Publications by authors named "Shyam Solanki"

12 Publications

  • Page 1 of 1

Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability.

PLoS Genet 2021 12 16;17(12):e1009473. Epub 2021 Dec 16.

Department of Crop and Soil Sciences, Washington State University, Pullman, Washington, United States of America.

Disease lesion mimic mutants (DLMMs) are characterized by the spontaneous development of necrotic spots with various phenotypes designated as necrotic (nec) mutants in barley. The nec mutants were traditionally considered to have aberrant regulation of programmed cell death (PCD) pathways, which have roles in plant immunity and development. Most barley nec3 mutants express cream to orange necrotic lesions contrasting them from typical spontaneous DLMMs that develop dark pigmented lesions indicative of serotonin/phenolics deposition. Barley nec3 mutants grown under sterile conditions did not exhibit necrotic phenotypes until inoculated with adapted pathogens, suggesting that they are not typical DLMMs. The F2 progeny of a cross between nec3-γ1 and variety Quest segregated as a single recessive susceptibility gene post-inoculation with Bipolaris sorokiniana, the causal agent of the disease spot blotch. Nec3 was genetically delimited to 0.14 cM representing 16.5 megabases of physical sequence containing 149 annotated high confidence genes. RNAseq and comparative analysis of the wild type and five independent nec3 mutants identified a single candidate cytochrome P450 gene (HORVU.MOREX.r2.6HG0460850) that was validated as nec3 by independent mutations that result in predicted nonfunctional proteins. Histology studies determined that nec3 mutants had an unstable cutin layer that disrupted normal Bipolaris sorokiniana germ tube development.
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http://dx.doi.org/10.1371/journal.pgen.1009473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8769293PMC
December 2021

Genome-Wide Association Mapping for Yield and Related Traits Under Drought Stressed and Non-stressed Environments in Wheat.

Front Genet 2021 22;12:649988. Epub 2021 Jun 22.

Institute of Plant Breeding, Genetics, and Genomics, University of Georgia, Griffin, GA, United States.

Understanding the genetics of drought tolerance in hard red spring wheat (HRSW) in northern USA is a prerequisite for developing drought-tolerant cultivars for this region. An association mapping (AM) study for drought tolerance in spring wheat in northern USA was undertaken using 361 wheat genotypes and Infinium 90K single-nucleotide polymorphism (SNP) assay. The genotypes were evaluated in nine different locations of North Dakota (ND) for plant height (PH), days to heading (DH), yield (YLD), test weight (TW), and thousand kernel weight (TKW) under rain-fed conditions. Rainfall data and soil type of the locations were used to assess drought conditions. A mixed linear model (MLM), which accounts for population structure and kinship (PC+K), was used for marker-trait association. A total of 69 consistent QTL involved with drought tolerance-related traits were identified, with ≤ 0.001. Chromosomes 1A, 3A, 3B, 4B, 4D, 5B, 6A, and 6B were identified to harbor major QTL for drought tolerance. Six potential novel QTL were identified on chromosomes 3D, 4A, 5B, 7A, and 7B. The novel QTL were identified for DH, PH, and TKW. The findings of this study can be used in marker-assisted selection (MAS) for drought-tolerance breeding in spring wheat.
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http://dx.doi.org/10.3389/fgene.2021.649988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8258415PMC
June 2021

Identification of Main-Effect and Environmental Interaction QTL and Their Candidate Genes for Drought Tolerance in a Wheat RIL Population Between Two Elite Spring Cultivars.

Front Genet 2021 17;12:656037. Epub 2021 Jun 17.

Institute of Plant Breeding, Genetics, and Genomics, University of Georgia, Griffin, GA, United States.

