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Genome editing provides a new therapeutic strategy to cure genetic diseases. The recently developed CRISPR-Cas9 base editing technology has shown great potential to repair the majority of pathogenic point mutations in the patient's DNA precisely. Base editor is the fusion of a Cas9 nickase with a base-modifying enzyme that can change a nucleotide on a single strand of DNA without generating double-stranded DNA breaks. However, a major limitation in applying such a system is the prerequisite of a protospacer adjacent motif sequence at the desired position relative to the target site. Progress has been made to increase the targeting scope of base editors by engineering SpCas9 protein variants, establishing systems with broadened editing windows, characterizing new SpCas9 orthologs, and developing prime editing technology. In this review, we discuss recent progress in the development of CRISPR base editing, focusing on its targeting scope, and we provide a workflow for selecting a suitable base editor based on the target nucleotide sequences.

Recent advances in genome sequencing have greatly improved our ability to understand and identify the causes of genetic diseases. However, there remains an urgent need for innovative, safe, and effective treatments for these diseases. CRISPR-based genome editing systems have become important and powerful tools in the laboratory, and efforts are underway to translate these into patient therapies. Therapeutic base editing is one form of genome engineering that has gained much interest because of its simplicity, specificity, and effectiveness. Base editors are a fusion of a partially deactivated Cas9 enzyme with nickase function, together with a base-modifying enzyme. They are capable of precisely targeting and repairing a pathogenic mutation to restore the normal function of a gene, ideally without disturbing the rest of the genome. In the past year, research has identified new safety concerns of base editors and sparked new innovations to improve their safety. In this review, we provide an overview of the recent advances in the safety and effectiveness of therapeutic base editors and prime editing.

This study investigated the relationship between acyl homoserine lactones (AHLs)-based quorum sensing (QS) and the properties of Anammox granular sludge at low temperatures (11-23°C). Results indicated that adding different concentrations of AHLs inhibitors reduced the content of N-hexanoyl-dl-homoserine lactone (C6-HSL) and N-octanoyl-dl-homoserinelactone (C8-HSL) in Anammox granules on different degrees at different operation temperatures, which led to the deterioration of granules stability and activity. The important role of endogenous C6-HSL and C8-HSL signals in maintaining Anammox granular sludge stability and activity in low-temperature conditions was revealed. In addition, in the process of reducing operation temperatures, another type of AHL signal (N-(3-oxooctanoyl)-l-homoserine lactone, 3OC8-HSL) was released by Anammox granules. The effects of exogenous C8-HSL on the strength, average diameter, and density of Anammox granules were closely related to the operation temperature. When the operation temperature ranged from 11°C to 16°C, the stability of granules could be significantly improved by exogenous C8-HSL. In addition, the addition of C6-HSL and 3OC8-HSL promoted the activity of Anammox granules when the operation temperatures of the reactors were 11-23°C. This study proposed a novel approach to improve the properties of Anammox granules at low temperatures from the perspective of QS. PRACTITIONER POINTS: Endogenous AHLs played an important role in maintaining the activity and stability of Anammox granules at 11-23°C. Exogenous C8-HSL improved the granules stability at the low temperature of 11-16°C. Exogenous C6-HSL or 3OC8-HSL promoted the granules activity at 11-23°C. Supply a novel way to improve the Anammox granules performance at low temperatures.

This study investigated the effects of denitrification sludge EPS enhanced (DS-EPSCN) by a fluctuating carbon and nitrogen ratio (C/N) cultivation strategy on the properties of Anammox granules under extreme acid or alkaline shock. The results showed that the DS-EPSCN significantly improved the nitrogen removal performance of low-density Anammox granular sludge (Granules-L) and high-density Anammox granular sludge (Granules-H) under extreme acid shock (pH 5.0). The contents of high-molecular-weight substances (such as aromatic proteins and polysaccharides) in the DS-EPSCN rose markedly, contributing to a substantial increase in the flocculation efficiency under acidic conditions and increasing the granule stability. In addition, abundant amounts of N-butyryl- dl -homoserine lactone (C4-HSL) and N-hexanoyl- dl -homoserine lactone (C6-HSL) in the DS-EPSCN promoted the granule activity. However, under extreme alkaline shock (pH 10.5), the flocculation efficiency of the DS-EPSCN was poor, and the addition of DS-EPSCN had no influence on the stability of the granules but improved the activity of the Granules-H. The reason was that the release mechanism of the endogenous acyl-homoserine lactone (AHL) signals in the Granules-H was activated by the exogenous C4-HSL and C6-HSL in the DS-EPSCN under alkaline conditions, leading to increased Granules-H activity. This research provides a novel approach to enhance the resistance of Anammox granular sludge to extreme pH shock.

