Mitochondrially targeted
            ZFN
            s for selective degradation of pathogenic mitochondrial genomes bearing large‐scale deletions or point mutations

AbstractThe mechanisms driving clonal heterogeneity and evolution in relapsed pediatric acute lymphoblastic leukemia (ALL) are not fully understood. We performed whole genome sequencing of samples collected at diagnosis, relapse(s) and remission from 29 Nordic patients. Somatic point mutations and large-scale structural variants were called using individually matched remission samples as controls, and allelic expression of the mutations was assessed in ALL cells using RNA-sequencing. We observed an increased burden of somatic mutations at relapse, compared to diagnosis, and at second relapse compared to first relapse. In addition to 29 known ALL driver genes, of which nine genes carried recurrent protein-coding mutations in our sample set, we identified putative non-protein coding mutations in regulatory regions of seven additional genes that have not previously been described in ALL. Cluster analysis of hundreds of somatic mutations per sample revealed three distinct evolutionary trajectories during ALL progression from diagnosis to relapse. The evolutionary trajectories provide insight into the mutational mechanisms leading relapse in ALL and could offer biomarkers for improved risk prediction in individual patients.

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SVFX: a machine-learning framework to quantify the pathogenicity of structural variants

10.1101/739474 ◽

2019 ◽

Cited By ~ 2

Author(s):

Sushant Kumar ◽

Arif Harmanci ◽

Jagath Vytheeswaran ◽

Mark B. Gerstein

Keyword(s):

Machine Learning ◽

Large Scale ◽

Three Dimensional ◽

Point Mutations ◽

Dimensional Structure ◽

Rapid Decline ◽

Ras Signaling ◽

Cancer Genes ◽

Structural Variations ◽

Pathogenic Variants

AbstractA rapid decline in sequencing cost has made large-scale genome sequencing studies feasible. One of the fundamental goals of these studies is to catalog all pathogenic variants. Numerous methods and tools have been developed to interpret point mutations and small insertions and deletions. However, there is a lack of approaches for identifying pathogenic genomic structural variations (SVs). That said, SVs are known to play a crucial role in many diseases by altering the sequence and three-dimensional structure of the genome. Previous studies have suggested a complex interplay of genomic and epigenomic features in the emergence and distribution of SVs. However, the exact mechanism of pathogenesis for SVs in different diseases is not straightforward to decipher. Thus, we built an agnostic machine-learning-based workflow, called SVFX, to assign a “pathogenicity score” to somatic and germline SVs in various diseases. In particular, we generated somatic and germline training models, which included genomic, epigenomic, and conservation-based features for SV call sets in diseased and healthy individuals. We then applied SVFX to SVs in six different cancer cohorts and a cardiovascular disease (CVD) cohort. Overall, SVFX achieved high accuracy in identifying pathogenic SVs. Moreover, we found that predicted pathogenic SVs in cancer cohorts were enriched among known cancer genes and many cancer-related pathways (including Wnt signaling, Ras signaling, DNA repair, and ubiquitin-mediated proteolysis). Finally, we note that SVFX is flexible and can be easily extended to identify pathogenic SVs in additional disease cohorts.

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Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance

Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques ◽

10.1017/s0317167100007733 ◽

2009 ◽

Vol 36 (4) ◽

pp. 409-428 ◽

Cited By ~ 25

Author(s):

Josef Finsterer

Keyword(s):

