Biochemical and structural characterization of quizalofop-resistant wheat acetyl-CoA carboxylase

A novel nucleotide mutation in ACC1 resulting in an alanine to valine amino acid substitution in acetyl-CoA carboxylase (ACCase) at position 2004 of the Alopecurus myosuroides reference sequence (A2004V) imparts quizalofop resistance in wheat. Genotypes endowed with the homozygous mutation in one or two ACC1 homoeologs are seven- and 68-fold more resistant to quizalofop than a wildtype winter wheat in greenhouse experiments, respectively. In vitro ACCase activities in soluble protein extracts from these varieties are 3.8- and 39.4-fold more resistant to quizalofop with the homozygous mutation in either one or two genomes, relative to the wildtype. The A2004V mutation does not alter the specific activity of wheat ACCase, suggesting that this resistance trait does not affect the catalytic functions of ACCase. Modeling of wildtype and quizalofop-resistant wheat ACCase demonstrates that the A2004V amino acid substitution causes a reduction in the volume of the binding pocket that hinders quizalofop’s interaction with ACCase. Docking studies confirm that the mutation reduces the binding affinity of quizalofop. Interestingly, the models suggest that the A2004V mutation does not affect haloxyfop binding. Follow up in vivo and in vitro experiments reveal that the mutation, in fact, imparts negative cross-resistance to haloxyfop, with quizalofop-resistant varieties exhibiting higher sensitivity to haloxyfop than the wildtype winter wheat line.

Loss-of-function mutation in Pcsk1 increases serum APOA1 level and LCAT activity in mice

Background The convertase subtilisin/kexin family 1 gene (PCSK1) has been associated in various human genetics studies with a wide spectrum of metabolic phenotypes, including early-onset obesity, hyperphagia, diabetes insipidus, and others. Despite the evident influence of PCSK1 on obesity and the known functions of other PCSKs in lipid metabolism, the role of PCSK1 specifically in lipid and cholesterol metabolism remains unclear. This study evaluated the effect of loss of PCSK1 function on high-density lipoprotein (HDL) metabolism in mice. Results HDL cholesterol, apolipoprotein A1 (APOA1) levels in serum and liver, and the activities of two enzymes (lecithin-cholesterol acyltransferase, LCAT and phospholipid transfer protein, PLTP) were evaluated in 8-week-old mice with a non-synonymous single nucleotide mutation leading to an amino acid substitution in PCSK1, which results in a loss of protein’s function. Mutant mice had similar serum HDL cholesterol concentration but increased levels of serum total and mature APOA1, and LCAT activity in comparison to controls. Conclusions This study presents the first evaluation of the role of PCSK1 in HDL metabolism using a loss-of-function mutant mouse model. Further investigations will be needed to determine the underlying molecular mechanism.

Case report of a de novo mutation of PCDH19 in Saudi family limited to females

Up to date more than 60 different mutations in PCDH19 have been identified. Most of PCDH19 gene is located in Xq22 and produces nonclustered delta protocadherin. This disorder primarily manifests in heterozygote females due to random X chromosome inactivation leading to somatic mosaicism and abnormal cellular interference between cells with and without delta-protocadherin., but we a heterozygous nucleotide mutation causing amino acid 561 to change from Pro to Ser (p.Pro561Ser). This mutation was de novo, and this alteration was not found in her parents. PCDH19-related epilepsy is a distinct childhood-onset epilepsy syndrome characterized by brief clusters of febrile and afebrile seizures with onset primarily before the age of three years, cognitive impairment, autistic traits, and behavioral abnormalities. We describe the features of a de novo mutation in 3 sibling, presented with early onset of seizure, two of them were controlled and wean off medication was at age of six year and her sister at age of 10 year .The youngest sister still partially controlled on medication, she had seizure only during febrile illness.