We describe neurotransmitter abnormalities in two patients with drug-resistant epilepsy resulting from deleterious de novo mutations in sodium channel genes. Whole exome sequencing identified a de novo SCN2A splice-site mutation (c.2379 +1G>A, p.G1u717Gly.fs*30) resulting in deletion of exon 14, in a 10-year old male with early onset global developmental delay, intermittent ataxia, autism, hypotonia, epileptic encephalopathy and cerebral/cerebellar atrophy. In the cerebrospinal fluid both homovanillic acid and 5-hydroxyindoleacetic acid were significantly decreased; extensive biochemical and genetic investigations ruled out primary neurotransmitter deficiencies and other known inborn errors of metabolism. In an 8-year old female with an early onset intractable epileptic encephalopathy, developmental regression, and progressive cerebellar atrophy, a previously unreported de novo missense mutation was identified in SCN8A (c.5615G>A; p.Arg1872GIn), affecting a highly conserved residue located in the C-terminal of the Na(v)1.6 protein. Aside from decreased homovanillic acid and 5-hydroxyindoleacetic acid, 5-methyltetrahydrofolate was also found to be low. We hypothesize that these channelopathies cause abnormal synaptic mono-amine metabolite secretion/uptake via impaired vesicular release and imbalance in electrochemical ion gradients, which in turn aggravate the seizures. Treatment with oral 5-hydroxytryptophan, L-Dopa/Carbidopa, and a dopa agonist resulted in mild improvement of seizure control in the male case, most likely via dopamine and serotonin receptor activated signal transduction and modulation of glutamatergic, GABA-ergic and glycinergic neurotransmission. Neurotransmitter analysis in other sodium channelopathy patients will help validate our findings, potentially yielding novel treatment opportunities. (C) 2015 Elsevier Inc. All rights reserved

Background: Previous clinical studies have reported that clinical value of high-frequency ultrasound combined computed tomography (HFUCT) is used for diagnosis of thyroid tumor (TT). However, no study has investigated this topic systematically. Therefore, this study will evaluate the clinical value of HFUCT for the diagnosis of TT. Methods: We will search the databases of Cochrane Library, EMBASE, PUBMED, SCOPUS, Web of Science, OpenGrey, Cumulative Index to Nursing and Allied Health Literature, Allied and Complementary Medicine Database, and China National Knowledge Infrastructure from any time period published to the present. We will consider all case-controlled studies that assessed the clinical value of HFUCT for diagnosis of TT. Two authors will independently scan titles and abstracts to check eligible studies, followed by full-text read. We will extract data and assess study quality using Quality Assessment of Diagnostic Accuracy Studies tool. RevMan 5.3 software will be utilized for data pooling and statistical analysis. Results: This study will be performed to assess the clinical value of HFUCT for the diagnosis of TT, and will provide an evidence-based synthesis for clinical application and further study. Conclusion: Summary of this study will provide the latest evidence to determine whether HFUCT can be used for TT diagnosis accurately. Study registration: INPLASY202040022.

Thermal gravimetric analysis is an important method in the study of biomass. In this paper, using this method on elmwood, through pyrolysis experimental analysis at different heating rates (10,30,50,80 °C / min), get the TG-DTG curves to investigate the pyrolysis characteristics of elmwood. The results show that the pyrolysis process of Elmwood are divided into three stages; With the increasing of heating rate, the major pyrolysis temperature range expansion, the temperature corresponding maximum weight loss rate rose; while taking advantage of Lyon integration algorithm for solving the activation energy of 78.47-271.58kj/mol. indicating Arrhenius first-order single-step reaction with biomass pyrolysis reaction has good linearity.

