Absence Of Known Mutations Research Articles

Metabolic Resistance to Protoporphyrinogen Oxidase-Inhibitor Herbicides in a Palmer amaranth Population from Kansas.

Palmer amaranth has evolved target and nontarget site resistance to protoporphyrinogen oxidase-inhibitor herbicides in the United States. Recently, a population (KCTR) from a long-term conservation tillage study in Kansas was found to be resistant to herbicides from six sites of action, including to PPO-inhibitors, even with this herbicide group being minimally used in this field. This research investigated the level of resistance to postemergence PPO-inhibitors, target- and nontarget-site resistance mechanism(s), and efficacy of pre-emergence chemistries. The greenhouse experiments confirmed 6.1- to 78.9-fold resistance to lactofen in KCTR, with the level of resistance increasing when KCTR was purified for the resistance trait. PPO2 sequences alignment revealed the absence of known mutations conferring resistance to PPO-inhibitors in KCTR Palmer amaranth, and differential expression of the PPO2 gene did not occur. KCTR metabolized fomesafen faster than the susceptible population, indicating that herbicide detoxification is the mechanism conferring resistance in this population. Further, treatment with the cytochrome P450-inhibitor malathion followed by lactofen restored the sensitivity of KCTR to this herbicide. Despite being resistant to POST applied PPO-inhibitors, KCTR Palmer amaranth was completely controlled by the labeled rate of the PRE applied PPO-inhibitors fomesafen, flumioxazin, saflufenacil, sulfentrazone, and oxadiazon. The overall results suggest that P450-mediated metabolism confers resistance to PPO-inhibitors in KCTR, rather than alterations in the PPO2, which were more commonly found in other Palmer amaranth populations. Future work will focus on identifying the fomesafen metabolites and on unravelling the genetic basis of metabolic resistance to PPO-inhibitor herbicides in KCTR Palmer amaranth.

Ectopic PLAG1 induces muscular dystrophy in the mouse

Even though various genetic mutations have been identified in muscular dystrophies (MD), there is still a need to understand the biology of MD in the absence of known mutations. Here we reported a new mouse model of MD driven by ectopic expression of PLAG1. This gene encodes a developmentally regulated transcription factor known to be expressed in developing skeletal muscle, and implicated as an oncogene in certain cancers including rhabdomyosarcoma (RMS), an aggressive soft tissue sarcoma composed of myoblast-like cells. By breeding loxP-STOP-loxP-PLAG1 (LSL-PLAG1) mice into the MCK-Cre line, we achieved ectopic PLAG1 expression in cardiac and skeletal muscle. The Cre/PLAG1 mice died before 6 weeks of age with evidence of cardiomyopathy significantly limiting left ventricle fractional shortening. Histology of skeletal muscle revealed dystrophic features, including myofiber necrosis, fiber size variation, frequent centralized nuclei, fatty infiltration, and fibrosis, all of which mimic human MD pathology. QRT-PCR and Western blot revealed modestly decreased Dmd mRNA and dystrophin protein in the dystrophic muscle, and immunofluorescence staining showed decreased dystrophin along the cell membrane. Repression of Dmd by ectopic PLAG1 was confirmed in dystrophic skeletal muscle and various cell culture models. In vitro studies showed that excess IGF2 expression, a transcriptional target of PLAG1, phenocopied PLAG1-mediated down-regulation of dystrophin. In summary, we developed a new mouse model of a lethal MD due to ectopic expression of PLAG1 in heart and skeletal muscle. Our data support the potential contribution of excess IGF2 in this model. Further studying these mice may provide new insights into the pathogenesis of MD and perhaps lead to new treatment strategies.

