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Rationale: The high clinical heterogeneity of hypermethioninemia caused by MAT1A gene defects has resulted in a paucity of studies examining the association between clinical phenotypes, biochemical characteristics, and gene mutations in this patient group. Furthermore, the indications for therapeutic interventions in patients remain unclear. The objective of this study is to provide a foundation for clinical diagnosis, genetic counseling, and follow-up management of hypermethioninemia caused by MAT1A gene defects., Patient Concerns: A retrospective analysis of children with hypermethioninemia at Jinan Maternal and Child Health Hospital from January 2016 to December 2023 was performed using tandem mass spectrometry (MS/MS). The screened and diagnosed children were tested for gene mutations using second-generation sequencing technology and confirmed using Sanger sequencing., Diagnoses: Newborn MS/MS screening for diseases demonstrated an elevated methionine level, which was outside the reference range. Upon recalling the newborns, the methionine levels remained elevated, necessitating further refinement of genetic testing. Ultimately, genetic testing confirmed hypermethioninemia, which was attributed to a mutation in the MAT1A gene., Interventions: The intervention for the patients in this study took the following forms: regular follow-up without treatment (n = 3), intake of methionine-free milk powder without any medication (n = 4), intake of methionine-free milk powder with some medication, and eventually liver transplantation (n = 1)., Outcomes: A total of 14 mutation types were detected, including 3 compound heterozygous mutation types (c.926G > T, c.37_38delCT, and c.316G > A) that have not been previously reported. One patient had monoheterozygous mutations, including the novel mutation c.550-1G > A. Eight cases were monitored over time, 7 of which demonstrated typical growth and development. One infant with growth retardation was fed a special formula lacking methionine. The patient underwent liver transplantation. Subsequent follow-up examinations showed methionine and homocysteine levels within normal limits and no further neurological manifestations., Lessons: Compound heterozygous mutations c.874C > T and c.896G > A may result in higher levels of methionine, affecting the central nervous system. For newborns with initial methionine levels of >500 µmol/L, treatment with a low-Met diet is recommended. Liver transplantation may be beneficial for children with severe hypermethioninemia, particularly in preventing central nervous system damage., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels 1 are essential for pacemaking activity and neural signalling 2,3 . Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain 4 and epileptic seizures 5 . The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor 6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs.  7,8 ). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Prion diseases are a family of infectious amyloid diseases affecting human and animals. Prion propagation in transmissible spongiform encephalopathies is associated with the unfolding and conversion of normal cellular prion protein into its pathogenic scrapie form. Understanding the fundamentals of prion protein aggregation caused by mutations is crucial to unravel the pathology of prion diseases. To help understand the contributions of individual residues to the stability of the human prion protein, we have carried out free energy simulations based on atomistic molecular dynamics trajectories. We focus on Met → Ala mutations at positions 205, 206 and 213, which are mostly buried residues located on helix 3 of the protein. The simulations predicted that all three mutations destabilize the prion protein. Changes in unfolding free energy upon mutation, ∆∆G, are 3.10 ± 0.79, 2.00 ± 0.26 and 3.06 ± 0.66 kcal/mol for M205A, M206A and M213A, respectively, in excellent agreement with the corresponding experimental values of 3.09 ± 0.28, 1.50 ± 0.34 and 3.12 ± 0.27 kcal/mol [T. Hart et al. (2009) PNAS 106, 5651-5656]. Component analysis indicates that the major contributions to the loss of protein stability arise from van der Waals interactions for the M205A and M206A mutations, and from van der Waals and covalent energy terms for M213A. Interestingly, while free energy contributions from a majority of residues neighboring the mutation sites tend to stabilize the wild type, there are a few residues stabilizing the mutant side chains. Our results show that this approach to free energy calculation can be very useful for understanding the detailed mechanism of human prion protein stability., (Copyright © 2021. Published by Elsevier B.V.)

Background: Hemoglobin (Hb) Alesha is a rare and very unstable Hb variant, resulting in disruption of the heme pocket and producing severe hemolysis in heterozygous statues. In this study, we describe the first report of this variant in an Iranian boy originated from south of Iran with severe hemolytic anemia and mild splenomegaly., Methods: A six-year-old boy from Khuzestan Province and his parents were studied. Gap-PCR and direct sequencing were performed to detect the a-globin gene deletions and β-globin gene mutations, respectively., Results: The subject had a sporadic mutation GTG to ATG (Val [valine]>Met [methionine]) at codon 67 in heterozygous form on β-globin gene, which was not detected in his parents., Conclusion: Since both parents proved to be normal, this Hb variant could be considered as a de novo mutation, which is highly useful for prenatal diagnosis.

Diffuse midline glioma (DMG), H3 K27M-mutant, is a new entity in the updated WHO classification grouping together diffuse intrinsic pontine gliomas and infiltrating glial neoplasms of the midline harboring the same canonical mutation at the Lysine 27 of the histones H3 tail.Two hundred and fifteen patients younger than 18 years old with centrally-reviewed pediatric high-grade gliomas (pHGG) were included in this study. Comprehensive transcriptomic (n = 140) and methylation (n = 80) profiling was performed depending on the material available, in order to assess the biological uniqueness of this new entity compared to other midline and hemispheric pHGG.Tumor classification based on gene expression (GE) data highlighted the similarity of K27M DMG independently of their location along the midline. T-distributed Stochastic Neighbor Embedding (tSNE) analysis of methylation profiling confirms the discrimination of DMG from other well defined supratentorial tumor subgroups. Patients with diffuse intrinsic pontine gliomas (DIPG) and thalamic DMG exhibited a similarly poor prognosis (11.1 and 10.8 months median overall survival, respectively). Interestingly, H3.1-K27M and H3.3-K27M primary tumor samples could be distinguished based both on their GE and DNA methylation profiles, suggesting that they might arise from a different precursor or from a different epigenetic reorganization.These differences in DNA methylation profiles were conserved in glioma stem-like cell culture models of DIPG which mimicked their corresponding primary tumor. ChIP-seq profiling of H3K27me3 in these models indicate that H3.3-K27M mutated DIPG stem cells exhibit higher levels of H3K27 trimethylation which are correlated with fewer genes expressed by RNAseq. When considering the global distribution of the H3K27me3 mark, we observed that intergenic regions were more trimethylated in the H3.3-K27M mutated cells compared to the H3.1-K27M mutated ones.H3 K27M-mutant DMG represent a homogenous group of neoplasms compared to other pediatric gliomas that could be further separated based on the type of histone H3 variant mutated and their respective epigenetic landscapes. As these characteristics drive different phenotypes, these findings may have important implication for the design of future trials in these specific types of neoplasms.

