Substitution Of Glycine Research Articles

Structure–function relationship of dynorphin B variants using naturally occurring amino acid substitutions

Dynorphins (Dyn) represent the subset of endogenous opioid peptides with the highest binding affinity to kappa opioid receptors (KOPrs). Activation of the G-protein-coupled pathway of KOPrs has strong anticonvulsant effects. Dyn also bind to mu (MOPrs) and delta opioid receptors (DOPrs) with lower affinity and can activate the β-arrestin pathway. To fully exploit the therapeutic potential of dynorphins and reduce potential unwanted effects, increased selectivity for KOPrs combined with reduced activation of the mTOR complex would be favorable. Therefore, we investigated a series of dynorphin B (DynB) variants, substituted in one or two positions with naturally occurring amino acids for differential opioid receptor activation, applying competitive radio binding assays, GTPγS assays, PRESTO-Tango, and Western blotting on single-opioid receptor-expressing cells. Seven DynB derivatives displayed at least 10-fold increased selectivity for KOPrs over either MOPrs or DOPrs. The highest selectivity for KOPrs over MOPrs was obtained with DynB_G3M/Q8H, and the highest selectivity for KOPrs over DOPrs was obtained with DynB_L5S. Increased selectivity for KOPr over MOPr and DOPr was based on a loss of affinity or potency at MOPr and DOPr rather than a higher affinity or potency at KOPr. This suggests that the investigated amino acid exchanges in positions 3, 5, and 8 are of higher importance for binding and activation of MOPr or DOPr than of KOPr. In tests for signal transduction using the GTPγS assay, none of the DynB derivatives displayed increased potency. The three tested variants with substitutions of glycine to methionine in position 3 displayed reduced efficacy and are, therefore, considered partial agonists. The two most promising activating candidates were further investigated for functional selectivity between the G-protein and the β-arrestin pathway, as well as for activation of mTOR. No difference was detected in the respective read-outs, compared to wild-type DynB. Our data indicate that the assessment of affinity to KOPr alone is not sufficient to predict either potency or efficacy of peptidergic agonists on KOPr. Further assessment of downstream pathways is required to allow more reliable predictions of in vivo effects.

Fungicide Sensitivity Survey of Pecan Scab in Texas Pecan Orchards

A fungicide sensitivity survey of pecan scab ( Venturia effusa) from eight different locations in seven pecan orchards in Texas was conducted during the summer of 2021. Using a rapid fungicide sensitivity assay based on single discriminatory concentrations, sensitivities to five fungicides (dodine, fentin hydroxide, thiophanate-methyl, propiconazole, and tebuconazole) were evaluated. Pecan orchards with frequent fungicide use tended to have greater levels of reduced sensitivities to demethylation inhibitor (propiconazole and tebuconazole; >40% of conidial germination at 1 µg/ml of active ingredient) and fentin hydroxide fungicides (>64% of conidial germination at 3 µg/ml of active ingredient) compared to those with fewer fungicide applications in the past. All populations tested in this survey exhibited a high level of resistance to the thiophanate-methyl fungicide but no resistance to dodine. Thiophanate-methyl did not inhibit conidial germination for six scab populations, while dodine consistently inhibited conidial germination for scab populations tested at the discriminatory concentration. The cytochrome b gene was sequenced for determining mutations associated with quinone outside inhibitor (QoI) fungicides. The mutation that results in a substitution of glycine 137 to serine (G137S) in the cytochrome b gene and is related to high resistance to QoI was not found in the representative isolates of scab populations tested in this survey. Our findings are the first report of fungicide resistance development in Texas pecan orchards. Information about fungicide sensitivities of scab populations in orchards will help pecan producers to prepare proper fungicide treatment plans.

A Novel Homozygous Variant in CPLANE1 Gene in a Patient with Developmental Deficits

Background: Oral-facial-digital syndrome (OFDS) type 6 is a rare subtype of Joubert syndrome characterized by orofacial anomalies and polydactyly with neurological features of Joubert syndrome. This rare syndrome is divided into thirteen subtypes, all of which demonstrate autosomal recessive inheritance, except for OFDS type 1 which demonstrates X-linked dominant inheritance. Case Presentation: A 19-year-old man with mild developmental delay was brought to a rural community clinic, as he had become irritable and angry, in the recent past. There was no history of prior medical conditions. In view of orofacial anomalies, and developmental deficits, a genetic analysis was requested. Karyotype analysis revealed a normal male karyotype (46,XY) in all 30 metaphase spreads analyzed. No numerical or structural chromosomal abnormalities were observed. Clinical exome sequencing and chromosomal microarray detected a variant of uncertain significance in exon 5 of CPLANE1 gene c.365T>G (p.Val122Gly) leading to substitution of Glycine for Valine. This was confirmed by Sanger sequencing. Parents were heterozygous, and the unaffected sibling was homozygous for the wild-type allele. This variant has not been reported earlier in the mutation databases or gnomAD. Runs of homozygosity (ROH) analysis showed a 3.2 Mb ROH around the CPLANE1 gene in the proband, which was absent in both parents and the unaffected sibling. Conclusion: We find a novel homozygous mutation in the CPLANE1 gene in a patient of non-consanguineous parentage with atypical orofacial features. This suggests that potentially deleterious, rare variants may occur in the heterozygous state in the population. Hence, sequencing of population samples might help understand the genetic epidemiology of rare syndromes.

