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G3 (Bethesda, Md.) Aug 2016To better understand maize endosperm filling and maturation, we used γ-irradiation of the B73 maize reference line to generate mutants with opaque endosperm and reduced...
To better understand maize endosperm filling and maturation, we used γ-irradiation of the B73 maize reference line to generate mutants with opaque endosperm and reduced kernel fill phenotypes, and created a population of 1788 lines including 39 Mo17 × F2s showing stable, segregating, and viable kernel phenotypes. For molecular characterization of the mutants, we developed a novel functional genomics platform that combined bulked segregant RNA and exome sequencing (BSREx-seq) to map causative mutations and identify candidate genes within mapping intervals. To exemplify the utility of the mutants and provide proof-of-concept for the bioinformatics platform, we present detailed characterization of line 937, an opaque mutant harboring a 6203 bp in-frame deletion covering six exons within the Opaque-1 gene. In addition, we describe mutant line 146 which contains a 4.8 kb intragene deletion within the Sugary-1 gene and line 916 in which an 8.6 kb deletion knocks out a Cyclin A2 gene. The publically available algorithm developed in this work improves the identification of causative deletions and its corresponding gaps within mapping peaks. This study demonstrates the utility of γ-irradiation for forward genetics in large nondense genomes such as maize since deletions often affect single genes. Furthermore, we show how this classical mutagenesis method becomes applicable for functional genomics when combined with state-of-the-art genomics tools.
Topics: Endosperm; Exome; Genetic Association Studies; Genome, Plant; Genomics; Phenotype; Plant Proteins; Sequence Deletion; Zea mays
PubMed: 27261000
DOI: 10.1534/g3.116.030528 -
Nature Aug 2004Mutations have pivotal functions in the onset of genetic diseases and are the fundamental substrate for evolution. However, present estimates of the spontaneous mutation...
Mutations have pivotal functions in the onset of genetic diseases and are the fundamental substrate for evolution. However, present estimates of the spontaneous mutation rate and spectrum are derived from indirect and biased measurements. For instance, mutation rate estimates for Caenorhabditis elegans are extrapolated from observations on a few genetic loci with visible phenotypes and vary over an order of magnitude. Alternative approaches in mammals, relying on phylogenetic comparisons of pseudogene loci and fourfold degenerate codon positions, suffer from uncertainties in the actual number of generations separating the compared species and the inability to exclude biases associated with natural selection. Here we provide a direct and unbiased estimate of the nuclear mutation rate and its molecular spectrum with a set of C. elegans mutation-accumulation lines that reveal a mutation rate about tenfold higher than previous indirect estimates and an excess of insertions over deletions. Because deletions dominate patterns of C. elegans pseudogene variation, our observations indicate that natural selection might be significant in promoting small genome size, and challenge the prevalent assumption that pseudogene divergence accurately reflects the spontaneous mutation spectrum.
Topics: Animals; Base Sequence; Caenorhabditis elegans; Cell Nucleus; DNA Mutational Analysis; Genome; Genomics; Kinetics; Mutagenesis, Insertional; Mutation; Phylogeny; Pseudogenes; Sequence Deletion
PubMed: 15295601
DOI: 10.1038/nature02697 -
BMC Medical Genetics Mar 2019Congenital cataract is the most common cause of blindness among children worldwide. The aim of this study was to identify causative mutations in a Chinese family with...
BACKGROUND
Congenital cataract is the most common cause of blindness among children worldwide. The aim of this study was to identify causative mutations in a Chinese family with isolated autosomal dominant posterior subcapsular cataract.
METHODS
The proband and her parents underwent full ophthalmological examinations. DNA was extracted from the participants' peripheral venous blood. The mutation was identified via panel-based next-generation sequencing (NGS) and was validated via Sanger sequencing.
RESULTS
Posterior subcapsular lenticular opacity was observed in both of the proband's eyes. The novel deletion mutation c.797_814del, p.Ser266_Ala271del in the PITX3 gene was identified in the proband and her father. This mutation is located within the otp/aristaless/rax (OAR) domain at the COOH-terminus of the protein, which functions in DNA binding and transactivation. This mutation would result in a deletion of 6 amino acid residues at the C terminal of the protein.
CONCLUSIONS
The mutation c.797_814del, p.Ser266_Ala271del is a novel mutation in the conserved DNA-binding OAR domain of PITX3 that causes congenital cataract.
Topics: Cataract; Female; Genetic Association Studies; Genetic Predisposition to Disease; High-Throughput Nucleotide Sequencing; Homeodomain Proteins; Humans; Male; Paternal Inheritance; Pedigree; Protein Domains; Sequence Analysis, DNA; Sequence Deletion; Transcription Factors
PubMed: 30894134
DOI: 10.1186/s12881-019-0782-2 -
Scientific Reports Feb 2022Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (P allele) comprised of a 212 bp...
Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (P allele) comprised of a 212 bp duplicated DNA sequence replacing a 10-bp sequence at the polled locus is associated with the hornless phenotype (polled) in cattle. To test the hypothesis that the 10 bp deletion alone is sufficient to result in polled, a CRISPR-Cas9 dual guide RNA approach was optimized to delete a 133 bp region including the 10 bp sequence. Timing of ribonucleoprotein complex injections at various hours post insemination (hpi) (6, 8, and 18 hpi) as well as in vitro transcribed (IVT) vs synthetic gRNAs were compared. Embryos injected 6 hpi had a significantly higher deletion rate (53%) compared to those injected 8 (12%) and 18 hpi (7%), and synthetic gRNAs had a significantly higher deletion rate (84%) compared to IVT gRNAs (53%). Embryo transfers were performed, and bovine fetuses were harvested between 3 and 5 months of gestation. All fetuses had mutations at the target site, with two of the seven having biallelic deletions, and yet they displayed horn bud development indicating that the 10 bp deletion alone is not sufficient to result in the polled phenotype.
Topics: Animals; CRISPR-Cas Systems; Cattle; Dairying; Embryo Transfer; Fetus; Genotype; Horns; Phenotype; RNA, Guide, CRISPR-Cas Systems; Sequence Deletion
PubMed: 35136148
DOI: 10.1038/s41598-022-06118-6 -
Genes Feb 2019Although gene expression can vary extensively within and among populations, the genetic basis of this variation and the evolutionary forces that maintain it are largely...
Although gene expression can vary extensively within and among populations, the genetic basis of this variation and the evolutionary forces that maintain it are largely unknown. In , a 49-bp insertion/deletion (indel) polymorphism in the () gene is associated with variation in expression and oxidative stress tolerance. To better understand the functional and evolutionary significance of this polymorphism, we investigated it in several worldwide populations. In a German population, the deletion was present at a high and stable frequency over multiple seasons and years, and was associated with increased expression. There was, however, no evidence that the polymorphism was maintained by overdominant, seasonally fluctuating, or sexually antagonistic selection. The deletion was rare in a population from the species' ancestral range in sub-Saharan Africa and is likely the result of non-African admixture, suggesting that it spread to high frequency following the species' out-of-Africa expansion. Using data from a North American population, we found that the deletion was associated with expression and tolerance to oxidative stress induced by menadione sodium bisulfite. Our results are consistent with the deletion being selectively favored in temperate populations due to the increased expression and oxidative stress tolerance that it confers.
Topics: Adaptation, Physiological; Africa South of the Sahara; Alleles; Animals; Drosophila melanogaster; Female; Gene Expression Regulation; Genetics, Population; INDEL Mutation; Male; Metallothionein; Oxidative Stress; Polymorphism, Genetic; Regulatory Sequences, Nucleic Acid; Sequence Deletion; Stress, Physiological
PubMed: 30769915
DOI: 10.3390/genes10020147 -
PLoS Genetics Apr 2017Preterm birth (PTB) affects ~12% of pregnancies in the US. Despite its high mortality and morbidity, the molecular etiology underlying PTB has been unclear. Numerous...
Preterm birth (PTB) affects ~12% of pregnancies in the US. Despite its high mortality and morbidity, the molecular etiology underlying PTB has been unclear. Numerous studies have been devoted to identifying genetic factors in maternal and fetal genomes, but so far few genomic loci have been associated with PTB. By analyzing whole-genome sequencing data from 816 trio families, for the first time, we observed the role of fetal de novo mutations in PTB. We observed a significant increase in de novo mutation burden in PTB fetal genomes. Our genomic analyses further revealed that affected genes by PTB de novo mutations were dosage sensitive, intolerant to genomic deletions, and their mouse orthologs were likely developmentally essential. These genes were significantly involved in early fetal brain development, which was further supported by our analysis of copy number variants identified from an independent PTB cohort. Our study indicates a new mechanism in PTB occurrence independently contributed from fetal genomes, and thus opens a new avenue for future PTB research.
Topics: Animals; Brain; DNA Copy Number Variations; Female; Fetal Development; Fetus; Frameshift Mutation; Genome, Human; Humans; Mice; Pregnancy; Premature Birth; Risk Factors; Sequence Deletion
PubMed: 28388617
DOI: 10.1371/journal.pgen.1006689 -
Scientific Reports Oct 2016FOXP2 is the major gene associated with severe, persistent, developmental speech and language disorders. While studies in the original family in which a FOXP2 mutation...