Rotavirus RNA chaperone mediates global transcriptome-wide increase in RNA backbone flexibility

Due to genome segmentation, rotaviruses must co-package a set of eleven distinct genomic RNAs. The packaging is mediated by virus-encoded RNA chaperones, such as the rotavirus (RV) NSP2 protein. While the activities of distinct viral RNA chaperones are well studied on synthetic RNA substrates, little is known about their global effect on the entire viral transcriptome. Here we used Selective 2′-hydroxyl Acylation Analyzed by Primer Extension and Mutational Profiling (SHAPE-MaP) to systematically examine the secondary structure of the RV transcriptome composed of eleven distinct transcripts in the absence and presence of increasing concentrations of RV NSP2. Surprisingly, SHAPE-MaP data reveals that despite the well-documented helix-unwinding activity of NSP2 in vitro, its incubation with cognate RV transcripts does not induce a significant change in the SHAPE reactivities. However, a quantitative analysis of the per nucleotide mutation rate measured by mutational profiling, from which SHAPE reactivities are derived, reveals a global five-fold rate increase in the presence of molar excess of NSP2. We demonstrate that the standard normalization procedure used in deriving SHAPE reactivities from mutation rates can mask an important global effect of an RNA chaperone activity. Further analysis of the mutation rate in the context of structural classification reveals a larger effect on stems rather than loop elements. Together, these data provide the first experimentally derived secondary structure model of the RV transcriptome and reveal that NSP2 acts by globally increasing RNA backbone flexibility in a concentration-dependent manner, consistent with its promiscuous RNA-binding nature.

Case Report of a de Novo Mutation of PCDH19 in Saudi Family Limited to Females

Up to date more than 60 different mutations in PCDH19 have been identified. Most of PCDH19 gene is located in Xq22 and produces nonclustered delta protocadherin. This disorder primarily manifests in heterozygote females due to random X chromosome inactivation leading to somatic mosaicism and abnormal cellular interference between cells with and without delta-protocadherin., but we a heterozygous nucleotide mutation causing amino acid 561 to change from Pro to Ser (p.Pro561Ser). This mutation was de novo, and this alteration was not found in her parents. PCDH19-related epilepsy is a distinct childhood-onset epilepsy syndrome characterized by brief clusters of febrile and afebrile seizures with onset primarily before the age of three years, cognitive impairment, autistic traits, and behavioral abnormalities. We describe the features of a de novo mutation in 3 sibling, presented with early onset of seizure, two of them were controlled and wean off medication was at age of six year and her sister at age of 10 year .The youngest sister still partially controlled on medication, she had seizure only during febrile illness.

A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient

A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR. We evaluated both models using minigene splicing reporters engineered to vary in secondary structure and/or cryptic splice site content. Analysis of splicing efficiency in transfected K562 cells suggested that the mutation-generated cryptic splice site in E44 was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site also had modest effects on IR and shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition among the sites. Further studies demonstrated that introduction of cryptic splice donor motifs at other positions in E44 did not promote IR, indicating that interference with the annotated site is context dependent. We conclude that mutant deep exon splice sites can interfere with proper splicing by inducing IR.

Insertion of the protective APP A673T mutation by CRISPR/Cas9 base editing or PRIME editing

There is currently no treatment for Alzheimer disease (AD). However, the Icelandic mutation in the APP gene (A673T) has been shown to confer a protection against the onset and development of AD (Jonsson et al. Nature 2012). This single nucleotide mutation in APP exon 16 reduces the cleavage of the APP protein by the beta-secretase by 40% thus preventing the development of AD even in persons more than 95 years old. Our research group has initially shown that the presence of the A673T mutation in an APP gene reduced the secretion of beta-amyloid peptides even if there is also a FAD mutation in the gene. This is the case for 14 different FAD mutations. We have used CRISPR/Cas9 base editing and PRIME editing technologies to insert the A673T mutation in the APP gene. We have compared several different cytidine base editor complexes to achieve the most effective and accurate genome modification possible in HEK293T cells and in SH-SY5Y neuroblastomas. The insertion of the A673T mutation in cells containing the London mutation reduced the secretion of beta-amyloid peptides. We are currently using lentiviral vectors to infect neurons from a mouse model and human neurons induced from fibroblasts of a patient with the London mutation. The insertion of the protective Icelandic mutation in the APP gene using these editing technologies opens a new potential therapeutic avenue not only for Familial Alzheimer’s diseases but also for sporadic Alzheimer’s disease.