6864 Genetic Profile And Identification Of A Novel Mutation In PHEX In X-Linked Hypophosphatemia (XLH) Patients At A Brazilian Research Center: Insights And Implications

Abstract Disclosure: M. Monseff Rodrigues da silva: None. I.M. de Araújo: None. F.J. de Paula: None. Background: X-Linked Hypophosphatemia (XLH) is a rare genetic disorder characterized by low phosphate levels in the blood, primarily caused by mutations in the PHEX gene, leading to bone skeletal deformities and reduced quality of life. XLH genetics is extensively studied with over 500 pathogenic mutations in the PHEXgene having been identified. Common mutations include missense, nonsense, and splicing, accounting for approximately 50% of cases. However, genotype-phenotype correlations remain unclear. Methods: We conducted a cross-sectional study using a convenience sample selected from the osteometabolism an outpatient clinic in Brazil. Genetic analysis was performed using next-generation sequencing (NGS; Ilumina) in collaboration with Mendelics Laboratory. Results: In our study, 9 out of 10 patients showed previously known and reported mutations, with 60% exhibiting common mutation types. The mutations occur in different codons; however, the most prevalent consequence was the substitution of glycine (G) with adenine (A), present in 40% of the patients. One patient, however, showed a unique missense mutation in PHEX gene (arginine to glutamate at codon 266), which leads to a premature stop codon, a mutation absent in over 183,000 X chromosomes from XLH individuals. Even though this mutation was novel, no significant diferences in the phenotype of this patient were seen in comparision with the rest of the cohort. Discussion: The findings of our study underscore the genetic heterogeneity characteristic of XLH, as evidenced by the fact that 90% of our patient cohort exhibited mutations previously identified in the literature, with a significant 60% showing common mutation types. This highlights the recurring genetic patterns in XLH, which can be instrumental for diagnostic and - perhaps in the future - therapeutic strategies. Particularly notable is the predominant mutation involving the substitution of glycine with adenine, found in 40% of our patients, underscoring its potential role as a key mutation in the pathogenesis of XLH. Furthermore, the discovery of a novel missense mutation in one patient, resulting in a premature stop codon, enriches the mutation spectrum of XLH and suggests a deeper complexity in the genetic landscape of the disease. This mutation, not previously documented in the extensive database of over 183,000 X chromosomes from XLH individuals, opens new avenues for research into the disease's molecular mechanisms and potential genotype-phenotype correlations. Conclusion: This study underlines the genetic diversity within XLH, emphasizing the need for comprehensive genetic analysis to understand the full spectrum of the disease. More studies are yet necessary to elucidate if there is a genotype-phenotype correlation. Presentation: 6/1/2024

Abstract A009: Mutant KRAS in Circulating Tumor DNA Assessed by Digit Droplet PCR as a Biomarker in Pancreatic Cancer in Patients Treated with Neoadjuvant Chemotherapy