Obesity-induced metabolic dysfunctions increase the risk for vascular diseases, including type II diabetes and stroke. Managing obesity is of interest to address the worldwide health problem; however, the role of genetic variability in human obesity development and specific targets for obesity-related metabolic disease have not been thoroughly studied. A SNP in the brain-derived neurotropic factor ( BDNF ) gene that results in the substitution of a valine with a methionine at codon 66 ( Val66Met ) occurs with a high frequency in humans. This study addressed the effect of genetic variability in developing obesity and the efficacy of the inhibition of cluster of differentiation 36 (CD36), a multifunctional receptor implicated in obesity and insulin resistance, in WT mice and mice with the BDNF Val66Met variant. CD36 inhibition by salvionolic acid B (SAB) in diet-induced obese WT mice reduced visceral fat accumulation and improved insulin resistance. The benefit of SAB was abrogated in CD36 knockout mice, showing the specificity of SAB. In addition, mice with the Val66Met variant in both alleles (BDNF M/M ) fed a high-fat diet exhibited extreme obesity with increased CD36 gene and protein levels in macrophages. Chronic SAB treatment in BDNF M/M mice significantly decreased visceral fat accumulation and improved insulin resistance. Notably, the effect of SAB was greater in the extremely obese BDNF M/M mice compared with the WT mice. The study demonstrated a link between BDNF Val66Met and elevated CD36 expression and suggested that CD36 inhibition may be a potential strategy to improve metabolic dysfunctions and to normalize risk factors for vascular diseases in the obese population., (© 2018 Yang et al.)

Adult brainstem gliomas are difficult to classify based on radiologic and histologic features. A K27M mutation in histone 3 has been described to identify high-grade midline gliomas associated with a particularly unfavorable prognosis. While initially considered a pediatric entity, it is now known that H3K27M-mutant brainstem gliomas occur in all age groups, but they are less well understood in adults. We studied clinical, radiologic, and pathologic features of 25 brainstem gliomas diagnosed at our institution between 1994 and 2017 in subjects at least 18 years old. Seven tumors (28%) were positive for the H3K27M mutation, and their median overall survival was significantly shorter than in the H3-wildtype group (p = 0.004). Although the mutation was invariably associated with a poor prognosis, our study also illustrates the radiologic and pathologic heterogeneity in this molecular tumor subtype. The results showed that H3K27M-mutant status and clinically aggressive course cannot be ruled out based on low-grade histology on the initial biopsy, exophytic growth, only focal or minimal enhancement or an extrapontine location, such as midbrain or medulla. These results favor an integrated approach employing a combination of clinical, radiologic, histologic features as well as H3K27M immunohistochemistry for the diagnostic subclassification of adult brainstem gliomas.

In comparison to sporadic Creutzfeldt-Jakob disease (sCJD) with MM1-type and MM2- cortical (MM2C)-type, genetic CJD with a prion protein gene V180I mutation (V180I gCJD) is clinically characterized by onset at an older age, slower progress, and the absence of visual disturbances or cerebellar symptoms. In terms of pathological characteristics, gliosis and neuronal loss are generally milder in degree, and characteristic spongiform change can be observed at both the early and advanced stages. However, little is known on the progress of spongiform change over time or its mechanisms. In this study, to elucidate the pathological course of V180I gCJD, statistical analysis of the size and dispersion of the major diameters of vacuoles in six V180I gCJD cases was performed, with five MM1-type sCJD and MM2C-type sCJD cases as controls. As a result, V180I gCJD showed no significant difference in vacuolar diameter regardless of disease duration. In addition, the dispersion of the major diameters of vacuoles in V180I gCJD was larger than that in the MM1-type, which was smaller than that in the MM2C-type. We speculated that the absence of difference in the size of the vacuoles regardless of disease duration suggests that tissue rarefaction does not result from the expansion of vacuole size and increase in number of vacuoles in V180Ig CJD. These features were considered to be significant pathological findings of V180I gCJD.

Hereditary transthyretin (ATTR) amyloidosis is a life-threatening, autosomal dominant, systemic amyloidosis caused by mutant transthyretin. In addition to ATTRV30M in endemic and non-endemic areas, more than 140 non-V30M mutations occur worldwide. The aim of this study was to analyze the clinical characteristics and genetic frequencies of hereditary ATTR amyloidosis. Diagnostic results and clinical manifestations of hereditary ATTR amyloidosis from April 1, 2012, to March 31, 2017, at Amyloidosis Medical Practice Center, Kumamoto University Hospital were analyzed. One hundred and four patients received a diagnosis of symptomatic hereditary ATTR amyloidosis. The following mutations of the TTR gene and their percentages were found: V30M in endemic areas, 10.6%; V30M in non-endemic areas, 51.0%; and non-V30M, 38.5%. The ages at onset of patients with ATTRV30M amyloidosis in non-endemic areas (66.6 ± 8.7 years) and those with non-V30M ATTR amyloidosis (55.8 ± 13.6 years) were significantly higher than those with ATTRV30M amyloidosis in endemic areas (37.0 ± 12.6 years). Of patients with ATTRV30M amyloidosis in endemic and non-endemic areas, and non-V30M ATTR amyloidosis, 63.6, 66.0, and 27.5% initially presented with polyneuropathy, respectively. Of patients with ATTRV30M amyloidosis in endemic areas, 81.8% had a family history of this disease. However, a significantly smaller percentage of patients with ATTRV30M amyloidosis (30.0%) in non-endemic areas and non-V30M ATTR amyloidosis (34.0%) had a family history. Patients with ATTRV30M amyloidosis in non-endemic areas and patients with non-V30M ATTR amyloidosis occurred more frequently than previously believed, and their clinical manifestations were diverse.

The objective of this study was to systematically investigate cardiac and peripheral vasomotor autonomic functions in late-onset transthyretin Val30Met familial amyloid polyneuropathy (FAP ATTR Val30Met) patients from non-endemic areas. The coefficient of variation of R-R intervals (CVR-R), responses to the Valsalva manoeuvre, head-up tilt test with impedance cardiography, noradrenaline infusion test, and (123)I-metaiodobenzylguanidine (MIBG) myocardial scintigraphy were assessed in eight patients. Although only four patients manifested orthostatic hypotension during the head-up tilt test, CVR-R, responses to the Valsalva manoeuvre, and myocardial MIBG uptake indicated a higher prevalence of cardiac sympathetic and parasympathetic dysfunction. Total peripheral resistance at 60° tilt did not increase from baseline values in five of six examined patients. An infusion of low-dose noradrenaline induced an increase in systolic blood pressure in all patients. The extent of the change in systolic blood pressure negatively correlated to that in total peripheral resistance (p < 0.05). Patients with poor vasoconstrictor responses to orthostatic stress tended to exhibit severe reduction of unmyelinated fibres in sural nerve biopsy specimens. In conclusion, both cardiac and peripheral vasomotor autonomic dysfunctions were prevalent in late-onset FAP ATTR Val30Met patients from non-endemic areas, even in those without orthostatic intolerance. However, vasoconstriction by alpha-adrenoceptor agonists was preserved even after denervation, carrying important implications for the management of orthostatic hypotension in FAP.