Understanding the genetics of drought tolerance can expedite the development of drought-tolerant cultivars in wheat. In this study, we dissected the genetics of drought tolerance in spring wheat using a recombinant inbred line (RIL) population derived from a cross between a drought-tolerant cultivar, 'Reeder' (PI613586), and a high-yielding but drought-susceptible cultivar, 'Albany.' The RIL population was evaluated for grain yield (YLD), grain volume weight (GVW), thousand kernel weight (TKW), plant height (PH), and days to heading (DH) at nine different environments. The Infinium 90 k-based high-density genetic map was generated using 10,657 polymorphic SNP markers representing 2,057 unique loci. Quantitative trait loci (QTL) analysis detected a total of 11 consistent QTL for drought tolerance-related traits. Of these, six QTL were exclusively identified in drought-prone environments, and five were constitutive QTL (identified under both drought and normal conditions). One major QTL on chromosome 7B was identified exclusively under drought environments and explained 13.6% of the phenotypic variation (PV) for YLD. Two other major QTL were detected, one each on chromosomes 7B and 2B under drought-prone environments, and explained 14.86 and 13.94% of phenotypic variation for GVW and YLD, respectively. One novel QTL for drought tolerance was identified on chromosome 2D. expression analysis of candidate genes underlaying the exclusive QTLs associated with drought stress identified the enrichment of ribosomal and chloroplast photosynthesis-associated proteins showing the most expression variability, thus possibly contributing to stress response by modulating the glycosyltransferase () and hexosyltransferase () unique genes present in QTL 21 and 24, respectively. While both parents contributed favorable alleles to these QTL, unexpectedly, the high-yielding and less drought-tolerant parent contributed desirable alleles for drought tolerance at four out of six loci. Regardless of the origin, all QTL with significant drought tolerance could assist significantly in the development of drought-tolerant wheat cultivars, using genomics-assisted breeding approaches.
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http://dx.doi.org/10.3389/fgene.2021.656037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8249774PMC
June 2021

Expansion of Internal Hyphal Growth in Fusarium Head Blight-Infected Grains Contributes to the Elevated Mycotoxin Production During the Malting Process.

Mol Plant Microbe Interact 2021 Jul 9;34(7):793-802. Epub 2021 Aug 9.

Department of Plant Sciences, North Dakota State University, Fargo, ND 58108, U.S.A.

Fusarium head blight (FHB) and the occurrence of mycotoxins is the largest food safety threat to malting and brewing grains. Worldwide surveys of commercial beers have reported that the trichothecene mycotoxin deoxynivalenol (DON) is the most frequent contaminant in beer. Although the DON content of grain generally declines during steeping due to its solubilization, spp. can continue to grow and produce DON from steeping through the early kilning stage of malting. DON present on malt is largely extracted into beer. The objective of the current study was to localize the growth of spp. within FHB-infected kernels by developing an improved method and to associate fungal growth with the production of DON during malting. FHB-infected barley, wheat, rye, and triticale grains that exhibited large increases in the amount of DNA and trichothecene mycotoxins following malting were screened for hyphal localization. The growth of fungal hyphae associated with grain and malt was imaged by scanning electron microscopy and confocal laser-scanning microscopy assisted with WGA-Alexa Fluor 488 staining, respectively. In barley, hyphae were present on or within the husk, vascular bundle, and pericarp cavities. Following malting, vast hyphal growth was observed not only in these regions but also in the aleurone layer, endosperm, and embryo. Extensive fungal growth was also observed following malting of wheat, rye, and triticale. However, these grains already had an extensive internal presence of hyphae in the unmalted grain, thus representing an enhanced chance of fungal expansion during the malting.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
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http://dx.doi.org/10.1094/MPMI-01-21-0024-RDOI Listing
July 2021

The Barley Transcription Factor Is Required for Resistance Against f. .

Front Genet 2020 15;11:601500. Epub 2021 Jan 15.

Department of Plant Pathology, North Dakota State University, Fargo, ND, United States.