In-Silico and In-vitro Analysis of Human SOS1 Protein Causing Noonan Syndrome – A Novel Approach to Explore the Molecular Pathways

Aims: Noonan syndrome (NS) is an autosomal dominant genetic disorder caused by single nucleotide mutation in PTPN11, SOS1, RAF1, and KRAS genes. Background: We hypothesize that in-silico analysis of human SOS1 mutations would be a promising predictor in identifying the pathogenic effect of NS. Methods: Here, we computationally analyzed the SOS1 gene to identify the pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs) to cause NS. The variant information of SOS1 was collected from the SNP database (dbSNP). The variants were further analyzed by in-silico tools I-Mutant, iPTREE-STAB, and MutPred to elucidate their structural and functional characteristics. Results: We found that 11 nsSNPs of SOS1 were more pathogenic to cause NS. The 3D modeling of the wild-type and the 11 nsSNPs were performed using I-TASSER and validated via ERRAT and RAMPAGE. SOS1 interacting proteins were analysed through STRING, which showed that SOS1 interacted with cardiac proteins GATA4, TNNT2, and ACTN2. During these interactions, GRB2 and HRAS act as an intermediate molecules between SOS1 and cardiac proteins. These in-silico analyses were validated using induced cardiomyocytes (iCMCs) derived from NS patients carrying SOS1 gene variant c.1654A>G (NS-iCMCs) and compared with control human skin fibroblast-derived iCMCs (C-iCMCs). Our in vitro data further confirmed that the SOS1, GRB2 and HRAS gene expressions as well as the activated ERK protein, were significantly decreased in NS-iCMCs compared to C-iCMCs. Conclusion: This is the first in-silico and in vitro study demonstrating that 11 nsSNPs of SOS1 were playing a deleterious pathogenic role in causing NS.

A SARS-CoV-2 Delta Variant Containing Mutation in the Probe Binding Region Used for qRT-PCR Test in Japan Exhibited Atypical PCR Amplification and Might Induce False Negative Result

A recent pandemic of SARS-CoV-2 infection has caused severe health problems and substantially restricted social and economic activities. To cope with such an outbreak, the identification of infected individuals with high accuracy is vital. qRT-PCR plays a key role in the diagnosis of SARS-CoV-2 infection. The N protein-coding region is widely analyzed in qRT-PCR for the diagnosis of SARS-CoV-2 infection in Japan. We recently encountered two cases of SARS-CoV-2-positive specimens showing atypical amplification curves in the qRT-PCR. We performed whole-genome sequencing and found that the virus was a Delta-type variant of SARS-CoV-2 with a single nucleotide mutation in the probe-binding site. To evaluate the extent of spread of the variant in the area, we performed whole viral genome sequencing of samples collected from 61 patients infected with SARS-CoV-2 during the same time and in the same area. There were no other cases with the same mutation, indicating that the variant had not spread in the area. Furthermore, we performed phylogenetic analysis with various SARS-CoV-2 sequences deposited in the public database. Hundreds of variants were reported globally, and one in Japan were found to contain the same mutation. Phylogenetic analysis showed that the variant was very close to other Delta variants endemic in Japan but quite far from the variants containing the same mutation reported from outside Japan, suggesting that the variant would have been sporadically generated in some domestic areas. These findings propose two key points: i) mutations in the region used for SARS-CoV-2 qRT-PCR can cause abnormal amplification curves; therefore, the qRT-PCR result should not just be judged in an automated manner, but also manually checked by the examiner to prevent false-negative results, and ii) various mutations can be generated sporadically and unpredictably; therefore, efficient and robust screening systems are needed to promptly monitor the emergence of de novo variants.