Diffuse intrinsic pontine glioma (DIPG) has proven to be one of the most challenging of all pediatric cancers. Owing to a historical reticence to obtain tumor tissue for study, and based on an erroneous assumption that the biology of DIPG would mirror that of supratentorial high-grade astrocytomas, innumerable studies have been undertaken-all of which have had a negligible impact on the natural history of this disease. More recently, improvements in neurosurgical techniques have allowed for the safe upfront biopsy of DIPG, which, together with a wider use of autopsy tissue, has led to an evolving understanding of the biology of this tumor. The discovery of a recurrent somatic gain-of-function mutation leading to lysine 27 to methionine (p.Lys27Met, K27M) substitution in histone 3 variants characterizes more than 85% of DIPG, suggesting for the first time the role of the epigenome and histones in the pathogenesis of this disease, and more unified diagnostic criteria. Along with further molecular insights into the pathogenesis of DIPG, rational targets are being identified and studied in the hopes of improving the otherwise dismal outcome for children with DIPG., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Mutations in the tail of histone H3 (K27M) are frequently found in pediatric midline high-grade glioma's but have rarely been reported in other malignancies. Recently, recurrent somatic nucleotide variants in histone H3 (H3 K27M) have been reported in group A posterior fossa ependymoma (EPN&#95;PFA), an entity previously described to have no recurrent mutations. However, the true incidence of H3 K27M mutations in EPN&#95;PFA is unknown., Methods: In order to discern the frequency of K27M mutations in histone H3 in EPN&#95;PFA, we analyzed 151 EPN&#95;PFA previously profiled with genome-wide methylation arrays using a validated droplet digital PCR assay., Results: We identified only 1 case out of 151 EPN&#95;PFA harboring the K27M mutation indicating that histone mutations are extremely rare in EPN&#95;PFA. Morphologically, this single mutated case is clearly consistent with an ependymoma, and the presence of the K27M mutation was confirmed using immunohistochemistry., Discussion: K27M mutations are extremely rare in EPN&#95;PFA. Routine evaluation of K27M mutations in EPN&#95;PFA is of limited utility, and is unlikely to have any bearing on prognosis and/or future risk stratification.

Introduction: Mutations in the C19orf12 gene cause mitochondrial membrane protein associated neurodegeneration (MPAN), an autosomal recessive form of neurodegeneration with brain iron accumulation (NBIA). A limited number of patients with C19orf12 mutations, particularly those with adult onset of symptoms, have been reported., Methods: We sequenced the entire coding region of C19orf12 in 15 Turkish adult probands with idiopathic NBIA. We also performed haplotype analysis in families with a recurrent C19orf12 mutation. Clinical features were collected using a standardized form., Results: Nine of our 15 probands (60%) carried the homozygous c.32C > T mutation in C19orf12 (predicted protein effect: p.Thr11Met). This homozygous mutation co-segregated with the disease in all affected relatives available for testing (16 homozygous subjects). Haplotypes across the C19orf12 locus were identical for a very small region, closest to the mutation, suggesting an old founder, or, two independent founders. The clinical phenotype was characterized by adult onset in most cases (mean 24.5 years, range 10-36), and broad spectrum, including prominent parkinsonism, pyramidal signs, psychiatric disturbances, cognitive decline, and motor axonal neuropathy, in various combinations. On T2- or susceptibility weighted-MRI images, all patients displayed bilateral hypointensities in globus pallidus and substantia nigra, without an eye-of-the-tiger sign; however, hyperintense streaking of the medial medullary lamina between the external and internal parts of globus pallidus was observed frequently., Conclusion: The C19orf12 p.Thr11Met mutation is frequent among adult Turkish patients with MPAN. These findings contribute to the characterization of this important NBIA form, and have direct implications for genetic testing of patients of Turkish origin., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Epigenetic modifications play critical roles in diverse biological processes. Histone Lys-to-Met (K-to-M) mutations act as gain-of-function mutations to inhibit a wide range of histone methyltransferases and are thought to promote tumorigenesis. However, it is largely unknown whether K-to-M mutations impact organismal development. Using Arabidopsis ( Arabidopsis thaliana ) as a model system, we discovered that a transgene exogenously expressing histone 3 Lys-36 to Met mutation (K36M) acts in a dominant-negative manner to cause global reduction of H3K36 methylation. Remarkably, this dominant repressive activity is dosage-dependent and causes strong developmental perturbations including extreme branching and early flowering by affecting the expression of genes involved in developmental and metabolic processes. Besides the established pathological roles of K-to-M mutations in tumor cells, we demonstrate a physiological outcome for K-to-M induced H3K36 hypomethylation. This study provides evidence for a conserved dominant-negative inhibitory role of histone K-to-M mutation across the plant and animal kingdoms. We also highlight the unique ability of K36M mutations to alter plant developmental processes leading to severe pleiotropic phenotypes. Finally, our data suggests K-to-M mutations may provide a useful strategy for altering epigenetic landscapes in organisms where histone methyltransferases are uncharacterized., (© 2017 The author(s). All Rights Reserved.)

The age of onset (AO) of hereditary ATTR amyloidosis (hATTR) is known to vary between populations, with differing characteristics reported according to AO in endemic/non-endemic foci. This was a retrospective study of patients with early AO (<50 years) and late AO (≥50 years) hATTR at our center in Mallorca. Data were collected on patient demographics, clinical disease manifestation, and physical symptoms. A total of 95 patients were analyzed, with mean follow-up of 9 years from diagnosis. The early AO group included 53 patients (33 male) and the late AO group included 42 patients (21 male). Neurologic involvement was the most common initial symptom, although it was significantly more frequent in the late AO vs. early AO group (p = 0.015). Autonomic involvement was observed in 26% of patients in the early AO group, but was rarely observed in the late AO group (5%). During follow up, cardiologic symptoms, renal involvement, and ophthalmologic symptoms were significantly more common in the late AO group (p < 0.05). This retrospective study demonstrates the variation in disease presentation and progression according to AO of hATTR at our Mallorcan center., (© 2016 Peripheral Nerve Society.)