Barley is an important cereal crop worldwide because of its use in the brewing and distilling industry. However, adequate supplies of quality malting barley are threatened by global climate change due to drought in some regions and excess precipitation in others, which facilitates epidemics caused by fungal pathogens. The disease net form net blotch caused by the necrotrophic fungal pathogen f. () has emerged as a global threat to barley production and diverse populations of have shown a capacity to overcome deployed genetic resistances. The barley line CI5791 exhibits remarkably effective resistance to diverse isolates from around the world that maps to two major QTL on chromosomes 3H and 6H. To identify genes involved in this effective resistance, CI5791 seed were γ-irradiated and two mutants, designated CI5791-γ3 and CI5791-γ8, with compromised resistance were identified from an M population. Phenotyping of CI5791-γ3 and -γ8 × Heartland F populations showed three resistant to one susceptible segregation ratios and CI5791-γ3 × -γ8 F individuals were susceptible, thus these independent mutants are in a single allelic gene. Thirty-four homozygous mutant (susceptible) CI5791-γ3 × Heartland F individuals, representing 68 recombinant gametes, were genotyped via PCR genotype by sequencing. The data were used for single marker regression mapping placing the mutation on chromosome 3H within an approximate 75 cM interval encompassing the 3H CI5791 resistance QTL. Sequencing of the mutants and wild-type (WT) CI5791 genomic DNA following exome capture identified independent mutations of the transcription factor located on chromosome 3H at ∼50.7 cM, within the genetically delimited region. Post transcriptional gene silencing of in barley line CI5791 resulted in susceptibility, confirming that it functions in NFNB resistance, validating it as the gene underlying the mutant phenotypes. Allele analysis and transcript regulation of from resistant and susceptible lines revealed sequence identity and upregulation upon pathogen challenge in all genotypes analyzed, suggesting a conserved transcription factor is involved in the defense against the necrotrophic pathogen. We hypothesize that functions as a conserved signaling component of defense mechanisms that restricts growth in barley.
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http://dx.doi.org/10.3389/fgene.2020.601500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844392PMC
January 2021

Visualization of spatial gene expression in plants by modified RNAscope fluorescent in situ hybridization.

Plant Methods 2020 15;16:71. Epub 2020 May 15.

1Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99163 USA.

Background: In situ analysis of biomarkers such as DNA, RNA and proteins are important for research and diagnostic purposes. At the RNA level, plant gene expression studies rely on qPCR, RNAseq and probe-based in situ hybridization (ISH). However, for ISH experiments poor stability of RNA and RNA based probes commonly results in poor detection or poor reproducibility. Recently, the development and availability of the RNAscope RNA-ISH method addressed these problems by novel signal amplification and background suppression. This method is capable of simultaneous detection of multiple target RNAs down to the single molecule level in individual cells, allowing researchers to study spatio-temporal patterning of gene expression. However, this method has not been optimized thus poorly utilized for plant specific gene expression studies which would allow for fluorescent multiplex detection. Here we provide a step-by-step method for sample collection and pretreatment optimization to perform the RNAscope assay in the leaf tissues of model monocot plant barley. We have shown the spatial distribution pattern of and the low expressed disease resistance gene in leaf tissue sections of barley and discuss precautions that should be followed during image analysis.

Results: We have shown the ubiquitous and predominantly stomatal guard cell associated subsidiary cell expressed expression pattern in barley leaf sections and described the improve RNAscope methodology suitable for plant tissues using confocal laser microscope. By addressing the problems in the sample collection and incorporating additional sample backing steps we have significantly reduced the section detachment and experiment failure problems. Further, by reducing the time of protease treatment, we minimized the sample disintegration due to over digestion of barley tissues.

Conclusions: RNAscope multiplex fluorescent RNA-ISH detection is well described and adapted for animal tissue samples, however due to morphological and structural differences in the plant tissues the standard protocol is deficient and required optimization. Utilizing barley specific and RNA probes we report an optimized method which can be used for RNAscope detection to determine the spatial expression and semi-quantification of target RNAs. This optimized method will be immensely useful in other plant species such as the widely utilized Arabidopsis.
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http://dx.doi.org/10.1186/s13007-020-00614-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229616PMC
May 2020

Transcriptome-wide association study identifies putative elicitors/suppressor of Puccinia graminis f. sp. tritici that modulate barley rpg4-mediated stem rust resistance.