Ataxia Telangiectasia ◽

Large Scale ◽

Autosomal Recessive ◽

Autosomal Dominant ◽

Late Onset ◽

Fragile X ◽

Axonal Neuropathy ◽

Friedreich Ataxia ◽

Point Mutations ◽

Spinocerebellar Ataxias

Heredoataxias are a group of genetic disorders with a cerebellar syndrome as the leading clinical manifestation. The current classification distinguishes heredoataxias according to the trait of inheritance into autosomal dominant, autosomal recessive, X-linked, and maternally inherited heredoataxias. The autosomal dominant heredoataxias are separated into spinocerebellar ataxias (SCA1-8, 10-15, 17-23, 25-30, and dentato-rubro-pallido-luysian atrophy), episodic ataxias (EA1-7), and autosomal dominant mitochondrial heredoataxias (Leigh syndrome, MIRAS, ADOAD, and AD-CPEO). The autosomal recessive ataxias are separated into Friedreich ataxia, ataxia due to vitamin E deficiency, ataxia due to Abeta-lipoproteinemia, Refsum disease, late-onset Tay-Sachs disease, cerebrotendineous xanthomatosis, spinocerebellar ataxia with axonal neuropathy, ataxia telangiectasia, ataxia telangiectasia-like disorder, ataxia with oculomotor apraxia 1 and 2, spastic ataxia of Charlevoix-Saguenay, Cayman ataxia, Marinesco-Sjögren syndrome, and autosomal recessive mitochondrial ataxias (AR-CPEO, SANDO, SCAE, AHS, IOSCA, MEMSA, LBSL CoQ-deficiency, PDC-deficiency). Only two of the heredoataxias, fragile X/tremor/ataxia syndrome, and XLSA/A are transmitted via an X-linked trait. Maternally inherited heredoataxias are due to point mutations in genes encoding for tRNAs, rRNAs, respiratory chain subunits or single large scale deletions/duplications of the mitochondrial DNA and include MELAS, MERRF, KSS, PS, MILS, NARP, and non-syndromic mitochondrial disorders. Treatment of heredoataxias is symptomatic and supportive and may have a beneficial effect in single patients.**Please see page 424 for abbreviation list.

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New Frontiers inSchistosomaGenomics and Transcriptomics

Journal of Parasitology Research ◽

10.1155/2012/849132 ◽

2012 ◽

Vol 2012 ◽

pp. 1-11 ◽

Cited By ~ 9

Author(s):

Laila A. Nahum ◽

Marina M. Mourão ◽

Guilherme Oliveira

Keyword(s):

Large Scale ◽

Biological Research ◽

Mitochondrial Genomes ◽

Future Directions ◽

Important Species ◽

Blood Flukes ◽

Causative Agents ◽

Mitochondrial Sequences ◽

Modern Technologies

Schistosomes are digenean blood flukes of aves and mammals comprising 23 species. Some species are causative agents of human schistosomiasis, the second major neglected disease affecting over 230 million people worldwide. Modern technologies including the sequencing and characterization of nucleic acids and proteins have allowed large-scale analyses of parasites and hosts, opening new frontiers in biological research with potential biomedical and biotechnological applications. Nuclear genomes of the three most socioeconomically important species (S. haematobium,S. japonicum, andS. mansoni) have been sequenced and are under intense investigation. Mitochondrial genomes of sixSchistosomaspecies have also been completely sequenced and analysed from an evolutionary perspective. Furthermore, DNA barcoding of mitochondrial sequences is used for biodiversity assessment of schistosomes. Despite the efforts in the characterization ofSchistosomagenomes and transcriptomes, many questions regarding the biology and evolution of this important taxon remain unanswered. This paper aims to discuss some advances in the schistosome research with emphasis on genomics and transcriptomics. It also aims to discuss the main challenges of the current research and to point out some future directions in schistosome studies.

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Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker

Journal of Bacteriology ◽

10.1128/jb.02384-14 ◽

2015 ◽

Vol 197 (6) ◽

pp. 1135-1144 ◽

Cited By ~ 21

Author(s):

Jennifer F. Carr ◽

Michael E. Danziger ◽

Athena L. Huang ◽

Albert E. Dahlberg ◽

Steven T. Gregory

Keyword(s):