Somatic mutations of the H3F3A and HIST1H3B genes encoding the histone H3 variants, H3.3 and H3.1, were recently identified in high-grade gliomas arising in the thalamus, pons and spinal cord of children and young adults. However, the complete range of patients and locations in which these tumors arise, as well as the morphologic spectrum and associated genetic alterations remain undefined. Here, we describe a series of 47 diffuse midline gliomas with histone H3-K27M mutation. The 25 male and 22 female patients ranged in age from 2 to 65 years (median = 14). Tumors were centered not only in the pons, thalamus, and spinal cord, but also in the third ventricle, hypothalamus, pineal region and cerebellum. Patients with pontine tumors were younger (median = 7 years) than those with thalamic (median = 24 years) or spinal (median = 25 years) tumors. A wide morphologic spectrum was encountered including gliomas with giant cells, epithelioid and rhabdoid cells, primitive neuroectodermal tumor (PNET)-like foci, neuropil-like islands, pilomyxoid features, ependymal-like areas, sarcomatous transformation, ganglionic differentiation and pleomorphic xanthoastrocytoma (PXA)-like areas. In this series, histone H3-K27M mutation was mutually exclusive with IDH1 mutation and EGFR amplification, rarely co-occurred with BRAF-V600E mutation, and was commonly associated with p53 overexpression, ATRX loss (except in pontine gliomas), and monosomy 10., (© 2015 International Society of Neuropathology.)

Lysine to methionine (K-to-M) mutations in genes encoding histone H3 are thought to drive a subset of pediatric brain and bone cancers. These high-frequency K-to-M mutations occur at sites of methylation on histone H3, and tumors containing the mutant histones exhibit a global loss of specific histone methylation marks. Previous studies showed that K-to-M mutant histones, also known as oncohistones, are potent orthosteric inhibitors of specific Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain methyltransferases. However, the biochemical and biophysical details of the interaction between K-to-M mutant histones and the respective SET domain methyltransferases are currently unknown. Here, we use the histone H3K9-directed methyltransferase G9a as a model to explore the mechanism of inhibition by K-to-M oncohistones. X-ray cocrystal structures revealed that the K9M residue of histone H3 occupies the active site cavity of G9a, and kinetic analysis indicates competitive inhibition of G9a by histone H3K9M. Additionally, we find that the cofactor S-adenosyl methionine (SAM) is necessary for stable interaction between G9a and H3K9M histone. Consistent with the formation of a ternary complex, we find that the inhibitory peptide is uncompetitive with regard to SAM. These data and others indicate that K-to-M oncohistones promote global loss of specific lysine methylation through sequestration and inhibition of SAM-bound SET domain methyltransferases.

Transient receptor potential (TRP) cation channels are highly conserved in humans and insects. Some of these channels are expressed in internal organs and their functions remain incompletely understood. By direct knock-in of the GAL4 gene into the trpA1 locus in Drosophila, we identified the expression of this gene in the subesophageal ganglion (SOGs) region. In addition, the neurites present in the dorsal posterior region as well as the drosophila insulin-like peptide 2 (dILP2)-positive neurons send signals to the SOGs. The signal is sent to the crop, which is an enlarged organ of the esophagus and functions as a storage place for food in the digestive system. To systematically investigate the role of TRPA1 in metabolism, we applied non-targeted metabolite profiling analysis together with gas-chromatography/time-of-flight mass spectrometry, with an aim to identify a wide range of primary metabolites. We effectively captured distinctive metabolomic phenotypes and identified specific metabolic dysregulation triggered by TRPA1 mutation based on reconstructed metabolic network analysis. Primarily, the network analysis pinpointed the simultaneous down-regulation of intermediates in the methionine salvation pathway, in contrast to the synchronized up-regulation of a range of free fatty acids. The gene dosage-dependent dynamics of metabolite levels among wild-type, hetero- and homozygous mutants, and their coordinated metabolic modulation under multiple gene settings across five different genotypes confirmed the direct linkages of TRPA1 to metabolism.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brain tumor with a median survival of 1 year after diagnosis. It has been reported recently that about 80% of DIPG cases and 70% of midline glioblastomas contain a mutation at one allele of the H3F3A gene (encoding histone H3 variant H3.3), replacing the lysine 27 with methionine (K27M). In order to facilitate diagnosis of DIPG patients, a quick and reliable method to identify the H3F3A K27M mutation is needed. Here, we describe a real-time PCR-based procedure involving a mutant-specific primer, a blocker oligonucleotide, and a reverse primer that can differentiate samples with H3F3A K27M mutation from those that do not. We first tested four different mutant-specific primers for their ability to selectively amplify H3F3A K27M-mutant allele and found that one primer amplified the mutant allele more efficiently than the rest. We then determined the optimal concentration of blocker oligo that significantly improved amplification of the H3F3A K27M-mutant allele. Using this optimized real-time PCR assay, we analyzed eleven samples, two of which containing H3F3A K27M mutation, and found that these two samples were differentially amplified from the nine others. In addition, we were able to discern the H3F3A K27M mutation in a newly obtained pediatric brainstem glioblastoma sample whose H3.3 status was not known previously, and in three other DIPG samples as well as paraffin embedded samples. These results demonstrate that we have developed a new reliable procedure for detecting the H3F3A K27M mutation in pediatric glioblastoma patient samples.

Although mistranslation is commonly believed to be deleterious, recent evidence indicates that mistranslation can be actively regulated and be beneficial in stress response. Methionine mistranslation in mammalian cells is regulated by reactive oxygen species where cells deliberately alter the proteome through incorporating Met at non-Met positions to enhance oxidative stress response. However, it was not known whether specific, mistranslated mutant proteins have distinct activities from the wild-type protein whose sequence is restrained by the genetic code. Here, we show that Met mistranslation with and without Ca(2+) overload generates specific mutant Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) proteins substituting non-Met with Met at multiple locations. Compared to the genetically encoded wild-type CaMKII, specific mutant CaMKIIs can have distinct activation profiles, intracellular localization and enhanced phenotypes. Our results demonstrate that Met-mistranslation, or "Met-scan" can indeed generate mutant proteins in cells that expand the activity profile of the wild-type protein, and provide a molecular mechanism for the role of regulated mistranslation.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases characterized by memory loss and cognitive impairment. Whereas most AD cases are sporadic, some are caused by mutations in early-onset familial AD (FAD) genes. One FAD gene encodes presenilin 1 (PS1), and a PS1 mutation in methionine 146 impairs homeostatic synaptic plasticity (HSP). We have previously shown that Ca(2+) and calcineurin activity are critical regulators of HSP. Here, we confirm that endoplasmic reticulum-mediated Ca(2+) signals are increased in mutant PS1 neurons. We further show that calcineurin activity is abnormally elevated in the mutant and that inhibition of increased calcineurin activity stabilizes GluA1 phosphorylation, promoting synaptic trafficking of Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, contributing to the recovery of impaired HSP found in the mutant. Because HSP is suggested to have roles during learning and memory formation, increased calcineurin activity-induced impairment of HSP can cause cognitive decline in FAD. Thus, reducing abnormally increased calcineurin activity in AD brain may be beneficial for improving AD-related cognitive decline., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Familial amyloid polyneuropathy (FAP) is a chronic debilitating multi-organic disorder, mainly assessed using ordinal-based impairment measures. To date, no outcome measure at the activity and participation level has been constructed in FAP. The current study aimed to design an interval activity/participation scale for FAP through Rasch methodology. A preliminary FAP Rasch-built overall disability scale (pre-FAP-RODS) containing 146 activity/participation items was assessed twice (interval: 2-4 week; test-retest reliability) in 248 patients with Val30Met FAP examined in Porto, Portugal, of which 65.7% have received liver transplantation. An ordinal-based 24-item FAP-symptoms inventory questionnaire (FAP-SIQ) was also assessed (validity purposes). The pre-FAP-RODS and FAP-SIQ data were subjected to Rasch analyses. The pre-FAP-RODS did not meet model's expectations. On the basis of requirements such as misfit statistics, differential item functioning, and local dependency, items were systematically removed until a final 34-item FAP-RODS(©) was constructed fulfilling all Rasch requirements. Acceptable reliability/validity scores were demonstrated. In conclusion, the 34-item FAP-RODS(©) is a disease-specific interval measure suitable for detecting activity and participation restrictions in patients with FAP. The use of the FAP-RODS(©) is recommended for future international clinical trials in patients with Val30Met FAP determining its responsiveness and its cross-cultural validation. Its expansion to other forms of FAP should also be focus of future clinical studies., (© 2015 Peripheral Nerve Society.)