Abstract Introduction: The detectability and prognostic significance of circulating tumor DNA (ctDNA) in patients with pancreatic ductal adenocarcinoma (PDAC) after neoadjuvant chemotherapy (NAC) is unclear. The objective of this study was to investigate the rate of detection and prognostic value of KRAS mutant ctDNA in PDAC patients after treatment with NAC as assessed by digital droplet polymerase chain reaction (ddPCR). Methods: Patients with newly diagnosed, localized, PDAC were enrolled in a prospective cohort study. Peripheral blood samples were collected at diagnosis, after NAC, and after resection. DNA was extracted from patient plasma samples using the QIAamp Circulating Nucleic Acid Kit #55114 (Qiagen N.V., Venlo Netherlands). Each sample was probed (catalog number 10049550) for KRAS codon 12 exon 2 glycine substitutions for aspartic acid (G12D), valine (G12V), and arginine (G12R). Kaplan-Meier, log-rank, and Cox regression survival analyses were performed. Results: 84 patients were included in the analysis. Patients were most commonly male (53.6%) and had tumors that were radiographically resectable (57.1%). After diagnosis, 79.8% of patients received NAC and 57.1% of patients received curative-intent surgical resection. Samples were collected for analysis from 59 (70.2%), 56 (83.6%), and 45 (93.8%) patients at diagnosis, after NAC, and after resection, respectively. Mutant KRAS ctDNA was detected in 49.3% of patients at diagnosis, 69.6% of patients after NAC, and 54.1% of patients after resection, respectively. The median OS of the cohort was 25.1 months (12.0-not reached [NR]). Detection (hazard ratio [HR] 36.8, 95% confidence interval [CI] 2.9-461.4), copy number (HR 4.0 95% CI 1.6-10.2), and percentage mutation (HR 2.9 95% CI 1.2-6.8) of KRAS G12V ctDNA after resection were all independently associated with shorter OS. Mutant KRAS ctDNA detection was not associated with and OS at other study timepoints. There were 15 (17.9%) patients that cleared mutational ctDNA over the course of treatment. Clearance of ctDNA during NAC was associated with improved overall survival (OS) (18.4 mo. vs NR). Conclusions: In patients with PDAC treated with NAC, KRAS mutations are detectable in ctDNA by ddPCR in the majority of patients over the course of treatment. Detection, copy number, and percentage mutation of KRAS G12V after NAC and resection were each independently associated with shorter OS. Clearance of ctDNA is associated with improved OS. The results demonstrate that KRAS mutant ctDNA predicts prognosis after NAC and resection. Citation Format: Dominic Vitello, Dhavan Shah, Amy Wells, Larissa Masnyk, Madison Cox, Lauren Janczewski, John Abad, Kevin Dawravoo, Arlene D'Souza, Grace Suh, Robert Bayer, Massimo Cristofanilli, David Bentrem, Yingzhe Liu, Hui Zhang, Lucas Santana-Santos, Lawrence Jennings, Qiang Zhang, Akhil Chawla. Mutant KRAS in Circulating Tumor DNA Assessed by Digit Droplet PCR as a Biomarker in Pancreatic Cancer in Patients Treated with Neoadjuvant Chemotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A009.

ГЕНЕТИЧЕСКАЯ ХАРАКТЕРИСТИКА Х-СЦЕПЛЕННОГО СИНДРОМА АЛЬПОРТА У ДЕТЕЙ: ДАННЫЕ ОДНОЦЕНТРОВОГО КОГОРТНОГО ИССЛЕДОВАНИЯ

X-linked Alport syndrome (XLAS) is a monogenic disease characterized by the progressive nephropathy development, sensorineural hearing loss and the lens and retina pathology. The clinical manifestations of the syndrome and the renal prognosis of patients are determined by the nature of the gene COL4A5 mutation. The range of COL4A5 genetic variants is different among populations. Information about the XLAS genetic structure in Russian pediatric cohort is insufficient. The purpose of this research was to determine the features of the spectrum of the gene COL4A5 genetic variants in children with XLAS. Materials and methods used: a single-center retrospective cohort study included children aged 2 to 17 y/o with genetically confirmed XLAS. Descriptions of variants in gene COL4A5 were performed using genome build GRCh37/hg19 in accordance with the reference sequence GRCh37/hg19 (Reference Sequence, HGVS) and the American College of Medical Genetics and Genomics (ACMG), AMP Clinical Practice Guidelines. Results: analysis of data from 174 probands from unrelated families showed that 108 (62%) children had missense variants in gene COL4A5 (62% v. 38%, missense v. non-missense variants, respectively, p=0.028), 24 (13.8%) had mutations leading to impaired RNA splicing, 17 (9.8%) had frameshift mutations, 14 (8%) had gene deletions and 10 (5.7%) had nonsense mutations. Missense variants were predominantly localized in the collagen domain compared to the non-collagen domain (n=102, 94.4%, p<0.001), in exons 18 to 41 (n=94; 94.8%) and were represented by glycine substitutions (n=90, 83.3%). Two common variants were identified in the cohort: NM_033380.3(COL4A5):c.1871G>A (p.Gly624Asp) in 28 (26%) and NM_033380.3(COL4A5):c.3319G>A (p.Gly1107Arg) in 4 (3.7%). Conclusion: the structure of COL4A5 genetic variants in the studied cohort was comparable with the current Worldwide data, such as two thirds of the examined had missense variants and ca. one-fifth had genetic variants leading to the protein synthesis absence. Two common missense variants in gene COL4A5 were identified, including the common variant NM_033380.3(COL4A5):c.1871G>A (p.Gly624Asp) that is of frequent occurrence in the European population.