Detection of a novel mutation in the rpoB gene in a multidrug resistant Mycobacterium tuberculosis isolate using whole genome next generation sequencing

BackgroundMycobacterium tuberculosis (Mtb) drug resistance is a global concern. Moreover, multiple drug resistant (MDR), extensively drug resistant (XDR), and totally drug resistant (TDR) Mtb cases are on the rise in developing countries like India. Most of these cases are identified only 3–6 months after initiation of treatment owing to incomplete/failed clinical response and incomplete information from phenotypic drug resistance assays and/or targeted Mtb mutation analysis. Here, we report the development of an in-house whole genome sequencing (WGS) assay and bioinformatics pipeline that helped resolve the phenotype-genotype discrepancy in a clinical isolate. Methodology and resultsA sample from a suspected drug resistant Mtb case tested by line probe assay (LPA) showed the absence of both the mutant and wild type alleles for an rpoB gene mutation site. An in-house next generation sequencing (NGS) assay was used for WGS of this isolate. Bioinformatics analysis revealed that the isolate harboured a novel insertional mutation in the 81-bp hotspot region of the rpoB gene and a S315T mutation in the katG gene, which could explain resistance to rifampicin and isoniazid, respectively. These results correlated with the clinical diagnosis, LPA, solid culture drug susceptibility testing, and pyrosequencing carried out on the sample. The WGS data also provided information regarding the isolate’s lineage and indicated an absence of known mutations conferring resistance to other antitubercular drugs. ConclusionWGS is a highly sensitive, specific, and unbiased approach for identification of all possible drug resistance-conferring mutations, which can help clinicians make more informed treatment-related decisions.

11C-PIB PET imaging reveals that amyloid deposition in cases with early-onset Alzheimer's disease in the absence of known mutations retains higher levels of PIB in the basal ganglia.

PurposeEarly-onset Alzheimer’s disease (EOAD) has a different pathologic burden and clinical features compared with late-onset Alzheimer’s disease (LOAD). We examined the effects of age at onset on the burden and distribution of β-amyloid in patients with EOAD, in whom well-characterized mutations associated with Alzheimer’s disease were absent.MethodsWe genotyped ApoE, APP, PSEN1 and PSEN2 in the patients with Alzheimer’s disease: 9 patients with EOAD (age <65), 11 with LOAD (age >70) and 8 normal controls (NCs), all of whom had undergone 11C-labeled Pittsburgh compound B-positron emission tomography imaging.ResultsPatients with EOAD exhibited higher z scores and larger cluster sizes, and retained higher levels of Pittsburgh compound B in the bilateral thalamus and in some parts of the globus pallidus (P<0.05, false discovery rate).ConclusionDistribution of amyloid deposition in EOAD outside the context of genetic mutations topographically showed some differences from that in LOAD.

Recurrent Loss of NFE2L2 Exon 2 Is a Mechanism for Nrf2 Pathway Activation in Human Cancers

The Nrf2 pathway is frequently activated in human cancers through mutations in Nrf2 or its negative regulator KEAP1. Using a cell-line-derived gene signature for Nrf2 pathway activation, we found that some tumors show high Nrf2 activity in the absence of known mutations in the pathway. An analysis of splice variants in oncogenes revealed that such tumors express abnormal transcript variants from the NFE2L2 gene (encoding Nrf2) that lack exon 2, or exons 2 and 3, and encode Nrf2 protein isoforms missing the KEAP1 interaction domain. The Nrf2 alterations result in the loss of interaction with KEAP1, Nrf2 stabilization, induction of a Nrf2 transcriptional response, and Nrf2 pathway dependence. In all analyzed cases, transcript variants were the result of heterozygous genomic microdeletions. Thus, we identify an alternative mechanism for Nrf2 pathway activation in human tumors and elucidate its functional consequences.

Insights into the Indian Peanut Genotypes for ahFAD2 Gene Polymorphism Regulating Its Oleic and Linoleic Acid Fluxes.

