Protein Modeling Studies Research Articles

Manzamine A reduces androgen receptor transcription and synthesis by blocking E2F8-DNA interactions and effectively inhibits prostate tumor growth in mice.

The androgen receptor (AR) is the main driver in the development of castration-resistant prostate cancer, where the emergence of AR splice variants leads to treatment-resistant disease. Through detailed molecular studies of the marine alkaloid manzamine A (MA), we identified transcription factor E2F8 as a previously unknown regulator of AR transcription that prevents AR synthesis in prostate cancer cells. MA significantly inhibited the growth of various prostate cancer cell lines and was highly effective in inhibiting xenograft tumor growth in mice without any pathophysiological perturbations in major organs. MA suppressed the full-length AR (AR-FL), its spliced variant AR-V7, and the AR-regulated prostate-specific antigen (PSA; also known as KLK3) and human kallikrein 2 (hK2; also known as KLK2) genes. RNA sequencing (RNA-seq) analysis and protein modeling studies revealed E2F8 interactions with DNA as a potential novel target of MA, suppressing AR transcription and its synthesis. This novel mechanism of blocking AR biogenesis via E2F8 may provide an opportunity to control therapy-resistant prostate cancer over the currently used AR antagonists designed to target different parts of the AR gene.

Homology modeling and molecular docking studies of INF1 protein of Phytophthora infestans in Potato

Homology modeling and molecular docking studies of INF1 protein of Phytophthora infestans in Potato

In silico characterization and identification of compound heterozygous variants in H/ACA Ribonucleoprotein Assembly Factor (SHQ1) from Indian population.

H/ACA small nucleolar ribonucleoproteins (snoRNP) form a complex with multiple proteins to accomplish the pseudouridylation of rRNA. The assembly of H/ACA small nucleolar ribonucleoproteins (snoRNP) is initiated by H/ACA ribonucleoprotein Assembly factor, that is, SHQ1. Mutations in SHQ1 have been reported to cause two disorders namely, dystonia-35 childhood onset (OMIM*619921) and neurodevelopmental disorder with seizures and dystonia (OMIM*619922), both of which are inherited in an autosomal recessive manner. Considering the high genetic and clinical diversity of SHQ1-related clinical features and the importance of SHQ1 in the assembly of the H/ACA snoRNP complex, it is important to take a systematic approach to delineate the genetic diagnosis and impact of mutations on protein structure and stability. Whole exome sequencing followed by Sanger validation was performed in an individual with the clinical features of neurodevelopmental disorder with seizures and dystonia (OMIM*619922). Protein modeling studies of all the reported SHQ1 variants to date were performed using freely available web servers Interactive Tree of Life, String, BioGrid, ShinyGO, DAVID, and Pathvix. Protein structures were visualized using Pymol. We identified compound heterozygous variants, one known frameshift deletion c. 828_831del, p.(Asp277Serfs*27) and the other novel missense variant c. 1157A>C, p.(Tyr386Ser) found in an individual with neurodevelopmental disorder, seizures, movement disorder, and hypomyelination leukodystrophy on neuroimaging. Protein-interactome studies identified potential genetic interactors that include GAR1, NAF1, TRUB1, UTP15, DKC1, NOP10, NPHOSPH 10, KRR1, NOP58, NOP56, FBL, RRP9, NHP2, RUVBL1, and RUVBL2. Ribosome biogenesis in eukaryotes, RNA polymerase, RNA transport, spliceosome, ribosome, cytosolic DNA-sensing pathway, DNA replication, mismatch repair, base excision repair, nucleotide excision repair, and basal transcription factors process were identified as the linked pathways with the prioritized genes. In conclusion, a sophisticated genotype and phenotype correlation followed by linking the genes to the key biological pathways opens new avenues to understand disease pathology and plan for therapeutic interventions.