Objective— High-density lipoproteins (HDL) are considered to protect against atherosclerosis in part by facilitating the removal of cholesterol from peripheral tissues. However, factors regulating lipid efflux are incompletely understood. We previously identified a variant in adenosine triphosphate–binding cassette transporter A8 ( ABCA8 ) in an individual with low HDL cholesterol (HDLc). Here, we investigate the role of ABCA8 in cholesterol efflux and in regulating HDLc levels. Approach and Results— We sequenced ABCA8 in individuals with low and high HDLc and identified, exclusively in low HDLc probands, 3 predicted deleterious heterozygous ABCA8 mutations (p.Pro609Arg [P609R], IVS17-2 A>G and p.Thr741Stop [T741X]). HDLc levels were lower in heterozygous mutation carriers compared with first-degree family controls (0.86±0.34 versus 1.17±0.26 mmol/L; P =0.005). HDLc levels were significantly decreased by 29% ( P =0.01) in Abca8b −/− mice on a high-cholesterol diet compared with wild-type mice, whereas hepatic overexpression of human ABCA8 in mice resulted in significant increases in plasma HDLc and the first steps of macrophage-to-feces reverse cholesterol transport. Overexpression of wild-type but not mutant ABCA8 resulted in a significant increase (1.8-fold; P =0.01) of cholesterol efflux to apolipoprotein AI in vitro. ABCA8 colocalizes and interacts with adenosine triphosphate–binding cassette transporter A1 and further potentiates adenosine triphosphate–binding cassette transporter A1–mediated cholesterol efflux. Conclusions— ABCA8 facilitates cholesterol efflux and modulates HDLc levels in humans and mice.

Clinical and laboratory data were collected from three Finnish patients including a sibling pair and another unrelated child with unexplained childhood hypoglycemia. Transient elevation of alanine transaminase, lactate and tricarboxylic acid cycle intermediates, especially fumarate, were noticed in urine organic acid analysis. Exome sequencing was performed for the patients and their parents. A novel homozygous PCK1 c.925G>A (p.G309R) mutation was detected in all affected individuals. COS-1 cells transfected with mutant PCK1 transcripts were used to study the pathogenic nature of the detected variant. The COS-1 transfected cells showed the mutant gene to be incapable of producing a normally functioning cytosolic phosphoenolpyruvate carboxykinase (PEPCK) enzyme. This report further delineates the clinical phenotype of isolated cytosolic PEPCK deficiency and offers a metabolic pattern helping to recognize these patients. Cytosolic PEPCK deficiency should be considered in the differential diagnosis of children presenting with hypoglycemia, hepatic dysfunction and elevated tricarboxylic acid intermediates in urinary organic acid analysis.

Primary 5-oxoprolinuria (pyroglutamic aciduria) is caused by a genetic defect in the gamma-glutamyl cycle, affecting either glutathione synthetase or 5-oxoprolinase. While several dozens of patients with glutathione synthetase deficiency have been reported, with hemolytic anemia representing the clinical key feature, 5-oxoprolinase deficiency due to OPLAH mutations is less frequent and so far has not attracted much attention. This has prompted us to investigate the clinical phenotype as well as the underlying genotype in patients from 14 families of various ethnic backgrounds who underwent diagnostic mutation analysis following the detection of 5-oxoprolinuria. In all patients with 5-oxoprolinuria studied, bi-allelic mutations in OPLAH were indicated. An autosomal recessive mode of inheritance for 5-oxoprolinase deficiency is further supported by the identification of a single mutation in all 9/14 parent sample sets investigated (except for the father of one patient whose result suggests homozygosity), and the absence of 5-oxoprolinuria in all tested heterozygotes. It is remarkable, that all 20 mutations identified were novel and private to the respective families. Clinical features were highly variable and in several sib pairs, did not segregate with 5-oxoprolinuria. Although a pathogenic role of 5-oxoprolinase deficiency remains possible, this is not supported by our findings. Additional patient ascertainment and long-term follow-up is needed to establish the benign nature of this inborn error of metabolism. It is important that all symptomatic patients with persistently elevated levels of 5-oxoproline and no obvious explanation are investigated for the genetic etiology. (C) 2016 Elsevier Inc. All rights reserved.

BACKGROUND: Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level.METHODS: To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes.RESULTS: We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%).CONCLUSIONS: Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.).