FOXP2 is the major gene associated with severe, persistent, developmental speech and language disorders. While studies in the original family in which a FOXP2 mutation was found showed volume reduction and reduced activation in core language and speech networks, there have been no imaging studies of different FOXP2 mutations. We conducted a multimodal MRI study in an eight-year-old boy (A-II) with a de novo FOXP2 intragenic deletion. A-II showed marked bilateral volume reductions in the hippocampus, thalamus, globus pallidus, and caudate nucleus compared with 26 control males (effect sizes from -1 to -3). He showed no detectable functional MRI activity when repeating nonsense words. The hippocampus is implicated for the first time in FOXP2 diseases. We conclude that FOXP2 anomaly is either directly or indirectly associated with atypical development of widespread subcortical networks early in life.
Topics: Child; Codon, Nonsense; Forkhead Transcription Factors; Hippocampus; Humans; Language; Language Disorders; Magnetic Resonance Imaging; Male; Mutation; Neuroimaging; Sequence Deletion; Speech
PubMed: 27734906
DOI: 10.1038/srep35192 -
International Journal of Molecular... Jun 2021Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed...
Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing.
Topics: Base Sequence; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Cleavage; DNA End-Joining Repair; HEK293 Cells; HeLa Cells; Humans; Mutation; Oligonucleotides; RNA, Guide, CRISPR-Cas Systems; Sequence Deletion
PubMed: 34203807
DOI: 10.3390/ijms22126397 -
BMC Genomics May 2010Osteopetrosis is a skeletal disorder of humans and animals characterized by the formation of overly dense bones, resulting from a deficiency in the number and/or...
BACKGROUND
Osteopetrosis is a skeletal disorder of humans and animals characterized by the formation of overly dense bones, resulting from a deficiency in the number and/or function of bone-resorbing osteoclast cells. In cattle, osteopetrosis can either be induced during gestation by viral infection of the dam, or inherited as a recessive defect. Genetically affected calves are typically aborted late in gestation, display skull deformities and exhibit a marked reduction of osteoclasts. Although mutations in several genes are associated with osteopetrosis in humans and mice, the genetic basis of the cattle disorder was previously unknown.
RESULTS
We have conducted a whole-genome association analysis to identify the mutation responsible for inherited osteopetrosis in Red Angus cattle. Analysis of >54,000 SNP genotypes for each of seven affected calves and nine control animals localized the defective gene to the telomeric end of bovine chromosome 4 (BTA4). Homozygosity analysis refined the interval to a 3.4-Mb region containing the SLC4A2 gene, encoding an anion exchanger protein necessary for proper osteoclast function. Examination of SLC4A2 from normal and affected animals revealed a approximately 2.8-kb deletion mutation in affected calves that encompasses exon 2 and nearly half of exon 3, predicted to prevent normal protein function. Analysis of RNA from a proven heterozygous individual confirmed the presence of transcripts lacking exons 2 and 3, in addition to normal transcripts. Genotyping of additional animals demonstrated complete concordance of the homozygous deletion genotype with the osteopetrosis phenotype. Histological examination of affected tissues revealed scarce, morphologically abnormal osteoclasts displaying evidence of apoptosis.
CONCLUSIONS
These results indicate that a deletion mutation within bovine SLC4A2 is associated with osteopetrosis in Red Angus cattle. Loss of SLC4A2 function appears to induce premature cell death, and likely results in cytoplasmic alkalinization of osteoclasts which, in turn, may disrupt acidification of resorption lacunae.
Topics: Animals; Anion Transport Proteins; Antiporters; Carrier State; Cattle; Chloride-Bicarbonate Antiporters; Female; Genetic Loci; Homozygote; Humans; Male; Mice; Osteopetrosis; Phenotype; RNA, Messenger; SLC4A Proteins; Sequence Deletion
PubMed: 20507629
DOI: 10.1186/1471-2164-11-337 -
Nature Communications Feb 2019Mitochondrial DNA (mtDNA) deletions are associated with mitochondrial disease, and also accumulate during normal human ageing. The mechanisms underlying mtDNA deletions...
Mitochondrial DNA (mtDNA) deletions are associated with mitochondrial disease, and also accumulate during normal human ageing. The mechanisms underlying mtDNA deletions remain unknown although several models have been proposed. Here we use deep sequencing to characterize abundant mtDNA deletions in patients with mutations in mitochondrial DNA replication factors, and show that these have distinct directionality and repeat characteristics. Furthermore, we recreate the deletion formation process in vitro using only purified mitochondrial proteins and defined DNA templates. Based on our in vivo and in vitro findings, we conclude that mtDNA deletion formation involves copy-choice recombination during replication of the mtDNA light strand.
Topics: Blotting, Southern; DNA Replication; DNA, Mitochondrial; Humans; Mitochondrial Proteins; Mutation; Sequence Deletion
PubMed: 30770810
DOI: 10.1038/s41467-019-08673-5