In peanut (Arachis hypogaea L.), the customization of fatty acid profile is an evolving area to fulfill the nutritional needs in the modern market. A total of 174 peanut genotypes, including 167 Indian cultivars, 6 advanced breeding lines and “SunOleic95R”—a double mutant line, were investigated using AS-PCRs, CAPS and gene sequencing for the ahFAD2 allele polymorphism, along with its fatty acid compositions. Of these, 80 genotypes were found having substitution (448G>A) mutation only in ahFAD2A gene, while none recorded 1-bp insertion (441_442insA) mutation in ahFAD2B gene. Moreover, 22 wild peanut accessions found lacking both the mutations. Among botanical types, the ahFAD2A mutation was more frequent in ssp. hypogaea (89%) than in ssp. fastigiata (17%). This single allele mutation, found affecting not only oleic to linoleic acid fluxes, but also the composition of other fatty acids in the genotypes studied. Repeated use of a few selected genotypes in the Indian varietal development programs were also eminently reflected in its ahFAD2 allele polymorphism. Absence of known mutations in the wild-relatives indicated the possible origin of these mutations, after the allotetraploidization of cultivated peanut. The SNP analysis of both ahFAD2A and ahFAD2B genes, revealed haplotype diversity of 1.05% and 0.95%, while Ka/Ks ratio of 0.36 and 0.39, respectively, indicating strong purifying selection pressure on these genes. Cluster analysis, using ahFAD2 gene SNPs, showed presence of both mutant and non-mutant genotypes in the same cluster, which might be due the presence of ahFAD2 gene families. This investigation provided insights into the large number of Indian peanut genotypes, covering various aspects related to O/L flux regulation and ahFAD2 gene polymorphism.

MicroRNAs: newcomers into the ALS picture.

Amyotrophic lateral sclerosis (ALS) causes neurodegeneration of both upper and lower motor neurons and progressive muscle impairment, atrophy and death within approximately five years from diagnosis. The aetiology is still not clear but evidence obtained in animal models of the disease indicates a non-cell-autonomous mechanism with the active contribution of non-neuronal cells such as microglia, astrocytes, muscle and T cells, which differently participate to the diverse phases of the disease. Clinically indistinguishable forms of ALS occur as sporadic disease in the absence of known mutation, or can be initiated by genetic mutations. About two-third of familial cases are triggered by mutations of four genes that are chromosome 9 open reading frame 72 (C9ORF72), Cu/Zn superoxide dismutase (SOD1), fused in sarcoma/translocated in liposarcoma (FUS/TLS), TAR-DNA binding protein 43 (TDP43). There is at present no succesfull treatment against ALS and the identification of novel signalling pathways, molecular mechanisms and cellular mediators are still a major task in the search for effective therapies. MiRNAs are conserved, endogenous, non-coding RNAs that post-transcriptionally regulate protein expression. Produced as long primary transcripts, they are exported to the cytoplasm and further modified to obtain the mature miRNAs, with each step of their biogenesis being a potential step of regulation. There are more than 1000 different known human miRNA sequences, and more than 20-30% of all human protein-coding genes are likely controlled by miRNAs. This earns to miRNAs the definition of fine regulators of genetic networks. The discovery of the involvement of ALS mutated proteins TDP43 and FUS/TLS in miRNAs biogenesis strongly suggests a role of miRNA dysregulation also in ALS and many efforts are thus directed toward understanding the role of these small RNA molecules in the pathogenesis of ALS. The overall objective of this review is thus to highlight the emerging involvement of miRNAs in ALS. After a brief description of miRNA biogenesis and function, we discuss the effects of miRNA dysregulation in cellular and molecular pathways that lead to ALS neuroinflammation and neurodegeneration. In the last part, we focus on the mechanistic insights of miRNAs that might have implications for the development of novel neuroprotective agents against ALS, and on recent attempts to establish new molecular miRNA-based therapies. Paving the way for more comparative studies on neuroinflammatory and neurodegenerative mechanisms, this strategy indeed promises a broader impact on ALS.