An exploratory open-label multicentre phase I/II trial evaluating the safety and efficacy of postnatal or prenatal and postnatal administration of allogeneic expanded fetal mesenchymal stem cells for the treatment of severe osteogenesis imperfecta in infants and fetuses: the BOOSTB4 trial protocol

IntroductionSevere osteogenesis imperfecta (OI) is a debilitating disease with no cure or sufficiently effective treatment. Mesenchymal stem cells (MSCs) have good safety profile, show promising effects and can form bone....

In silico evidence that substitution of glycine for valine (p.G8V) in a common variant of TMPRSS2 isoform 1 increases accessibility to an endocytic signal: Implication for SARS-cov-2 entry into host cells and susceptibility to COVID-19

The TMPRSS2 protease plays a key role in the entry of the SARS-CoV-2 into cells. The TMPRSS2 gene is highly polymorphic in humans, and some polymorphisms may affect the susceptibility to COVID-19 or disease severity. rs75603675 (c.23G > T) is a missense variant that causes the replacement of glycine with valine at position 8 (p.G8V) in the TMPRSS2 isoform 1. According to GnomAD v4.0.0 database, the allele frequency of the rs75603675 on a global scale is 38.10 %, and range from 0.92 % in East Asian to 40.77 % in non-Finnish European (NFE) population. We analyzed the occurrence of the rs75603675 in two cohorts of patients, the first with severe/critical COVID-19 enrolled in a French hospital (42 patients), and the second with predominantly asymptomatic/pauci-symptomatic/mild COVID-19 enrolled in an Italian hospital (69 patients). We found that the TMPRSS2-c.23T minor allele frequency was similar in the two cohorts, 46.43 % and 46.38 %, respectively, and higher than the frequency in the NFE population (40.77 %). Chi-square test provided significant results (p < 0.05) when the genotype data (TMPRSS2-c.23T/c.23T homozygotes + TMPRSS2-c.23G/c.23T heterozygotes vs. TMPRSS2-c.23G/c.23G homozygotes) of the two patient groups were pooled and compared to the expected data for the NFE population, suggesting a possible pathogenetic mechanism of the p.G8V substitution. We explored the possible effects of the p.G8V substitution and found that the N-terminal region of the TMPRSS2 isoform 1 contains a signal for clathrin/AP-2-dependent endocytosis. In silico analysis predicted that the p.G8V substitution may increase the accessibility to the endocytic signal, which could help SARS-CoV-2 enter cells.

A novel hemizygous CD40L mutation of X-linked hyper IgM syndromes and compound heterozygous DOCK8 mutations of hyper IgE syndromes in two Chinese families.

X-linked hyper-immunoglobulin M (X-HIGM) syndrome and autosomal recessive hyper-immunoglobulin E syndrome (HIES) are rare inborn errors of immunity characterized by recurrent infections due to immune system impairment. In this study, we identified a novel hemizygous CD40 ligand (CD40L) mutation and compound heterozygous dedicator of cytokinesis-8 (DOCK8) mutations in two Han Chinese families with X-HIGM and HIES, respectively. We aimed to investigate the association between their genotypes and phenotypes. Genomic DNA was extracted from peripheral blood samples obtained from the families. Whole exome sequencing and Sanger sequencing were performed to identify and verify pathogenic variants in the two families. Clinical analyses of the probands were also performed. A novel hemizygous mutation of CD40L in exon 2 (c.257delA) was identified in the first proband, resulting in the substitution of glycine with glutamic acid at codon 86 of the protein. This leads to premature termination of translation at downstream codon 9 (p.E86Gfs*9). Sanger sequencing confirmed that the variant was inherited from the mother. The second proband carried two novel compound heterozygous mutations in DOCK8: one at exon 14 (c.1546C > G) inherited from the father, and the other at intron 41 (c.5355 + 6C > T; splicing) inherited from the mother. This study enhances our understanding of the pathogenetic mutation spectrum of CD40L and DOCK8 genes, facilitating the prenatal diagnosis of X-HIGM and HIES and enabling timely treatment of patients.