Contains fulltext : 167925.pdf (Publisher’s version ) (Open Access) See Cannon (doi:10.1093/brain/awv400) for a scientific commentary on this article.Congenital myopathies are a clinically and genetically heterogeneous group of muscle disorders characterized by congenital or early-onset hypotonia and muscle weakness, and specific pathological features on muscle biopsy. The phenotype ranges from foetal akinesia resulting in in utero or neonatal mortality, to milder disorders that are not life-limiting. Over the past decade, more than 20 new congenital myopathy genes have been identified. Most encode proteins involved in muscle contraction; however, mutations in ion channel-encoding genes are increasingly being recognized as a cause of this group of disorders. SCN4A encodes the alpha-subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4). This channel is essential for the generation and propagation of the muscle action potential crucial to muscle contraction. Dominant SCN4A gain-of-function mutations are a well-established cause of myotonia and periodic paralysis. Using whole exome sequencing, we identified homozygous or compound heterozygous SCN4A mutations in a cohort of 11 individuals from six unrelated kindreds with congenital myopathy. Affected members developed in utero- or neonatal-onset muscle weakness of variable severity. In seven cases, severe muscle weakness resulted in death during the third trimester or shortly after birth. The remaining four cases had marked congenital or neonatal-onset hypotonia and weakness associated with mild-to-moderate facial and neck weakness, significant neonatal-onset respiratory and swallowing difficulties and childhood-onset spinal deformities. All four surviving cohort members experienced clinical improvement in the first decade of life. Muscle biopsies showed myopathic features including fibre size variability, presence of fibrofatty tissue of varying severity, without specific structural abnormalities. Electrophysiology suggested a myopathic process, without myotonia. In vitro functional assessment in HEK293 cells of the impact of the identified SCN4A mutations showed loss-of-function of the mutant Nav1.4 channels. All, apart from one, of the mutations either caused fully non-functional channels, or resulted in a reduced channel activity. Each of the affected cases carried at least one full loss-of-function mutation. In five out of six families, a second loss-of-function mutation was present on the trans allele. These functional results provide convincing evidence for the pathogenicity of the identified mutations and suggest that different degrees of loss-of-function in mutant Nav1.4 channels are associated with attenuation of the skeletal muscle action potential amplitude to a level insufficient to support normal muscle function. The results demonstrate that recessive loss-of-function SCN4A mutations should be considered in patients with a congenital myopathy.

We report a patient from a consanguineous family who presented with transient acute liver failure and biochemical patterns suggestive of disturbed urea cycle and mitochondrial function, for whom conventional genetic and metabolic investigations for acute liver failure failed to yield a diagnosis. Whole exome sequencing revealed a homozygous 12-bp deletion in PCK1 (MIM 614168) encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK); enzymatic studies subsequently confirmed its pathogenic nature. We propose that PEPCK deficiency should be considered in the young child with unexplained liver failure, especially where there are marked, accumulations of TCA cycle metabolites on urine organic acid analysis and/or an amino acid profile with hyperammonaemia suggestive of a proximal urea cycle defect during the acute episode. If suspected, intravenous administration of dextrose should be initiated. Long-term management comprising avoidance of fasting with the provision of a glucose polymer emergency regimen for illness management may be sufficient to prevent future episodes of liver failure. This case report provides further insights into the (patho-)physiology of energy metabolism, confirming the power of genomic analysis of unexplained biochemical phenotypes. (C) 2016 Elsevier Inc. All rights reserved

Introduction Mutations in the family of SCN genes encoding sodium channels are responsible for several disorders affecting the central and peripheral nervous systems and muscle. Disease arising from sodium channel mutants range from the relatively benign (e.g. mild myotonia) to the fatal (e.g. long-QT syndrome), with a wide variety of disorders spanning the spectrum of severity. Identified SCN4a mutations to date have been consistently autosomal dominant and associated with paramyotonia congenita, potassium-mediated periodic paralysis or aggravated myotonia due to defects altering the biophysical properties of sodium channels that mediate membrane hyper- or hypo-excitability. Here we describe a newly recognised autosomal-recessive syndrome comprising severe congenital hypotonia with respiratory failure in a family of Punjabi descent, with 2 of 3 children affected. Methods and results Using whole exome sequencing we identified two new mutations (g. 62025363 C >T, D1069N and g. 62025425 T >G, splice site) in the SCN4A gene, confirmed via Sanger sequencing. Reverse transcriptase polymerase chain reaction shows that the splice-site mutation in SCN4A leads to altered RNA. To investigate the impact of the missense mutation, c.3205G >A, Chinese hamster ovary (CHOk1) cells transfected with either a WT or D1069N SCN4A were examined for their biophysical properties. A set of depolarizing test pulses was used to measure the voltage dependence of activation and indicated biophysical changes in the encoded voltage-gated sodium channel (NaV1.4). Conclusions Together, our findings characterise the first reported evidence of an autosomal recessive SCN4a sodium channelopathy comprising severe congenital neuromuscular hypotonia and respiratory failure with biophysical dysfunction of NaV1.4 attributable to SCN4a compound heterozygous gene mutation.