A Case of Reye’s-Like Syndrome in a 68-Day Old Infant: Water Damaged Home, Mold, Bacteria and Aflatoxins.

Introduction: Reye’s Syndrome is characterized by acute encephalopathy, hepatic injury accompanied with elevated serum ammonia, serum fatty acids, amino acids and triglycerides; hypoglycemia, prolonged prothrombin time, fatty infiltration of the liver, and mitochondrial pathology. We present an infant that died at 68 days of age with Reye ’s syndrome. The infant and parents were exposed to airborne fungi, bacteria and toxic bioaerosols resulting from water intrusion in the home. The purpose of this study was to investigate and, if possible, to determine the cause of death of the infant with respect to the fungi, bacteria and their toxins present in the families water-damaged home. Materials and Methods: Health and genetic history was done on the family (father, mother and two siblings). Environmental evaluation was carried out to identify airborne fungi and bacteria in the home. Clinical testing of the baby while in the hospital provided data on blood chemistry and EEG results. Mitochondrial studies on skin fibroblasts and skeletal muscle were carried out testing for functions of Complexes I-IV and mitochondrial DNA mutations. Light and E.M. microscopy were done on liver biopsy material. Immunoaffinity column and fluorometry were used to detect aflatoxins (B1, B2, G1 and G2) in liver autopsy material. The mother’s breast milk and urine was tested for mycotoxins: trichothecenes, aflatoxins and ochratoxin. Results: Medical and genetic history were negative for familial diseases similar to Reye’s syndrome and for mitochondrial DNA mutations. Aortic and pulmonic valve abnormalities were observed. Environmental testing revealed the presence of elevated levels of several species of fungi and bacteria in the infant’s bedroom and other rooms of the home. Clinical diagnostic tests of the infant revealed metabolic acidosis, elevated serum ammonia, triglycerides, pyruvic and lactic acids, serum alanine, and beta-hydroxybutyrate. Mitochondrial studies showed decreased function of complexes I-IV and the absence of known mutations associated with mitochondrial diseases. Microscopy (light and E.M.) of biopsies demonstrated the accumulation of glycogen in muscle and fatty droplets in the liver. Aflatoxins were detected in the infant’s liver (2.1 ppb), and the mother’s breast milk (15 ppb), while maternal urine was positive for trichothecenes (4.76 ppb) and ochratoxin (3.4 ppb). Conclusions: After a review of the peer reviewed literature, we conclude that the infant died of a Reye’s-Like Syndrome at the age of 68 days (all 22 cited criteria were met). Clinical and autopsy findings were consistent with this disease process. The valvular abnormalities found are associated with actinomycetes exposure. The medical and genetic histories were negative for any familial diseases of a similar nature. The infant had mitochondrial dysfunction of complexes I-IV, suggesting mitochondrial disease consistent with alflatoxin toxicity. The presence of aflatoxins in the liver supports the causal role of this mycotoxin in the illness of this infant. We have discussed the role of other factors in the indoor environment that may also lead to similar conditions.

Presenilins: A novel class of calcium modulators

A disease (AD) is the most common form of dementia, affecting more than 5 mi Americans. As such, AD poses a significant burden on the affected individual, caregivers and society. Most cases of AD are attributed to the sporadic form, which is believed to be of multifactorial origin. However, several genetic loci etiological for the rare familial form of the disease have been identified. One of the loci is the group of presenilin proteins, which form the enzymatic core of the γ-secretase complex. Most of the almost 200 identified familial AD mutations in presenilins are located in the gene encoding presenilin-1, while presenilin-2 mutations typically cause later onset familial AD. Recent evidence identified the group of presenilin proteins as potent modulators of intracellular calcium signaling, through potentiation of the intracellular ryanodine receptor, which likely underlies this phenomenon. This potentiation occurs via the highly evolutionarily conserved N-terminal region of presenilin, resulting in differential modulation of the ryanodine receptor by presenilin-1 and presenilin-2. The proposed mechanism is in accordance with previous studies identifying elevated Ca2+ concentrations in the endoplasmic reticulum during AD, and the critical role of ryanodine receptors in regulating calcium via calcium-induced calcium release. Furthermore, ryanodine receptors contribute to the pathologic, elevated intracellular Ca2+ concentrations observed in AD. Intriguingly, similar Ca2+ dyshomeostasis occurs during healthy aging, in the absence of known mutations. Utilizing preclinical models for healthy aging, we have implicated presenilin proteins in the etiology of age-related changes in synaptic signaling and, ultimately, agerelated deficits in memory and motor coordination. In this keynote talk, the author will summarize the evidence for the group of presenilin proteins as a novel class of calcium modulators, and discuss the opportunities for targeting presenilin proteins as novel drug targets for age-related and neurodegenerative diseases, incl. AD.