Resistance Gene ◽

Large Scale ◽

Genetic Manipulation ◽

Thermus Thermophilus ◽

Point Mutations ◽

Trna Synthetase ◽

Biological Research ◽

Lethal Mutant ◽

Α Subunit ◽

Content Type

ABSTRACTThermus thermophilusis an extremely thermophilic bacterium that is widely used as a model thermophile, in large part due to its amenability to genetic manipulation. Here we describe a system for the introduction of genomic point mutations or deletions using a counterselectable marker consisting of a conditionally lethal mutant allele ofpheSencoding the phenylalanyl-tRNA synthetase α-subunit. Mutant PheS with an A294G amino acid substitution renders cells sensitive to the phenylalanine analogp-chlorophenylalanine. Insertion of the mutantpheSallele via a linked kanamycin resistance gene into a chromosomal locus provides a gene replacement intermediate that can be removed by homologous recombination usingp-chlorophenylalanine as a counterselective agent. This selection is suitable for the sequential introduction of multiple mutations to produce a final strain unmarked by an antibiotic resistance gene. We demonstrated the utility of this method by constructing strains bearing either a point mutation in or a precise deletion of therrsBgene encoding 16S rRNA. We also used this selection to identify spontaneous, large-scale deletions in the pTT27 megaplasmid, apparently mediated by either of theT. thermophilusinsertion elements ISTth7and ISTth8. One such deletion removed 121 kb, including 118 genes, or over half of pTT27, including multiple sugar hydrolase genes, and facilitated the development of a plasmid-encoded reporter system based on β-galactosidase. The ability to introduce mutations ranging from single base substitutions to large-scale deletions provides a potentially powerful tool for engineering the genome ofT. thermophilusand possibly other thermophiles as well.IMPORTANCEThermus thermophilusis an extreme thermophile that has played an important part in the development of both biotechnology and basic biological research. Its suitability as a genetic model system is established by its natural competence for transformation, but the scarcity of genetic tools limits the kinds of manipulations that can currently be performed. We have developed a counterselectable marker that allows the introduction of unmarked deletions and point mutations into theT. thermophilusgenome. We find that this marker can also be used to select large chromosomal deletions apparently resulting from aberrant transposition of endogenous insertion sequences. This system has the potential to advance the genetic manipulation of this important model organism.

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Mitochondrial DNA-related diseases associated with single large-scale deletions and point mutations

The Human Mitochondrial Genome ◽

10.1016/b978-0-12-819656-4.00014-0 ◽

2020 ◽

pp. 353-374

Author(s):

Robert D.S. Pitceathly ◽

Shamima Rahman

Keyword(s):

Mitochondrial Dna ◽

Large Scale ◽

Point Mutations

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Denaturing-HPLC-Based Assay for Detection of ABL Mutations in Chronic Myeloid Leukemia Patients Resistant to Imatinib

Clinical Chemistry ◽

10.1373/clinchem.2004.031112 ◽

2004 ◽

Vol 50 (7) ◽

pp. 1205-1213 ◽

Cited By ~ 85

Author(s):

Simona Soverini ◽

Giovanni Martinelli ◽

Marilina Amabile ◽

Angela Poerio ◽

Michele Bianchini ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Large Scale ◽

Kinase Inhibitor ◽

Direct Sequencing ◽

Point Mutations ◽

Kinase Domain ◽

Imatinib Treatment ◽

Abl Tyrosine Kinase ◽

Reverse Transcription Pcr

Abstract Background: Despite the efficacy of the BCR-ABL tyrosine kinase inhibitor Imatinib mesylate for the treatment of chronic myeloid leukemia (CML), resistance has been observed in a proportion of cases, especially those with advanced stages of the disease. Point mutations within the ABL kinase domain are emerging as the most frequent mechanism for reactivation of kinase activity within the leukemic clone. Methods: We developed a denaturing-HPLC (D-HPLC)-based assay for screening for ABL point mutations. For each sample, two partially overlapping fragments of 393 and 482 bp corresponding to the kinase domain were amplified by nested reverse transcription-PCR and analyzed under selected temperature and acetonitrile gradient conditions. Fifty-one bone marrow and/or peripheral blood specimens from 27 CML patients who showed cytogenetic resistance to Imatinib were screened in parallel by D-HPLC and by direct sequencing. Results: In 12 of 27 (44%) patients, D-HPLC showed an abnormal elution profile suggesting the presence of a nucleotide change. Direct sequencing confirmed the presence of a point mutation in all cases. Conversely, all samples scored as wild type by D-HPLC showed no evidence of mutations by direct sequencing. In two cases, novel amino acid substitutions at codons already known for being hot-spots of mutation were identified (F311I and E355D). Conclusions: The proposed D-HPLC-based assay is highly specific and at least as sensitive as sequencing; with respect to the latter, it provides a much faster and less expensive semiautomated system for mutational screening. It may therefore potentially be a valuable tool for regular, large-scale testing of patients undergoing Imatinib treatment.

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