Epidermolysis Bullosa: Two rare case reports of COL7A1 and EBS-GEN SEV KRT14 variants with review of literature

EpidermolysisBullosa is a rare hereditary skin condition that causes blisters. Genes encoding structural proteins at or near the dermal-epidermal junction are mutated recessively or dominantly, and this is the primary cause of EB. Herein, two Chinese boys were diagnosed with the condition, each with a different variant in a gene that serves as a reference for EB genetic counseling. Skincare significantly impacted their prognosis and quality of life.Case presentationTwo Chinese boys, with phenotypically normal parents, have been diagnosed with distinct blister symptoms, one with Dominant Dystrophic Epidermolysis Bullosa and the other with a severe form of Epidermolysis Bullosa Simplex. The first patient had a G-to-A variant in the COL7A1 allele, at nucleotide position 6163 which was named “G2055A”. The proband is heterozygous for Dystrophic Epidermolysis Bullosa due to a COL7A1 allele with a glycine substitution at the triple helix domain. A similar variant has been discovered in his mother, indicating its potential transmission to future generations. Another patient had severe Epidermolysis Bullosa Simplex with a rare c.377T > A variant resulting in substitution of amino acid p.Leu126Arg (NM_000526.5 (c.377T > G, p.Leu126Arg) in the Keratin 14 gene. In prior literature, Keratin 14 has been associated with an excellent prognosis. However, our patient with this infrequent variant tragically died from sepsis at 21 days old. There has been a reported occurrence of the variant only once.ConclusionOur study reveals that Epidermolysis Bullosa patients with COL7A1 c.6163G > A and KRT14 c.377T>A variants have different clinical presentations, with dominant forms of Dystrophic EB having milder phenotypes than recessive ones. Thus, the better prognosis in the c.6163G > A patient. Furthermore, c.377T>A patient was more prone to infection than the patient with c.6163G>A gene variant. Genetic testing is crucial for identifying the specific variant responsible and improving treatment options.

Optimized allele-specific silencing of the dominant-negative COL6A1 G293R substitution causing collagen VI-related dystrophy

Collagen VI-related dystrophies (COL6-RDs) are a group of severe, congenital-onset muscular dystrophies for which there is no effective causative treatment. Dominant-negative mutations are common in COL6A1, COL6A2, and COL6A3 genes, encoding the collagen α1, α2, and α3 (VI) chains. They act by incorporating into the hierarchical assembly of the three α (VI) chains and consequently produce a dysfunctional collagen VI extracellular matrix, while haploinsufficiency for any of the COL6 genes is not associated with disease. Hence, allele-specific transcript inactivation is a valid therapeutic strategy, although selectively targeting a pathogenic single nucleotide variant is challenging. Here, we develop a small interfering RNA (siRNA) that robustly, and in an allele-specific manner, silences a common glycine substitution (G293R) caused by a single nucleotide change in COL6A1 gene. By intentionally introducing an additional mismatch into the siRNA design, we achieved enhanced specificity toward the mutant allele. Treatment of patient-derived fibroblasts effectively reduced the levels of mutant transcripts while maintaining unaltered wild-type transcript levels, rescuing the secretion and assembly of collagen VI matrix by reducing the dominant-negative effect of mutant chains. Our findings establish a promising treatment approach for patients with the recurrent dominantly negative acting G293R glycine substitution.

Abstract 6344: Engineering of ICAM-1 refractory antibodies for the development of therapeutic antibodies and antibody drug conjugates (ADCs) in ICAM-1 over-expressing cancers

Abstract Human cancers employ a number of mechanisms to evade host immune responses against novel antigens generated from aberrant over-expression, mutations and/or epigenetic alterations. Humoral immunity utilizes antibodies and immune-effector cells as well as molecular immune complexes involving the complement system to mediate the killing of dysregulated cancer cells. We refer to these anti-cancer mechanisms as Humoral Immuno-Oncology (HIO). HIO suppression is mediated by tumor-produced proteins called HIO factors. One such factor is CA125, which was previously shown to bind IgG-type antibodies and inhibit their immune-effector activities, including antibody dependent (ADCC) and complement dependent (CDC) cellular cytotoxicity. Using a combination of experimental screening and literature searches, a second protein produced by tumors and associated with a variety of cancer indications was discovered and found to be soluble ICAM-1 (sICAM-1). Through various functional studies we reported that sICAM-1 and membrane ICAM-1 (mICAM-1) are capable of binding IgG1-type antibodies that in turn inhibits their immune-effector activity. Through a combination of truncation and substitution mutagenesis, we identified a four amino acid motif within the CH3 domain of IgG1 essential for ICAM-1 binding, resulting in the inhibition of ADCC activity. While traditional glycine and alanine substitutions in this region abrogated ICAM-1 binding, these modifications caused tertiary structural changes in the Fc domain that resulted in loss of ADCC activity. Through a combinatorial amino acid substitution approach, we identified a four amino acid combination within the 407YSKL410 to 407FARV410 motif that resulted in antibodies refractory to sICAM-1 binding with robust ADCC activity. Additionally, isogenic wildtype and ICAM-1 knockdown target cell lines showed mICAM-1 inhibited target cell killing of a saporin antibody drug conjugate (ADC) and this was the direct result of mICAM-1 reducing antibody internalization, a requisite for maximal ADC target cell killing. The inhibitory activity of mICAM-1 could be overcome employing antibodies containing the 407FARV410 substitution. These findings highlight yet another mechanism by which tumors can suppress the host’s immune system for survival and offer new concepts for developing antibody-based therapies that can aid in the treatment of various cancer indications, especially those over-expressing ICAM-1. Moreover, the findings here offer diagnostic and therapeutic clinical design opportunities to improve upon existing approved immune-mediated therapies for which this factor is present. Citation Format: Brad Kline, Luigi Grasso, Nicolas Nicolaides. Engineering of ICAM-1 refractory antibodies for the development of therapeutic antibodies and antibody drug conjugates (ADCs) in ICAM-1 over-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6344.