BMC Genomics 2019 Dec 16;20(1):985. Epub 2019 Dec 16.

Department of Crop and Soil Sciences, Washington State University, Pullman, WA, USA.

Background: Stem rust is an economically important disease of wheat and barley. However, studies to gain insight into the molecular basis of these host-pathogen interactions have primarily focused on wheat because of its importance in human sustenance. This is the first extensive study utilizing a transcriptome-wide association mapping approach to identify candidate Puccinia graminis f. sp. tritici (Pgt) effectors/suppressors that elicit or suppress barley stem rust resistance genes. Here we focus on identifying Pgt elicitors that interact with the rpg4-mediated resistance locus (RMRL), the only effective source of Pgt race TTKSK resistance in barley.

Results: Thirty-seven Pgt isolates showing differential responses on RMRL were genotyped using Restriction Site Associated DNA-Genotyping by Sequencing (RAD-GBS), identifying 24 diverse isolates that were used for transcript analysis during the infection process. In planta RNAseq was conducted with the 24 diverse isolates on the susceptible barley variety Harrington, 5 days post inoculation. The transcripts were mapped to the Pgt race SCCL reference genome identifying 114 K variants in predicted genes that would result in nonsynonymous amino acid substitutions. Transcriptome wide association analysis identified 33 variants across 28 genes that were associated with dominant RMRL virulence, thus, representing candidate suppressors of resistance. Comparative transcriptomics between the 9 RMRL virulent -vs- the 15 RMRL avirulent Pgt isolates identified 44 differentially expressed genes encoding candidate secreted effector proteins (CSEPs), among which 38 were expressed at lower levels in virulent isolates suggesting that they may represent RMRL avirulence genes. Barley transcript analysis after colonization with 9 RMRL virulent and 15 RMRL avirulent isolates inoculated on the susceptible line Harrington showed significantly lower expression of host biotic stress responses specific to RMRL virulent isolates suggesting virulent isolates harbor effectors that suppress resistance responses.

Conclusions: This transcriptomic study provided novel findings that help fill knowledge gaps in the understanding of stem rust virulence/avirulence and host resistance in barley. The pathogen transcriptome analysis suggested RMRL virulence might depend on the lack of avirulence genes, but evidence from pathogen association mapping analysis and host transcriptional analysis also suggested the alternate hypothesis that RMRL virulence may be due to the presence of suppressors of defense responses.
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http://dx.doi.org/10.1186/s12864-019-6369-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6915985PMC
December 2019

Characterization of genes required for both Rpg1 and rpg4-mediated wheat stem rust resistance in barley.

BMC Genomics 2019 Jun 14;20(1):495. Epub 2019 Jun 14.

Department of Plant Pathology, North Dakota State University, Fargo, ND, 58108-6050, USA.

Background: Puccinia graminis f. sp. tritici (Pgt) race TTKSK and its lineage pose a threat to barley production world-wide justifying the extensive efforts to identify, clone, and characterize the rpg4-mediated resistance locus (RMRL), the only effective resistance to virulent Pgt races in the TTKSK lineage. The RMRL contains two nucleotide-binding domain and leucine-rich repeat (NLR) resistance genes, Rpg5 and HvRga1, which are required for resistance. The two NLRs have head-to-head genome architecture with one NLR, Rpg5, containing an integrated C-terminal protein kinase domain, characteristic of an "integrated sensory domain" resistance mechanism. Fast neutron mutagenesis of line Q21861 was utilized in a forward genetics approach to identify genetic components that function in the RMRL or Rpg1 resistance mechanisms, as Q21861 contains both genes. A mutant was identified that compromises both RMRL and Rpg1-mediated resistances and had stunted seedling roots, designated required for P. graminis resistance 9 (rpr9).