Molecular and clinical characterization of two unrelated families with factor V deficiency, including a novel nonsense variant (p.Gln1532*)

BackgroundFactor V (FV) is an essential cofactor in the coagulation cascade. The characterization of novel mutations is advantageous for the clinical management of FV-deficient patients. MethodsCoagulation screening and thrombin generation assay were performed with the plate-poor plasma. All 25 exons of the F5 gene were amplified and sequenced. The ClustalX-2.1 software was applied to the multiple sequence alignment. The possible adverse effects of mutations were investigated with online bioinformatics software and protein modeling. ResultsTwo unrelated families with FV deficiency were under investigation. Proband A was an 18-year-old youth with recurrent epistaxis. Proband B was a 29-year-old woman who did not present with any bleeding symptoms. Three heterozygous mutations (p.Gln1532*, p.Phe218Ser, and p.Asp2222Gly) were detected. Interestingly, they were compound heterozygotes and both contained the p.Asp2222Gly, a polymorphism. The thrombin generation assay showed that both patients had impaired ability of thrombin generation, and in particular, proband A was more severe. Conservation, pathogenicity and protein modeling studies all indicated that these three mutations could cause deleterious effects on the function and structure of FV. ConclusionThese three mutations are responsible for the FV-deficient in two pedigrees. Moreover, the nonsense variant p.Gln1532* is first reported in the world.

In Silico Characterization of RNASEH2A Pathogenic Variants and Identification of Novel Splice Site Donor Variant c.549+1G>T in Indian Population.

Background Aicardi-Goutieres syndrome (AGS) is a genetic disorder that has variable manifestations including neurological, immunological, and sometimes other system involvement in various combinations. Considering the high genetic and clinical diversity of AGS and the importance of RNASEH2 complex in the biological system, it is important to take a systematic approach to delineate the genetic diagnosis and impact of missense mutations. Methods Clinical targeted gene sequencing followed by Sanger validation was performed in an individual with the clinical features of AGS. Protein modeling studies of all the reported RNASEH2A missense variants till date were performed using freely available web servers BioGrid, ShinyGO. Protein structures were visualized using Pymol. Results and discussion We identified a novel homozygous splice site donor variant c.549+1G>T in RNASEH2A. Furthermore protein-interactome studies identifiedpotential genetic interactors that include RNASEH2A, RNASEH2B, TYMS, RNASEH2C, RPA1, ORC3, ORC2, CDC6, PCNA, LIG1, PRIM1, RFC2, DUT, GINS1, MCM7, FEN1, MCM4, GINS2, CDK4, and MCM5. Identified genes were mapped to specific pathways using SHINY GO. DNA replication and cell cycle, centrosome cycle, post-replication repair, nucleic acid and metabolic process, cellular response to stress, DNA metabolic process, nucleic acid phosphodiester bond hydrolysis, RNA phosphodiester bond hydrolysis, and DNA biosynthetic process were identified as the linked pathways with the prioritized genes. Conclusion In conclusion, a sophisticated genotype and phenotype correlation followed by linking the genes to the key biological pathways opens new avenues to understand disease pathology and plan for therapeutic interventions.

Mitochondrial Genome Alterations, Cytochrome C Oxidase Activity, and Oxidative Stress: Implications in Primary Open-angle Glaucoma.

To evaluate mitochondrial genome alterations, cytochrome c oxidase (COX) activity, and oxidative stress in primary open-angle glaucoma (POAG). Whole mitochondrial genome was screened in 75 POAG cases and 105 controls by polymerase chain reaction (PCR) sequencing. COX activity was measured from peripheral blood mononuclear cells (PBMCs). A protein modeling study was done to evaluate the impact of G222E variant on protein function. Levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), 8-isoprostane (8-IP), and total antioxidant capacity (TAC) were also measured. A total of 156 and 79 mitochondrial nucleotide variations were found in the cohort of 75 POAG patients and 105 controls, respectively. Ninety-four (60.26%) variations spanned the coding region, and 62 (39.74%) variations spanned noncoding regions (D-loop, 12SrRNA, and 16SrRNA) of mitochondrial genome in POAG patients. Out of 94 nucleotide changes in coding region, 68 (72.34%) were synonymous changes, 23 (24.46%) non-synonymous, and three (3.19%) were found in the region coding for transfer ribonucleic acid (tRNA). Three changes (p.E192K in ND1, p.L128Q in ND2, and p.G222E in COX2) were found to be pathogenic. Twenty-four (32.0%) patients were positive for either of these pathogenic mitochondrial deoxyribonucleic acid (mtDNA) nucleotide changes. Majority of cases (18.7%) had pathogenic mutation in COX2 gene. Patients who harbored pathogenic mtDNA change in COX2 gene had significantly lower levels of COX activity (p < 0.0001) and TAC (p = 0.004), and higher levels of 8-IP (p = 0.01) as compared to patients who did not harbor this mtDNA. G222E changed the electrostatic potential and adversely impacted protein function of COX2 by affecting nonpolar interactions with neighboring subunits. Pathogenic mtDNA mutations were present in POAG patients, which were associated with reduced COX activity and increased levels of oxidative stress. POAG patients should be evaluated for mitochondrial mutations and oxidative stress and may be managed accordingly with antioxidant therapies. Mohanty K, Mishra S, Dada R, et al. Mitochondrial Genome Alterations, Cytochrome C Oxidase Activity, and Oxidative Stress: Implications in Primary Open-angle Glaucoma. J Curr Glaucoma Pract 2022;16(3):158-165.