A novel mutation in IAA16 is associated with dicamba resistance in Chenopodium album.

Resistance to dicamba in Chenopodium album was first documented over a decade ago, however, the molecular basis of dicamba resistance in this species has not been elucidated. In this research, the resistance mechanism in a dicamba-resistant C. album phenotype was investigated using a transcriptomics (RNA-sequence) approach. The dose-response assay showed that the resistant (R) phenotype was nearly 25-fold more resistant to dicamba than a susceptible (S) phenotype of C. album. Also, dicamba treatment significantly induced transcription of the known auxin-responsive genes, Gretchen Hagen 3 (GH3), small auxin-up RNAs (SAURs), and 1-aminocyclopropane-1-carboxylate synthase (ACS) genes in the susceptible phenotype. Comparing the transcripts of auxin TIR/AFB receptors and auxin/indole-3-acetic acid (AUX/IAA) proteins identified from C. album transcriptomic analysis revealed that the R phenotype contained a novel mutation at the first codon of the GWPPV degron motif of IAA16, resulting in an amino acid substitution of glycine (G) with aspartic acid (D). Sequencing the IAA16 gene in other R and S individuals further confirmed that all the R individuals contained the mutation. In this research, we describe the dicamba resistance mechanism in the only case of dicamba-resistant C. album reported to date. Prior work has shown that the dicamba resistance allele confers significant growth defects to the R phenotype investigated here, suggesting that dicamba-resistant C. album carrying this novel mutation in the IAA16 gene may not persist at high frequencies upon removal of dicamba application. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Association of dopamine receptor D3 polymorphism with Levodopa-induced Dyskinesia: A study on Parkinson’s disease patients from India

IntroductionLevodopa-induced dyskinesia (LID) is a debilitating motor feature in a subset of patients with Parkinson’s disease (PD) after prolonged therapeutic administration of levodopa. Preliminary animal and human studies are suggestive of a key role of dopamine type 3 (D3) receptor polymorphism (Ser9Gly; rs6280) in LID. Its contribution to development of LID among Indian PD patients has remained relatively unexplored and merits further investigation. Methods and materials200 well-characterised PD patients (100 without LID and 100 with LID) and 100 age-matched healthy controls were recruited from the outpatient department of Institute of Neurosciences Kolkata. MDS-UPDRS (Unified Parkinson’s Disease Rating Scale from International Movement Disorder Society) Part III and AIMS (abnormal involuntary movement scale) were performed for estimation of severity of motor features and LID respectively in the ON state of the disease. Participants were analysed for the presence of Ser9Gly single nucleotide variant (SNV) (rs6280) by polymerase chain reaction followed by restriction fragment length polymorphism techniques. ResultsThe frequency of AA genotype (serine type) was more frequently present in PD patients with LID compared to PD patients without LID (50 % vs 28 %; P = 0.002; OR = 2.57, 95 % CI: 1.43 – 4.62). The abnormal involuntary movement scale score was significantly higher in PD patients with AA genotype compared to carriers of glycine allele (AG + GG) (4.08 ± 3.35; P = 0.002). ConclusionWe observed a significant association of serine type SNV (rs6280) in D3 receptor gene in a cohort of PD patients with LID from India. More severe motor severity was found in patients with glycine substitution of the same SNV. The current study emphasised the role of D3 receptor in the pathogenesis of LID.

Revealing a New Homozygous Variant in CYP17A1 c.908G>A (p. Gly303Asp) by Genotyping a Chinese Patient with 46, XY 17a-Hydroxylase/17,20-Lyase Deficiency and Adrenal Space-Occupying Lesion.