Biosynthesis of aspartate (Asp)-derived amino acids lysine (Lys), methionine (Met), threonine (Thr), and isoleucine involves monofunctional Asp kinases (AKs) and dual-functional Asp kinase-homoserine dehydrogenases (AK-HSDHs). Four-week-old loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in the AK-HSDH2 gene had increased amounts of Asp and Asp-derived amino acids, especially Thr, in leaves. To explore mechanisms behind this phenotype, we obtained single mutants for other AK and AK-HSDH genes, generated double mutants from ak-hsdh2 and ak mutants, and performed free and protein-bound amino acid profiling, transcript abundance, and activity assays. The increases of Asp, Lys, and Met in ak-hsdh2 were also observed in ak1-1, ak2-1, ak3-1, and ak-hsdh1-1. However, the Thr increase in ak-hsdh2 was observed in ak-hsdh1-1 but not in ak1-1, ak2-1, or ak3-1. Activity assays showed that AK2 and AK-HSDH1 are the major contributors to overall AK and HSDH activities, respectively. Pairwise correlation analysis revealed positive correlations between the amount of AK transcripts and Lys-sensitive AK activity and between the amount of AK-HSDH transcripts and both Thr-sensitive AK activity and total HSDH activity. In addition, the ratio of total AK activity to total HSDH activity negatively correlates with the ratio of Lys to the total amount of Met, Thr, and isoleucine. These data led to the hypothesis that the balance between Lys-sensitive AKs and Thr-sensitive AK-HSDHs is important for maintaining the amounts and ratios of Asp-derived amino acids., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Familial fatal insomnia (FFI) is fatal disorder characterized by damage to select thalamic nuclei, together with progressive insomnia and dysautonomia. In subjects carrying the D178N prion protein (PRNP) mutation, distinct phenotypes can be observed, depending on the methionine (Met) /valine (Val) codon 129 polymorphism. We report here a Chinese case of FFI with a D178N/Met129 genotype of the PRNP gene, who exhibited rapidly progressive dementia combined with behavioral disturbances and paroxysmal limb myoclonus. Our patient did not show refractory insomnia early in the disease course, nor demonstrate typical MRI and EEG alterations. There was remarkable family history of similar symptoms.

Background: Mutations within exons 16 and 17 of the amyloid-β protein precursor (AβPP) gene were the first known causes of early-onset familial Alzheimer's disease (EOFAD). Since the first AβPP mutation was reported, 39 different AβPP variations have been discovered in EOFAD., Objective: We described a novel AβPP M722K mutation found in a Chinese familial Alzheimer's disease pedigree and confirmed its effects on amyloid-β (Aβ) secretion and tau phosphorylation., Methods: We performed direct sequencing of exons 16 and 17 of the AβPP gene and coding exons 3-12 of the PSEN1 and PSEN2 genes for genetic analysis. N2a cells were transfected with wild-type AβPP, AβPP constructs harboring the M722K mutation, or AβPP constructs harboring the Swedish mutation to demonstrate the effects of the AβPP M722K mutation on Aβ secretion and tau phosphorylation., Results: Different phenotypes of patients carrying the AβPP M722K mutation maybe were related to different apolipoprotein E genotypes. The expression of AβPP M722K in mouse neuroblastoma N2a cells induced a 1.7-fold increased ratio of Aβ 42 to Aβ 40 without changes in sAβPPα and sAβPPβ. Tau phosphorylation at the AT8 sites was also increased., Conclusion: Maybe the AβPP M722K mutation contributed to the cause of EOFAD in this Chinese pedigree mediated by increased Aβ 42/Aβ 40. Further studies should be conducted to validate the pathogenicity of AβPP M722K and the interactions among γ-secretase, APOE, and AβPP.

Studies in pediatric high-grade astrocytomas (HGA) by our group and others have uncovered recurrent somatic mutations affecting highly conserved residues in histone 3 (H3) variants. One of these mutations leads to analogous p.Lys27Met (K27M) mutations in both H3.3 and H3.1 variants, is associated with rapid fatal outcome, and occurs specifically in HGA of the midline in children and young adults. This includes diffuse intrinsic pontine gliomas (80 %) and thalamic or spinal HGA (>90 %), which are surgically challenging locations with often limited tumor material available and critical need for specific histopathological markers. Here, we analyzed formalin-fixed paraffin-embedded tissues from 143 pediatric HGA and 297 other primary brain tumors or normal brain. Immunohistochemical staining for H3K27M was compared to tumor genotype, and also compared to H3 tri-methylated lysine 27 (H3K27me3) staining, previously shown to be drastically decreased in samples carrying this mutation. There was a 100 % concordance between genotype and immunohistochemical analysis of H3K27M in tumor samples. Mutant H3K27M was expressed in the majority of tumor cells, indicating limited intra-tumor heterogeneity for this specific mutation within the limits of our dataset. Both H3.1 and H3.3K27M mutants were recognized by this antibody while non-neoplastic elements, such as endothelial and vascular smooth muscle cells or lymphocytes, did not stain. H3K27me3 immunoreactivity was largely mutually exclusive with H3K27M positivity. These results demonstrate that mutant H3K27M can be specifically identified with high specificity and sensitivity using an H3K27M antibody and immunohistochemistry. Use of this antibody in the clinical setting will prove very useful for diagnosis, especially in the context of small biopsies in challenging midline tumors and will help orient care in the context of the extremely poor prognosis associated with this mutation.