Acarbose May Function as a Competitive Exclusion Agent for the Producing Bacteria.

Acarbose is a well-known microbial specialized metabolite used clinically to treat type 2 diabetes. This natural pseudo-oligosaccharide (PsOS) shows potent inhibitory activity toward various glycosyl hydrolases, including α-glucosidases and α-amylases. While acarbose and other PsOSs are produced by many different bacteria, their ecological or biological role in microbial communities is still an open question. Here, we show that several PsOS-producing actinobacteria, i.e., Actinoplanes sp. SE50/110 (acarbose producer), Streptomyces glaucescens GLA.O (acarbose producer), and Streptomyces dimorphogenes ATCC 31484 (trestatin producer), can grow in the presence of acarbose, while the growth of the non-PsOS-producing organism Streptomyces coelicolor M1152 was suppressed when starch is the main source of energy. Further investigations using recombinant α-amylases from S. coelicolor M1152 and the PsOS-producing actinobacteria revealed that the S. coelicolor α-amylase was inhibited by acarbose, whereas those from the PsOS-producing bacteria were not inhibited by acarbose. Bioinformatic and protein modeling studies suggested that a point mutation in the α-amylases of the PsOS-producing actinobacteria is responsible for the resistance of those enzymes toward acarbose. Converting the acarbose-resistant α-amylase AcbE to its A304H variant diminished its acarbose-resistance property. Taken together, the results suggest that acarbose is used by the producing bacteria as a competitive exclusion agent to suppress the growth of other microorganisms in their natural environment, while the producing organisms equip themselves with α-amylase variants that are resistant to acarbose.

Integrated Genomic Analysis Identifies ANKRD36 Gene as a Novel and Common Biomarker of Disease Progression in Chronic Myeloid Leukemia.

Simple SummaryChronic myeloid leukemia is a type of blood cancer that is regarded as a success story in determining the exact biological origin, pathogenesis and development of a molecularly targeted (mutation-specific) therapy that has led to successful treatment of this fatal cancer. It is caused by the BCR-ABL fusion gene, which is formed from the translocation between chromosomes 9 and 22. Anti-BCR-ABL drugs, known as tyrosine kinase inhibitors (TKIs), have led to long-term remissions in more than 80% of CML patients and even cure in about one-third of patients. Nevertheless, many patients face drug resistance, and disease progression occurs in about 30% of CML patients, leading to morbidities and mortality. Unfortunately, no biomarkers of CML progression are available due to a poor understanding of the mechanism of progression. Therefore, finding reliable molecular biomarkers of CML progression is one of the most attractive research areas in 21st-century cancer research. In this study, we report novel genomic variants exclusively found in all our advanced-phase CML patients. This study will help in identifying CML patients at risk of disease progression and timely therapeutic interventions to avoid or at least delay fatal disease progression in this cancer.Background: Chronic myeloid leukemia (CML) is initiated in bone marrow due to chromosomal translocation t(9;22) leading to fusion oncogene BCR-ABL. Targeting BCR-ABL by tyrosine kinase inhibitors (TKIs) has changed fatal CML into an almost curable disease. Despite that, TKIs lose their effectiveness due to disease progression. Unfortunately, the mechanism of CML progression is poorly understood and common biomarkers for CML progression are unavailable. This study was conducted to find novel biomarkers of CML progression by employing whole-exome sequencing (WES). Materials and Methods: WES of accelerated phase (AP) and blast crisis (BC) CML patients was carried out, with chronic-phase CML (CP-CML) patients as control. After DNA library preparation and exome enrichment, clustering and sequencing were carried out using Illumina platforms. Statistical analysis was carried out using SAS/STAT software version 9.4, and R package was employed to find mutations shared exclusively by all AP-/BC-CML patients. Confirmation of mutations was carried out using Sanger sequencing and protein structure modeling using I-TASSER followed by mutant generation and visualization using PyMOL. Results: Three novel genes (ANKRD36, ANKRD36B and PRSS3) were mutated exclusively in all AP-/BC-CML patients. Only ANKRD36 gene mutations (c.1183_1184 delGC and c.1187_1185 dupTT) were confirmed by Sanger sequencing. Protein modeling studies showed that mutations induce structural changes in ANKRD36 protein. Conclusions: Our studies show that ANKRD36 is a potential common biomarker and drug target of early CML progression. ANKRD36 is yet uncharacterized in humans. It has the highest expression in bone marrow, specifically myeloid cells. We recommend carrying out further studies to explore the role of ANKRD36 in the biology and progression of CML.