17α-hydroxylase/17,20-lyase deficiency (17OHD) is an autosomal recessive genetic disorder caused by a mutation of the cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1). This study reports the case of a 22-year-old Chinese patient (46, XY) with 17OHD and a unilateral adrenal space-occupying lesion. The patient underwent serological, radiographic, genetic, and molecular analyses including whole-genome exome sequencing through high-throughput sequencing (HTS) technology to analyze the genetic conditions of both the patient and her parents. Additionally, chromosomal karyotype analysis was performed. The impact of the novel mutation on protein conformation was investigated by examining the three-dimensional structure of human CYP17A1 using the SWISS-MODEL website tool (PDB code 3RUK). The patient had a chromosomal karyotype 46, XY, and presented with hypertension, hypokalemia, and male pseudohermaphroditism. Furthermore, decreased levels of testosterone, dehydroepiandrosterone sulfate, and estradiol, along with increased levels of progesterone, luteinizing hormone, and follicle-stimulating hormone (FSH), were observed. DNA sequencing revealed a homozygous mutation (c.908G>A, p.G303A) in the fifth exon of the CYP17A1. Both parents carried a heterozygous c.908G>A mutation in the same exon, confirming the inheritance of the patient's exonic mutation. For the first time, this study reports a novel homozygous mutation (c.908G>A in the fifth exon) in CYP17A1. Modeling analysis of CYP17A1 suggested that the substitution of glycine with aspartic acid at position 303 induces alterations in the number, structure, and electrostatic potential of the protein's local binding sites. The p.G303A mutation may possess pathogenic properties. Our study expands the mutation spectrum of CYP17A1.

Identification of Potential Modulators of a Pathogenic G Protein-Gated Inwardly Rectifying K+ Channel 4 Mutant: In Silico Investigation in the Context of Drug Discovery for Hypertension.

Genetic abnormalities have been associated with primary aldosteronism, a major cause of secondary hypertension. This includes mutations in the KCNJ5 gene, which encodes G protein-gated inwardly rectifying K+ channel 4 (GIRK4). For example, the substitution of glycine with glutamic acid gives rise to the pathogenic GIRK4G151E mutation, which alters channel selectivity, making it more permeable to Na+ and Ca2+. While tertiapin and tertiapin-Q are well-known peptide inhibitors of the GIRK4WT channel, clinically, there is a need for the development of selective modulators of mutated channels, including GIRK4G151E. Using in silico methods, including homology modeling, protein-peptide docking, ligand-binding site prediction, and molecular docking, we aimed to explore potential modulators of GIRK4WT and GIRK4G151E. Firstly, protein-peptide docking was performed to characterize the binding site of tertiapin and its derivative to the GIRK4 channels. In accordance with previous studies, the peptide inhibitors preferentially bind to the GIRK4WT channel selectivity filter compared to GIRK4G151E. A ligand-binding site analysis was subsequently performed, resulting in the identification of two potential regions of interest: the central cavity and G-loop gate. Utilizing curated chemical libraries, we screened over 700 small molecules against the central cavity of the GIRK4 channels. Flavonoids, including luteolin-7-O-rutinoside and rutin, and the macrolides rapamycin and troleandomycin bound strongly to the GIRK4 channels. Similarly, xanthophylls, particularly luteoxanthin, bound to the central cavity with a strong preference towards the mutated GIRK4G151E channel compared to GIRK4WT. Overall, our findings suggest potential lead compounds for further investigation, particularly luteoxanthin, that may selectively modulate GIRK4 channels.

Dual Role of Dysfunctional Asc-1 Transporter in Distinct Human Pathologies, Human Startle Disease, and Developmental Delay.

Human startle disease is associated with mutations in distinct genes encoding glycine receptors, transporters or interacting proteins at glycinergic synapses in spinal cord and brainstem. However, a significant number of diagnosed patients does not carry a mutation in the common genes GLRA1, GLRB, and SLC6A5 Recently, studies on solute carrier 7 subfamily 10 (SLC7A10; Asc-1, alanine-serine-cysteine transporter) knock-out (KO) mice displaying a startle disease-like phenotype hypothesized that this transporter might represent a novel candidate for human startle disease. Here, we screened 51 patients from our patient cohort negative for the common genes and found three exonic (one missense, two synonymous), seven intronic, and single nucleotide changes in the 5' and 3' untranslated regions (UTRs) in Asc-1. The identified missense mutation Asc-1G307R from a patient with startle disease and developmental delay was investigated in functional studies. At the molecular level, the mutation Asc-1G307R did not interfere with cell-surface expression, but disrupted glycine uptake. Substitution of glycine at position 307 to other amino acids, e.g., to alanine or tryptophan did not affect trafficking or glycine transport. By contrast, G307K disrupted glycine transport similar to the G307R mutation found in the patient. Structurally, the disrupted function in variants carrying positively charged residues can be explained by local structural rearrangements because of the large positively charged side chain. Thus, our data suggest that SLC7A10 may represent a rare but novel gene associated with human startle disease and developmental delay.