Pediatric glioblastomas (GBM) are highly aggressive and lethal tumors. Recent sequencing studies have shown that ~30 % of pediatric GBM and ~80 % of diffuse intrinsic pontine gliomas show K27M mutations in the H3F3A gene, a variant encoding histone H3.3. H3F3A K27M mutations lead to global reduction in H3K27me3. Our goal was to develop biomarkers for the histopathologic detection of these tumors. Therefore, we evaluated the utility of measuring H3K27me3 global reduction as a histopathologic and prognostic biomarker and tested an antibody directed specifically against the H3.3 K27M mutation in 290 samples. The study cohort included 203 pediatric (including 38 pediatric high-grade astrocytomas) and 38 adult brain tumors of various subtypes and grades and 49 non-neoplastic reactive brain tissues. Detection of H3.3 K27M by immunohistochemistry showed 100 % sensitivity and specificity and was superior to global reduction in H3K27me3 as a biomarker in diagnosing H3F3A K27M mutations. Moreover, cases that stained positive for H3.3 K27M showed a significantly poor prognosis compared to corresponding negative tumors. These results suggest that immunohistochemical detection of H3.3 K27M is a sensitive and specific surrogate for the H3F3A K27M mutation and defines a prognostically poor subset of pediatric GBM.

Objective: To evaluate the long-term efficacy and safety of diflunisal in late-onset familial amyloid polyneuropathy (FAP) in a Japanese endemic area., Methods: Consecutive six FAP patients (mean age: 65.8 ± 7.3 years) with a transthyretin (TTR) Val30Met mutation from an endemic area of late-onset FAP were prospectively recruited to an open label study with oral diflunisal (250 mg twice a day). We evaluated clinical symptoms, Kumamoto FAP score, modified body mass index (mBMI), Medical Research Council sum score, nerve conduction studies (NCS), electrocardiogram (ECG), ECG Holter monitor test, echocardiography, and (123)iodine-metaiodobenzylguanidine ((123)I-MIBG) myocardial scintigraphy., Results: One patient ceased to take diflunisal because of hematuria which was reversible. The other five patients were treated with diflunisal for 3-5 (4.4 ± 0.9 years) years. Autonomic symptoms (orthostatic hypotension and gastrointestinal symptoms) disappeared after treatment in two of the four patients with the symptoms. Delayed heart to mediastinum ratio on (123)I-MIBG imaging, a marker of cardiac postganglionic sympathetic nerve function, increased during the three-year treatment. mBMI was maintained through observation period. While, motor and sensory symptoms, Kumamoto FAP scores, and data on NCS gradually deteriorated., Conclusion: Diflunisal might be effective especially for autonomic dysfunction in late-onset FAP with a TTR Val30Met mutation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: To investigate the effect of amino acids around two Leu-based motifs (Leu 7/Ile 8 and Met 16/Leu 17) in the cytosolic tail of porcine Ia-associated invariant chain (Ii) on endomembrane system positioning function., Methods: At first, two Leu-based motifs and amino acids around motifs were mutated through site-directed mutagenesis of the megaprimer PCR method, and the mutant genes were ligated to the vector pEGFP-C1, respectively. Twenty-one mutations of Ii recombinant plasmid were obtained and transiently transfected into the COS-7 cells by Lipofectamine(TM) 2000. Fluorescence microscopy was used to detect the intracellular localization of mutation Ii., Results: Leu 7/Ile 8 and Met 16/Leu 17 independently mediated the intracellular localization of mutation Ii. When one leucine motif was kept and the amino acid around the other leucine motif was mutated, fusion protein GFP-Ii was located in the endomembrane system or distributed in the whole cell., Conclusion: pig Ii comprises two independent sorting signals, and the functional Leu-based sorting signal requires specific neighboring residues.

A 73-year-old man with recurrent periodic paralytic episodes lasting for two weeks each admitted to our hospital because of the leg weakness and the elevated value of serum creatine kinase. On admission, weakness in the proximal legs and mild eye lid myotonia were noted. Needle electromyography revealed abundant myotonic discharges. The prolonged exercise test showed a continuous reduction of compound muscle action potentials in the abductor digiti minimi muscle. Direct sequencing of SCN4A in the proband showed a G-to-A alteration at position 4774 that results in a change of 1592(nd) methionine to valine (M1592V). Cosegregation regarding the M1592V mutation and paralytic phenotype in this family was confirmed. Two cardinal features in this family were longer paralytic episodes compared to classical hyperkalemic/normokalemic periodic paralysis and the normal potassium value during the paralytic episodes. This study together with antecedent reports indicates that M1592V mutation shares a much greater clinical diversity ranging from congenital paramyotonia to periodic paralysis with a longer duration.

Mutations in PSEN1 are the most common cause of autosomal dominant familial Alzheimer's disease (FAD). We describe an African-American woman with a family history consistent with FAD who began to experience cognitive decline at age 50. Her clinical presentation, MRI, FDG-PET, and PIB-PET scan findings were consistent with AD and she was found to have a novel I238M substitution in PSEN1. As this mutation caused increased production of Aβ42 in an in vitro assay, was not present in two population databases, and is conserved across species, it is likely to be pathogenic for FAD.

H3F3A mutations are seen in ∼30% of pediatric glioblastoma (GBMs) and involve either the lysine residue at position 27 (K27M) or glycine at position 34 (G34R/V). Sixteen genes encode histone H3, each variant differing in only a few amino acids. Therefore, how mutations in a single H3 gene contribute to carcinogenesis is unknown. H3F3A K27M mutations are predicted to alter methylation of H3K27. H3K27me3 is a repressive mark critical to stem cell maintenance and is mediated by EZH2, a member of the polycomb-group (PcG) family. We evaluated H3K27me3 and EZH2 expression using immunohistochemistry in 76 pediatric brain tumors. H3K27me3 was lowered/absent in tumor cells but preserved in endothelial cells and infiltrating lymphocytes in six out of 20 GBMs. H3K27me3 showed strong immunoreactivity in all other tumor subtypes. Sequencing of GBMs showed H3F3A K27M mutations in all six cases with lowered/absent H3K27me3. EZH2 expression was high in GBMs, but absent/focal in other tumors. However, no significant differences in EZH2 expression were observed between H3F3A K27M mutant and wild type GBMs, suggesting that EZH2 mediated trimethylation of H3K27 is inhibited in GBM harboring K27M mutations. Our results indicate that H3F3A K27M mutant GBMs show decreased H3K27me3 that may be of both diagnostic and biological relevance., (© 2013 International Society of Neuropathology.)

One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5'-GGAGGC-3' sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5'-GGAGGA-3', by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein.