DLG4-related synaptopathy: a new rare brain disorder

PurposePostsynaptic density protein-95 (PSD-95), encoded by DLG4, regulates excitatory synaptic function in the brain. Here we present the clinical and genetic features of 53 patients (42 previously unpublished) with DLG4 variants. MethodsThe clinical and genetic information were collected through GeneMatcher collaboration. All the individuals were investigated by local clinicians and the gene variants were identified by clinical exome/genome sequencing. ResultsThe clinical picture was predominated by early onset global developmental delay, intellectual disability, autism spectrum disorder, and attention deficit–hyperactivity disorder, all of which point to a brain disorder. Marfanoid habitus, which was previously suggested to be a characteristic feature of DLG4-related phenotypes, was found in only nine individuals and despite some overlapping features, a distinct facial dysmorphism could not be established. Of the 45 different DLG4 variants, 39 were predicted to lead to loss of protein function and the majority occurred de novo (four with unknown origin). The six missense variants identified were suggested to lead to structural or functional changes by protein modeling studies. ConclusionThe present study shows that clinical manifestations associated with DLG4 overlap with those found in other neurodevelopmental disorders of synaptic dysfunction; thus, we designate this group of disorders as DLG4-related synaptopathy.

Discovery and Protein Modeling Studies of Novel Compound Mutations Causing Resistance to Multiple Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia.

Objective:BCR-ABL fusion oncogene is the hallmark of chronic myeloid leukemia (CML), causing genomic instability which leads to accumulation of mutations in BCR-ABL as well as other genes. BCR-ABL mutations are the cause of tyrosine kinase inhibitors (TKIs) resistance in CML. Recently, compound BCR-ABL mutations have been reported to resist all FDA approved TKIs. Therefore, finding novel compound BCR-ABL mutations can help and clinically manage CML. Therefore, our objective was to find out novel drug-resistant compound BCR-ABL mutations in CML and carry out their protein modelling studies. Methodology:Peripheral blood samples were collected from ten imatinib resistant CML patients receiving nilotinib treatment. BCR-ABL transcript mutations were investigated by employing capillary sequencing. Patient follow-up was carried out using European LeukemiaNet guidelines. Protein modeling studies were carried out for new compound mutations using PyMol to see the effects of mutations at structural level. Results:A novel compound mutation (K245N mutation along with G250W mutation) and previously known T351I utation was detected in two of the nilotinib resistance CML patients respectively while in the rest of 8 nilotinib responders, no resistant mutations were detected. Protein modelling studies indicated changes in BCR-ABL mutant protein which may have negatively impacted its binding with nilotinib leading to drug resistance. Conclusion:We report a novel nilotinib resistant BCR-ABL compound mutation (K245N along with G250W mutation) which impacts structural modification in BCR-ABL mutant protein leading to drug resistance. As compound mutations pose a new threat by causing resistance to all FDA approved tyrosine kinase inhibitors in BCR-ABL+ leukemias, our study opens a new direction for in vitro characterization of novel BCR-ABL compound mutations and their resistant to second generation and third generation TKIs.

Identification of novel mutations in the methyltransferase complex (Nsp10-Nsp16) of SARS-CoV-2

A recent outburst of the pandemic caused by a member of the coronaviridae family identified as SARS-CoV-2. The highly contagious nature of the virus allows it to spread rapidly worldwide and caused severe healthcare and economic distress. So far, no proper line of treatment or vaccines has been available against SARS-CoV-2. Since, the infected people rapidly increased, causing the saturation of healthcare systems with coronavirus disease (COVID-19) patients. As the virus spread to new locations it also acquired various mutations. Here, in this study, we focused on identifying mutations in one of the crucial complex of SARS-CoV-2, the Nsp10-Nsp16 2′-O-methyltransferase complex. This complex plays indispensable role in the post-transcriptional modifications of viral RNA by its capping. We analysed 208 sequences of Nsp10-Nsp16 reported from India and compared with first reported sequence from Wuhan, China. Our analysis revealed a single mutation in Nsp10 and five mutations in Nsp16 protein. We also show that these mutations are leading to alteration in the secondary structure of Nsp10-Nsp16. Further, the protein modelling studies revealed that the mutation of both Nsp10-Nsp16 impacts the protein dynamicity and stability. Altogether, this study provides novel insights into the variations observed in the proteins of SARS-CoV-2 that might have functional consequences.