Aberrant Localization of FUS and TDP43 Is Associated with Misfolding of SOD1 in Amyotrophic Lateral Sclerosis

BackgroundAmyotrophic lateral sclerosis (ALS) is incurable and characterized by progressive paralysis of the muscles of the limbs, speech and swallowing, and respiration due to the progressive degeneration of voluntary motor neurons. Clinically indistinguishable ALS can be caused by genetic mutations of Cu/Zn superoxide dismutase (SOD1), TAR-DNA binding protein 43 (TDP43), or fused in sarcoma/translocated in liposarcoma (FUS/TLS), or can occur in the absence of known mutation as sporadic disease. In this study, we tested the hypothesis that FUS/TLS and TDP43 gain new pathogenic functions upon aberrant accumulation in the cytosol that directly or indirectly include misfolding of SOD1.Methodology/Principal FindingsPatient spinal cord necropsy immunohistochemistry with SOD1 misfolding-specific antibodies revealed misfolded SOD1 in perikarya and motor axons of SOD1-familial ALS (SOD1-FALS), and in motor axons of R521C-FUS FALS and sporadic ALS (SALS) with cytoplasmic TDP43 inclusions. SOD1 misfolding and oxidation was also detected using immunocytochemistry and quantitative immunoprecipitation of human neuroblastoma SH-SY5Y cells as well as cultured murine spinal neural cells transgenic for human wtSOD1, which were transiently transfected with human cytosolic mutant FUS or TDP43, or wtTDP43.Conclusion/SignificanceWe conclude that cytosolic mislocalization of FUS or TDP43 in vitro and ALS in vivo may kindle wtSOD1 misfolding in non-SOD1 FALS and SALS. The lack of immunohistochemical compartmental co-localization of misfolded SOD1 with cytosolic TDP43 or FUS suggests an indirect induction of SOD1 misfolding followed by propagation through template directed misfolding beyond its site of inception. The identification of a final common pathway in the molecular pathogenesis of ALS provides a treatment target for this devastating disease.

Western Pacific ALS-PDC: a prototypical neurodegenerative disorder linked to DNA damage and aberrant proteogenesis?

Western Pacific ALS-PDC: a prototypical neurodegenerative disorder linked to DNA damage and aberrant proteogenesis?

Papillary thyroid carcinoma shows elevated levels of 2-hydroxyglutarate

Elevated levels of D: -2-hydroxyglutarate (D: -2-HG) occur in gliomas and myeloid leukemias associated with mutations of IDH1 and IDH2. L: -2-Hydroxyglutaric aciduria, an inherited metabolic disorder, predisposes to brain tumors. Therefore, we asked whether sporadic cancers, without IDH1 or IDH2 hot-spot mutations, show elevated 2-hydroxyglutarate levels. We retrieved 15 pairs of frozen papillary thyroid carcinoma (PTC) and adjacent non-neoplastic thyroid, and 14 pairs of hyperplastic nodule (HN) and adjacent non-hyperplastic thyroid. In all lesions, exon 4 sequencing confirmed the absence of known mutations of IDH1 and IDH2. We measured 2-hydroxyglutarate by liquid chromatography-tandem mass spectrometry. Compared to normal thyroid, PTCs had significantly higher D: -2-HG and L: -2-hydroxyglutarate (L: -2-HG) levels, and compared to HNs, PTCs had significantly higher D: -2-HG levels. D: -2-HG/L: -2-HG levels were not significantly different between HNs and normal thyroid. Further studies should clarify if elevated 2-hydroxyglutarate in PTC may be useful as cancer biomarker and evaluate the role of 2-hydroxyglutarate in cancer biology.

