASDs) have mostly focused on large-scale population samples, but examination of rare variations in isolated populations may provide additional insights into the disease pathogenesis.MethodsAs a first step in the genetic analysis of ASD in Croatia, we characterized genetic variation in a sample of 103 subjects with ASD and 203 control individuals, who were genotyped using the Illumina HumanHap550 BeadChip. We analyzed the genetic diversity of the Croatian population and its relationship to other populations, the degree of relatedness via Runs of Homozygosity (ROHs), and the distribution of large (>500 Kb) copy number variations.ResultsCombining the Croatian cohort with several previously published populations in the FastME analysis (an alternative to Neighbor Joining) revealed that Croatian subjects cluster, as expected, with Southern Europeans; in addition, individuals from the same geographic region within Europe cluster together. Whereas Croatian subjects could be separated from a sample of healthy control subjects of European origin from North America, Croatian ASD cases and controls are well mixed. A comparison of runs of homozygosity indicated that the number and the median length of regions of homozygosity are higher for ASD subjects than for controls (p = 6 × 10-3). Furthermore, analysis of copy number variants found a higher frequency of large chromosomal rearrangements (>2 Mb) in ASD cases (5/103) than in ethnically matched control subjects (1/197, p = 0.019).ConclusionsOur findings illustrate the remarkable utility of high-density genotype data for subjects from a limited geographic area in dissecting genetic heterogeneity with respect to population and disease related variation.