Dystrophic epidermolysis bullosa: genotype-phenotype correlations

Dystrophic epidermolysis bullosa is caused by mutations in the COL7A1 gene. The disease characterized by clinical heterogeneity. To date, scientific findings allow to evaluate correlations between the severity of clinical manifestations and genetic defects underlying in the development of the disease. A systematic literature search was performed using PubMed and RSCI, and keywords including dystrophic epidermolysis bullosa, collagen VII, COL7A1. The review includes description of clinical findings of dystrophic epidermolysis bullosa. The types and localization of pathogenic mutations of the COL7A1 gene, their influence on the protein synthesis, structure and functioning are characterized. The correlation between severe course of dystrophic epidermolysis bullosa and mutations resulting in premature termination codons generation which associate with the absence of type VII collagen at the dermo-epidermal junction has been described. Nevertheless, genotype-phenotype correlations should be analyzed carefully due to mechanisms which enable to restore protein expression as well as the possibility of the formation of premature termination codons associated with a more severe course of the disease, when replacing nucleotides in case of their influence on splicing.

Effects of single-point mutations on translocation of non-interacting heteropolymer protein chains

We analyse the role of single-point mutations on the translocation of heteropolymer protein chains with helical folding. We propose a statistical mechanics model that computes the equilibrium partition function of a given protein by incorporating experimental helix propensities of amino acids. From that we evaluate the free energy barrier for translocation and the average translocation time for single-point protein mutants and compared with wild-type proteins used as reference. In our calculations, single-point mutations that increase energy of the folded state in increase the energy barrier for translocation up to , , in consequence, the translocation time undergoes variations between and with the largest variations associated to substitutions of amino acids glycine and proline by alanine. In fact, substitutions of amino acids with the lowest helix propensities by amino acids with the largest helix propensities induced the largest mutational effects. These results indicate that effects of single-point mutations become evident when mutations increase energy of the folded state at least in . Our model predicts translocation time delays for proline to alanine single-point mutations which agree with deficiencies of protein translocation observed recently in experiments.

Development of a PNA-LNA-LAMP Assay to Detect an SNP Associated with QoI Resistance in Erysiphe necator.

The repetitive use of quinone outside inhibitor fungicides (QoIs, strobilurins; Fungicide Resistance Action Committee [FRAC] 11) to manage grape powdery mildew has led to development of resistance in Erysiphe necator. While several point mutations in the mitochondrial cytochrome b gene are associated with resistance to QoI fungicides, the substitution of glycine to alanine at codon 143 (G143A) has been the only mutation observed in QoI-resistant field populations. Allele-specific detection methods such as digital droplet PCR and TaqMan probe-based assays can be used to detect the G143A mutation. In this study, a peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA-LAMP) assay consisting of an A-143 reaction and a G-143 reaction, was designed for rapidly detecting QoI resistance in E. necator. The A-143 reaction amplifies the mutant A-143 allele faster than the wild-type G-143 allele, while the G-143 reaction amplifies the G-143 allele faster than the A-143 allele. Identification of resistant or sensitive E. necator samples was determined by which reaction had the shorter time to amplification. Sixteen single-spore QoI-resistant and -sensitive E. necator isolates were tested using both assays. Assay specificity in distinguishing the single nucleotide polymorphism (SNP) approached 100% when tested using purified DNA of QoI-sensitive and -resistant E. necator isolates. This diagnostic tool was sensitive to one-conidium equivalent of extracted DNA with an R2 value of 0.82 and 0.87 for the G-143 and A-143 reactions, respectively. This diagnostic approach was also evaluated against a TaqMan probe-based assay using 92 E. necator samples collected from vineyards. The PNA-LNA-LAMP assay detected QoI resistance in ≤30 min and showed 100% agreement with the TaqMan probe-based assay (≤1.5 h) for the QoI-sensitive and -resistant isolates. There was 73.3% agreement with the TaqMan probe-based assay when samples had mixed populations with both G-143 and A-143 alleles present. Validation of the PNA-LNA-LAMP assay was conducted in three different laboratories with different equipment. The results showed 94.4% accuracy in one laboratory and 100% accuracy in two other laboratories. The PNA-LNA-LAMP diagnostic tool was faster and required less expensive equipment relative to the previously developed TaqMan probe-based assay, making it accessible to a broader range of diagnostic laboratories for detection of QoI resistance in E. necator. This research demonstrates the utility of the PNA-LANA-LAMP for discriminating SNPs from field samples and its utility for point-of-care monitoring of plant pathogen genotypes.