Silk from Indian paper wasp: Structure prediction and secondary conformational analysis

The pupae of Indian paper wasp, Ropalidia marginata, produce silk that covers the opening of each cells in their hives, which probably helps in thermoregulation and providing mechanical stability. In this study an attempt has been made to investigate the amino acid composition and protein structure of Indian wasp silk using bioinformatics and experimental tools. The amino acid analysis of the regenerated solubilized wasp silk revealed high alanine and serine content that increases the propensity of α-helical and random coil and/or β-sheet structure respectively. The Matrix Assisted Laser Desorption/Ionization-Time Of Flight/Time Of Flight based protein fingerprinting analysis of the regenerated solubilized wasp silk revealed its short-range sequences. The protein modeling studies indicated the presence of predominant coiled coil α-helical and random coil structure in the protein isolated from wasp hive caps, similar to previous reports from the Vespoidea family. Fourier transform infrared spectroscopic analysis and X-ray diffraction analysis of the native paper wasp cap revealed the presence of mix of coiled coil α-helix and β-sheet structure in outer regions; whereas inner region contains tannin/intermolecularly bonded hydroxyl groups of polyphenols. The present study is first report to generate valuable insights about the chemical composition and secondary structure prediction of Indian paper wasp silk and highlight on nature's novel modus operandi to engineer unique functional fibrous matrix.

Identification of novel mutations in RNA-dependent RNA polymerases of SARS-CoV-2 and their implications on its protein structure.

The rapid development of the SARS-CoV-2 mediated COVID-19 pandemic has been the cause of significant health concern, highlighting the immediate need for effective antivirals. SARS-CoV-2 is an RNA virus that has an inherently high mutation rate. These mutations drive viral evolution and genome variability, thereby facilitating viruses to have rapid antigenic shifting to evade host immunity and to develop drug resistance. Viral RNA-dependent RNA polymerases (RdRp) perform viral genome duplication and RNA synthesis. Therefore, we compared the available RdRp sequences of SARS-CoV-2 from Indian isolates and the ‘Wuhan wet sea food market virus’ sequence to identify, if any, variation between them. Our data revealed the occurrence of seven mutations in Indian isolates of SARS-CoV-2. The secondary structure prediction analysis of these seven mutations shows that three of them cause alteration in the structure of RdRp. Furthermore, we did protein modelling studies to show that these mutations can potentially alter the stability of the RdRp protein. Therefore, we propose that RdRp mutations in Indian SARS-CoV-2 isolates might have functional consequences that can interfere with RdRp targeting pharmacological agents.

Delineation of phenotypes and genotypes related to cohesin structural protein RAD21

RAD21 encodes a key component of the cohesin complex, and variants in RAD21 have been associated with Cornelia de Lange Syndrome (CdLS). Limited information on phenotypes attributable to RAD21 variants and genotype–phenotype relationships is currently published. We gathered a series of 49 individuals from 33 families with RAD21 alterations [24 different intragenic sequence variants (2 recurrent), 7 unique microdeletions], including 24 hitherto unpublished cases. We evaluated consequences of 12 intragenic variants by protein modelling and molecular dynamic studies. Full clinical information was available for 29 individuals. Their phenotype is an attenuated CdLS phenotype compared to that caused by variants in NIPBL or SMC1A for facial morphology, limb anomalies, and especially for cognition and behavior. In the 20 individuals with limited clinical information, additional phenotypes include Mungan syndrome (in patients with biallelic variants) and holoprosencephaly, with or without CdLS characteristics. We describe several additional cases with phenotypes including sclerocornea, in which involvement of the RAD21 variant is uncertain. Variants were frequently familial, and genotype–phenotype analyses demonstrated striking interfamilial and intrafamilial variability. Careful phenotyping is essential in interpreting consequences of RAD21 variants, and protein modeling and dynamics can be helpful in determining pathogenicity. The current study should be helpful when counseling families with a RAD21 variation.