P37 Routine use of PCR for PGD in translocation carriers: New developments to differentiate balanced carriers from non-carriers

at KFSHRC in order to increase the confidence of the diagnosis and to test for diseases with unidentified mutations. Materials and Methods: Couples were counseled and consented. WGA from single blastomeres was achieved using multiple displacement amplification (MDA) followed by disease specific mutation detection and/or PGH. Results: Fifty cycles were performed for 31 couples at risk of autosomal recessive or dominant, or X-linked disorders. A total of 328 embryos were tested, 301 of the samples were successfully amplified by MDA with an amplification efficiency of 92%. From the MDA product, 1462 PCR reaction were set and 47 (3.2%) amplification failure were observed. Allele drop out (ADO) were observed in 101 alleles (3.5%). The confirmations of PGH results with the mutation testing were available for 269 embryos. Of those, 239 (89%) showed full concordance. There was a discrepancy between the PGH and mutation analysis from 12 embryos (4.5%). From the remaining, 4.5% were though concordant; PGH results showed a haploid pattern indicating that such embryos are being haploid or monosomic for that specific chromosome. Consequently, such embryos were not considered for transfer. There were 6 (2%) embryos on which diagnosis could not be predicted either due to maternal cell contamination or extensive ADO with PGH analysis. For the cycles only PGH were performed either due to unknown mutation or gene expansion, there were 8 cycles for 4 couples. A total of 56 embryos were tested by PGH analysis and 53 (95%) give conclusive results to make a clear diagnosis. Of the 50 cycles, 42 had embryo transfers resulting 15 pregnancies (35.7%) in 13 couples. One of the pregnancies resulted in abortion while the remaining is either ongoing or delivered. Conclusions: PGH helps to confirm the diagnosis and increase the confidence, as it predicts the origin of inheritance of each chromosome. It can also be safely used to make diagnosis in the absence of known mutation.

A new mutation in exon 7 of NEMO gene: late skewed X-chromosome inactivation in an incontinentia pigmenti female patient with immunodeficiency

Incontinentia pigmenti is an X-linked genodermatosis, lethal in males. Affected females survive because of X-chromosome dizygosity and negative selection of cells carrying the mutant X-chromosome, and for this reason the skewed X inactivation pattern is often used to confirm the diagnosis. The most frequent mutation is a deletion of part of the NEMO gene (NEMODelta4-10), although other mutations have been reported. Mutations of NEMO which do not abolish NF-kappaB activity totally permit male survival, causing an allelic variant of IP called hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID). We present a non-classical IP female patient who also suffered transient immunodeficiency because of a late and progressive selection against peripheral blood cells carrying an active mutated X-chromosome. This finding suggests that in the absence of known mutation the X-inactivation studies used in genetic counselling can induce mistakes with some female patients. At the age of 3 years and 6 months, all immunodeficiency signs disappeared, and the X-chromosome inactivation pattern was completely skewed. The low T cell proliferation and CD40L expression corroborate the important role of NEMO/ NF-kappaB pathway in T cell homeostasis. The decreased NEMO protein amount and the impaired IkBalpha degradation suggest that this new mutation, NM_003639: c.1049dupA, causes RNA or protein instability. To our knowledge, this is the first time that selection against the mutated X-chromosome in X-linked disease has been documented in vivo.