The corpus callosum is the principal cerebral commissure connecting the right and left hemispheres. The development of the corpus callosum is under tight genetic control, as demonstrated by abnormalities in its development in more than 1,000 genetic syndromes. We recruited more than 25 families in which members affected with corpus callosum hypoplasia (CCH) lacked syndromic features and had consanguineous parents, suggesting recessive causes. Exome sequence analysis identified C12orf57 mutations at the initiator methionine codon in four different families. C12orf57 is ubiquitously expressed and encodes a poorly annotated 126 amino acid protein of unknown function. This protein is without significant paralogs but has been tightly conserved across evolution. Our data suggest that this conserved gene is required for development of the human corpus callosum., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Misfolding of the natively α-helical prion protein into a β-sheet rich isoform is related to various human diseases such as Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. In humans, the disease phenotype is modified by a methionine/valine polymorphism at codon 129 of the prion protein gene. Using a combination of hydrogen/deuterium exchange coupled to NMR spectroscopy, hydroxyl radical probing detected by mass spectrometry, and site-directed mutagenesis, we demonstrate that stop mutants of the human prion protein have a conserved amyloid core. The 129 residue is deeply buried in the amyloid core structure, and its mutation strongly impacts aggregation. Taken together the data support a critical role of the polymorphic residue 129 of the human prion protein in aggregation and disease.

In the current study we aimed to show the common YMDD motif mutations in viral polymerase gene in chronic hepatitis B patients during lamivudine and adefovir therapy. Forty-one serum samples obtained from chronic hepatitis B patients (24 male, 17 female; age range: 34-68 years) were included in the study. HBV-DNA was extracted from the peripheral blood of the patients using an extraction kit (Invisorb, Instant Spin DNA/ RNA Virus Mini Kit, Germany). A line probe assay and direct sequencing analyses (INNO-LIPA HBV DR v2; INNOGENETICS N.V, Ghent, Belgium) were applied to determine target mutations of the viral polymerase gene in positive HBV-DNA samples. A total of 41 mutations located in 21 different codons were detected in the current results. In 17 (41.5%) patients various point mutations were detected leading to lamivudin, adefovir and/ or combined drug resistance. Wild polymerase gene profiles were detected in 24 (58.5%) HBV positive patients of the current cohort. Eight of the 17 samples (19.5%) having rtM204V/I/A missense transition and/or transversion point mutations and resistance to lamivudin. Six of the the mutated samples (14.6%) having rtL180M missense transversion mutation and resistance to combined adefovir and lamivudin. Three of the mutated samples (7.5%) having rtG215H by the double base substituation and resistance to adefovir. Three of the mutated samples (7.5%) having codon rtL181W due to the missense transversion point mutations and showed resistance to combined adefovir and lamivudin. Unreported novel point mutations were detected in the different codons of polymerase gene region in the current HBV positive cohort fromTurkish population. The current results provide evidence that rtL180M and rtM204V/I/A mutations of HBV-DNA may be associated with a poor antiviral response and HBV chronicity during conventional therapy in Turkish patients.

We investigated the association between TOMM40 rs10524523, age of onset, and memory performance in patients with the PSEN1 M146L mutation in a large familial Alzheimer's disease Calabrian kindred, with a wide variability of onset not attributable to APOE. APOE33/TOMM40VL/VL patients showed a tendency for an earlier age at onset compared to those with APOE33/TOMM40VL/S and APOE33/TOMM40S/S. Moreover, TOMM40VL/VL patients had better memory performance, when compared to TOMM40S/S but not to TOMM40VL/S patients, so there is not a dose-dependent effect. Our results suggest that, in the presence of the PSEN1 mutation, the slight difference in age of onset together with memory performance could be influenced by TOMM40 genotypes.

Our objective was to elucidate the genetic epidemiology of familial amyotrophic lateral sclerosis (FALS) and sporadic ALS (SALS) with OPTN mutations in the Japanese population. Mutational analysis of OPTN was conducted in 18 FALS pedigrees in whom mutations in other causative genes have been excluded and in 218 SALS patients by direct nucleotide sequence analysis. Novel non-synonymous variants identified in ALS patients were further screened in 271 controls. Results showed that although no mutations were identified in the FALS pedigrees, a novel heterozygous non-synonymous variant c.481G > A (p.V161M) was identified in one SALS patient, who originated from the southernmost part of the Kii Peninsula. The mutation was not present in 271 controls. As the clinical feature, the patient carrying V161M showed predominantly upper motor neuron signs with slow progression. This study suggests that mutations in OPTN are not the main cause of ALS in the Japanese population.

Genetic deletion or mutations of presenilin genes (PS1/PS2) cause familial Alzheimer's disease and calcium (Ca²⁺) signaling abnormalities. PS1/PS2 act as endoplasmic reticulum (ER) Ca²⁺ leak channels that facilitate passive Ca²⁺ leak across ER membrane. Studies with PS1/PS2 double knockout (PS1/PS2-DKO) mouse embryonic fibroblasts showed that PS1/PS2 were responsible for 80% of passive Ca²⁺ leak from the lumen of endoplasmic reticulum to cytosol. Transient transfection of the wild type PS1 expression construct increased cytoplasmic Ca²⁺ as a result of Ca²⁺ leak across ER membrane whereas the FADPS1 (PS1-M146V) mutation construct alone or in combination with the wild type PS1 expression construct abrogated Ca²⁺ leak in SK-N-SH cells. Inhibition of basal c-jun-NH2-terminal kinase (JNK) activity by JNK inhibitor SP600125 repressed PS1 transcription and PS1 protein expression by augmenting p53 protein level in SK-N-SH cells (Lee and Das 2008). In this report we also showed that repression of PS1 transcription by JNK inhibitor SP600125 inhibited passive Ca²⁺ leak across ER membrane which could be rescued by expressing PS1 wild type and not by expressing FADPS1 (PS1-M146V) under a SP600125 non-responsive promoter. Treatment of SK-N-SH cells with SP600125 also triggered InsP3R-mediated Ca²⁺ release from the ER by addition of 500 nM bradykinin, an agonist of InsP3 receptor (InsP3R1) without changing the expression of InsP3R1. This data confirms that SP600125 increases the Ca²⁺ store in the ER by inhibiting PS1-mediated Ca²⁺ leak across ER membrane. p53, ZNF237 and Chromodomain helicase DNA-binding protein 3 which are repressors of PS1 transcription, also reduced Ca²⁺ leak across ER membrane in SK-N-SH cells but γ-secretase inhibitor or dominant negative γ-secretase-specific PS1 mutant (PS1-D257A) had no significant effect. Therefore, p53, ZNF237, and Chromodomain helicase DNA-binding protein 3 inhibit the function ER Ca²⁺ leak channels to regulate both ER and cytoplasmic Ca²⁺ levels and may potentially control Ca²⁺-signaling function of PS1., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Objective: This study aimed to determine the natural prevalence of variants of tyrosine-methionine-aspartic acid-aspartic acid (YMDD) motif in patients with chronic hepatitis B (CHB), and to explore its relation with demographic and clinical features, hepatitis B virus (HBV) genotypes, and HBV DNA levels., Methods: A total of 1,042 antiviral treatment naïve CHB patients (including with lamivudine [LAM]) in the past year were recruited from outpatient and inpatient departments of six centers from December 2008 to June 2010. YMDD variants were analyzed using the HBV drug resistance line probe assay (Inno-Lipa HBV-DR). HBV genotypes were detected with polymerase chain reaction (PCR) microcosmic nucleic acid cross-ELISA, and HBV deoxyribonucleic acid (DNA) was quantitated with real-time PCR. All serum samples underwent tests for HBV, HCV, and HDV with ELISA., Results: YMDD variants were detected in 23.3% (243/1042) of CHB patients. YMDD mutation was accompanied by L180M mutation in 154 (76.9%) patients. Both wild-type HBV and YMDD variant HBV were present in 231 of 243 patients. Interestingly, 12 patients had only YIDD and/or YVDD variants without wild YMDD motif. In addition, 27.2% (98/359) of HbeAg-positive patients had YMDD mutations, which was higher than that in HbeAg-negative patients (21.2%, 145/683). The incidence of YMDD varied among patients with different HBV genotypes, but the difference was not significant. Moreover, the incidence of YMDD in patients with high HBV DNA level was significantly higher than that in those with low HBV DNA level., Conclusion: Mutation of YMDD motif was detectable at a high rate in CHB patients in this study. The incidence of YMDD may be correlated with HBeAg and HBV DNA level.