Results: The rpr9 mutant generated in the Q21861 background was crossed with the Swiss landrace Hv584, which carries RMRL but contains polymorphism across the genome compared to Q21861. To map Rpr9, a Hv584 x rpr9 F recombinant inbred line (RIL) population was developed. The RIL population was phenotyped with Pgt race QCCJB. The Hv584 x rpr9 RIL population was genotyped with the 9 k Illumina Infinium iSelect marker panel, producing 2701 polymorphic markers. A robust genetic map consisting of 563 noncosegregating markers was generated and used to map Rpr9 to an ~ 3.4 cM region on barley chromosome 3H. The NimbleGen barley exome capture array was utilized to capture rpr9 and wild type Q21861 exons, followed by Illumina sequencing. Comparative analysis, resulting in the identification of a 1.05 Mbp deletion at the chromosome 3H rpr9 locus. The identified deletion contains ten high confidence annotated genes with the best rpr9 candidates encoding a SKP1-like 9 protein and a F-box family protein.

Conclusion: Genetic mapping and exome capture rapidly identified candidate gene/s that function in RMRL and Rpg1 mediated resistance pathway/s. One or more of the identified candidate rpr9 genes are essential in the only two known effective stem rust resistance mechanisms, present in domesticated barley.
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http://dx.doi.org/10.1186/s12864-019-5858-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570958PMC
June 2019

Shedding Light on Penetration of Cereal Host Stomata by Wheat Stem Rust Using Improved Methodology.

Sci Rep 2019 05 28;9(1):7939. Epub 2019 May 28.

Department of Plant Pathology, North Dakota State University, Fargo, ND, 58108-6050, USA.

Asexual urediniospore infection of primary cereal hosts by Puccinia graminis f. sp. tritici (Pgt), the wheat stem rust pathogen, was considered biphasic. The first phase, spore germination and appressoria formation, requires a dark period and moisture. The second phase, host entry by the penetration peg originating from the appressoria formed over the guard cells, was thought to require light to induce natural stomata opening. Previous studies concluded that inhibition of colonization by the dark was due to lack of penetration through closed stomata. A sensitive WGA-Alexa Fluor 488 fungal staining, surface creation and biovolume analysis method was developed enabling visualization and quantification of fungal growth in planta at early infection stages surpassing visualization barriers using previous methods. The improved method was used to investigate infection processes of Pgt during stomata penetration and colonization in barley and wheat showing that penetration is light independent. Based on the visual growth and fungal biovolume analysis it was concluded that the differences in pathogen growth dynamics in both resistant and susceptible genotypes was due to light induced pathogen growth after penetration into the substomatal space. Thus, light induced plant or pathogen cues triggers pathogen growth in-planta post penetration.
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http://dx.doi.org/10.1038/s41598-019-44280-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538696PMC
May 2019

T cell inhibitory mechanisms in a model of aggressive Non-Hodgkin's Lymphoma.

Oncoimmunology 2017;7(1):e1365997. Epub 2017 Sep 21.

Tumor Immunology, Department of Clinical Research, University of Bern, Bern, Switzerland.

A reduced immune surveillance due to immune deficiency or treatment with immunosuppressive drugs is associated with a higher risk to develop aggressive Non-Hodgkin's lymphoma (NHL). Nevertheless, NHL also develops in immunocompetent patients indicating an escape from the immune system. T cell function in advanced aggressive lymphoma is not well characterized and the molecular mechanisms how malignant B cells influence T cell function are ill-defined. We therefore studied T cell function in Eμ-myc transgenic mice that develop an aggressive B cell lymphoma with some similarities to human Burkitt-lymphoma (BL). In advanced lymphoma, the number of T cells was severely reduced and the remaining CD4 and CD8 T cells lost the capacity to produce effector cytokines and expand upon re-stimulation. T cells in lymphoma-bearing mice were characterized by the expression of the immune inhibitory molecules programmed death (PD)-1, 2B4 and lymphocyte activation protein (LAG)-3. The proto-oncogene c-Myc not only drives cell proliferation and disease progression but also induces apoptosis of the malignant cells. We found that apoptotic lymphoma cells release purine metabolites that inhibit T cell function. Taken together, our data document that the characteristic high cell turnover and apoptotic rate in aggressive NHL induce a severe T cell dysfunction mediated by several immune-inhibitory mechanisms including ligation of inhibitory ligands and purine metabolites. Blocking a single mechanism only partially restored T cell function and did not increase survival of lymphoma mice.
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http://dx.doi.org/10.1080/2162402X.2017.1365997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739580PMC
September 2017

A Systems Oncology Approach Identifies NT5E as a Key Metabolic Regulator in Tumor Cells and Modulator of Platinum Sensitivity.

J Proteome Res 2016 Jan 17;15(1):280-90. Epub 2015 Dec 17.

Division of Cancer, Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital , London W12 0NN, United Kingdom.

Altered metabolism in tumor cells is required for rapid proliferation but also can influence other phenotypes that affect clinical outcomes such as metastasis and sensitivity to chemotherapy. Here, a genome-wide association study (GWAS)-guided integration of NCI-60 transcriptome and metabolome data identified ecto-5'-nucleotidase (NT5E or CD73) as a major determinant of metabolic phenotypes in cancer cells. NT5E expression and associated metabolome variations were also correlated with sensitivity to several chemotherapeutics including platinum-based treatment. NT5E mRNA levels were observed to be elevated in cells upon in vitro and in vivo acquisition of platinum resistance in ovarian cancer cells, and specific targeting of NT5E increased tumor cell sensitivity to platinum. We observed that tumor NT5E levels were prognostic for outcomes in ovarian cancer and were elevated after treatment with platinum, supporting the translational relevance of our findings. In this work, we integrated and analyzed a plethora of public data, demonstating the merit of such a systems oncology approach for the discovery of novel players in cancer biology and therapy. We experimentally validated the main findings of the NT5E gene being involved in both intrinsic and acquired resistance to platinum-based drugs. We propose that the efficacy of conventional chemotherapy could be improved by NT5E inhibition and that NT5E expression may be a useful prognostic and predictive clinical biomarker.
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http://dx.doi.org/10.1021/acs.jproteome.5b00793DOI Listing
January 2016

COX10 mutations resulting in complex multisystem mitochondrial disease that remains stable into adulthood.

JAMA Neurol 2013 Dec;70(12):1556-61

Medical Research Council Centre for Neuromuscular Diseases, University College London Institute of Neurology and National Hospital for Neurology and Neurosurgery, London, England13Department of Molecular Neuroscience, University College London Institute o.

Importance: Isolated cytochrome-c oxidase (COX) deficiency is one of the most frequent respiratory chain defects seen in human mitochondrial disease. Typically, patients present with severe neonatal multisystem disease and have an early fatal outcome. We describe an adult patient with isolated COX deficiency associated with a relatively mild clinical phenotype comprising myopathy; demyelinating neuropathy; premature ovarian failure; short stature; hearing loss; pigmentary maculopathy; and renal tubular dysfunction.

Observations: Whole-exome sequencing detected 1 known pathogenic and 1 novel COX10 mutation: c.1007A>T; p.Asp336Val, previously associated with fatal infantile COX deficiency, and c.1015C>T; p.Arg339Trp. Muscle COX holoenzyme and subassemblies were undetectable on immunoblots of blue-native gels, whereas denaturing gels and immunocytochemistry showed reduced core subunit MTCO1. Heme absorption spectra revealed low heme aa3 compatible with heme A:farnesyltransferase deficiency due to COX10 dysfunction. Both mutations demonstrated respiratory deficiency in yeast, confirming pathogenicity. A COX10 protein model was used to predict the structural consequences of the novel Arg339Trp and all previously reported substitutions.

Conclusions And Relevance: These findings establish that COX10 mutations cause adult mitochondrial disease. Nuclear modifiers, epigenetic phenomenon, and/or environmental factors may influence the disease phenotype caused by reduced COX activity and contribute to the variable clinical severity related to COX10 dysfunction.
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http://dx.doi.org/10.1001/jamaneurol.2013.3242DOI Listing
December 2013
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