Background: Continuous lamivudine therapy is associated with high rates of YMDD mutations, which are the main causes of drug resistance. The current study explores the association of the emergence of YMDD mutations with pretherapy HBV genotype, HBV-DNA levels, HBeAg status, and serum alanine aminotransferase (ALT) levels in Chinese patients receiving lamivudine therapy for chronic hepatitis B., Methods: A total of 319 chronic hepatitis B patients who received lamivudine therapy for more than a year were enrolled in this study. YMDD mutations, HBV genotype, HBV-DNA levels, HBeAg status, and ALT levels were determined prior to their lamivudine treatment and every three months for a year of this therapy., Results: Among the 319 patients, 137 (42.95%) were infected with genotype B and 182 (57.05%) with genotype C. Up to 94 patients (29.47%) developed YMDD mutations within one year of lamivudine therapy. Furthermore, 50 patients with HBV genotype B and 44 patients with genotype C developed YMDD mutations (36.50% vs 24.18%, P<0.05). Logistic regression analysis showed that pretherapy HBV genotype, HBV-DNA levels, and HBeAg status are independent factors for the emergence of YMDD mutations (HBV genotype: OR=2.159, 95% CI 1.291-3.609, P=0.003; HBV-DNA: OR=1.653, 95% CI 1.231-2.218, P=0.001; HBeAg: OR=2.021, 95% CI 1.201-3.399, P=0.008)., Conclusions: HBV genotype, HBV-DNA levels, and HBeAg status at baseline are the independent factors associated with the emergence of YMDD mutations among Chinese patients receiving lamivudine therapy for chronic hepatitis B. These findings are helpful to the development of therapeutic strategies for these patients.

Background: Mutations in LRRK2 are associated with familial and sporadic Parkinson's disease (PD). Subjects with PD caused by LRRK2 mutations show pleiotropic pathology that can involve inclusions containing α-synuclein, tau or neither protein. The mechanisms by which mutations in LRRK2 lead to this pleiotropic pathology remain unknown., Objectives: To investigate mechanisms by which LRRK2 might cause PD., Methods: We used systems biology to investigate the transcriptomes from human brains, human blood cells and Caenorhabditis elegans expressing wild-type LRRK2. The role of autophagy was tested in lines of C. elegans expressing LRRK2, V337M tau or both proteins. Neuronal function was measured by quantifying thrashing., Results: Genes regulating autophagy were coordinately regulated with LRRK2. C. elegans expressing V337M tau showed reduced thrashing, as has been noted previously. Coexpressing mutant LRRK2 (R1441C or G2019S) with V337M tau increased the motor deficits. Treating the lines of C. elegans with an mTOR inhibitor that enhances autophagic flux, ridaforolimus, increased the thrashing behavior to the same level as nontransgenic nematodes., Conclusion: These data support a role for LRRK2 in autophagy, raise the possibility that deficits in autophagy contribute to the pathophysiology of LRRK2, and point to a potential therapeutic approach addressing the pathophysiology of LRRK2 in PD., (Copyright © 2011 S. Karger AG, Basel.)

Mutations in the Presenilin 2 gene (PSEN2) represent the less frequent genetic cause of familial Alzheimer's disease (FAD). Only eight PSEN2 mutations, reported in approximately 27 families, satisfied strict criteria of pathogenicity. We reported a patient with early-onset FAD and the PSEN2 p.Met239Ile mutation, presenting with severe executive dysfunction and myoclonic tremor, associated with memory loss. Brain SPECT study showed an early hypoperfusion of the frontal cortex. We confirmed the pathogenicity of PSEN2 p.Met239Ile mutation and its heterogeneous phenotypic expression. The modulating effect of the Apolipoprotein E and Prion Protein gene polymorphisms on the phenotypic variability was not confirmed.

The frequency with which the yeast [PSI(+)] prion form of Sup35 arises de novo is controlled by a number of genetic and environmental factors. We have previously shown that in cells lacking the antioxidant peroxiredoxin proteins Tsa1 and Tsa2, the frequency of de novo formation of [PSI(+)] is greatly elevated. We show here that Tsa1/Tsa2 also function to suppress the formation of the [PIN(+)] prion form of Rnq1. However, although oxidative stress increases the de novo formation of both [PIN(+)] and [PSI(+)], it does not overcome the requirement of cells being [PIN(+)] to form the [PSI(+)] prion. We use an anti-methionine sulfoxide antibody to show that methionine oxidation is elevated in Sup35 during oxidative stress conditions. Abrogating Sup35 methionine oxidation by overexpressing methionine sulfoxide reductase (MSRA) prevents [PSI(+)] formation, indicating that Sup35 oxidation may underlie the switch from a soluble to an aggregated form of Sup35. In contrast, we were unable to detect methionine oxidation of Rnq1, and MSRA overexpression did not affect [PIN(+)] formation in a tsa1 tsa2 mutant. The molecular basis of how yeast and mammalian prions form infectious amyloid-like structures de novo is poorly understood. Our data suggest a causal link between Sup35 protein oxidation and de novo [PSI(+)] prion formation.