Homology Modeling and Virtual Screening Studies of Antigen MLAA-42 Protein: Identification of Novel Drug Candidates against Leukemia-An In Silico Approach.

Monocytic leukemia-associated antigen-42 (MLAA-42) is associated with excessive cell division and progression of leukemia. Thus, human MLAA-42 is considered as a promising target for designing of new lead molecules for leukemia treatment. Herein, the 3D model of the target was generated by homology modeling technique. The model was then evaluated using various cheminformatics servers. Moreover, the virtual screening studies were performed to explore the possible binding patterns of ligand molecules to MLAA's active site pocket. Thirteen ligand molecules from the ChemBank™ database were identified as they showed good binding affinities, scaffold diversity, and preferential ADME properties which may act as potent drug candidates against leukemia. The study provides the way to identify novel therapeutics with optimal efficacy, targeting MLAA-42.

A novel missense variant in the RASGRP2 gene in patients with moderate to severe bleeding disorder

Inherited platelet function disorder-18 (IPD-18) is a relatively new non-syndromic autosomal recessive bleeding disorder. It is characterized by deficient or dysfunctional CalDAG-GEFI protein. The distinctive feature of the disease is impaired platelet aggregation in response to multiple physiologic agonists. We here report a family with a platelet-type bleeding disorder and a novel mutation in the RASGRP2 gene. The overall bleeding score for the affected individuals was 15 and 12. Based on the initial diagnosis of Glanzmann thrombasthenia, targeted sequencing of integrin subunit alpha 2b and integrin subunit beta 3 encoding genes ITGA2B and ITGB3 were carried out in both affected members of a family. Sequence alignment failed to identify the disease-causing variant(s) in both genes. Therefore, whole exome sequencing in one affected individual was performed. Data analysis detected a novel homozygous missense variant (c.956C>T; p.Pro319Leu) in the exon 9 of the RASGRP2 gene. Five additional individuals of a family including both parents, an affected individual and two asymptomatic individuals were Sanger sequenced for the variant (c.956C>T). The variant segregates in the family in an autosomal recessive manner. Several in silico tools predicted the variant as pathogenic. Protein modeling studies suggest that the mutation (p.Pro319Leu) cause a conformational change in the loop structure of the RasGEF domain of the CalDAG-GEFI protein. Reported variants in the RasGEF domain impair expression and/or nucleotide exchange activity of CalDAG-GEFI protein and thus inhibit the activation of Rap1 protein required for platelet adhesion and hemostatic plug formation.

High Functioning Autism with Missense Mutations in Synaptotagmin-Like Protein 4 (SYTL4) and Transmembrane Protein 187 (TMEM187) Genes: SYTL4- Protein Modeling, Protein-Protein Interaction, Expression Profiling and MicroRNA Studies.

We describe a 7-year-old male with high functioning autism spectrum disorder (ASD) and maternally-inherited rare missense variant of Synaptotagmin-like protein 4 (SYTL4) gene (Xq22.1; c.835C>T; p.Arg279Cys) and an unknown missense variant of Transmembrane protein 187 (TMEM187) gene (Xq28; c.708G>T; p. Gln236His). Multiple in-silico predictions described in our study indicate a potentially damaging status for both X-linked genes. Analysis of predicted atomic threading models of the mutant and the native SYTL4 proteins suggest a potential structural change induced by the R279C variant which eliminates the stabilizing Arg279-Asp60 salt bridge in the N-terminal half of the SYTL4, affecting the functionality of the protein’s critical RAB-Binding Domain. In the European (Non-Finnish) population, the allele frequency for this variant is 0.00042. The SYTL4 gene is known to directly interact with several members of the RAB family of genes, such as, RAB27A, RAB27B, RAB8A, and RAB3A which are known autism spectrum disorder genes. The SYTL4 gene also directly interacts with three known autism genes: STX1A, SNAP25 and STXBP1. Through a literature-based analytical approach, we identified three of five (60%) autism-associated serum microRNAs (miRs) with high predictive power among the total of 298 mouse Sytl4 associated/predicted microRNA interactions. Five of 13 (38%) miRs were differentially expressed in serum from ASD individuals which were predicted to interact with the mouse equivalent Sytl4 gene. TMEM187 gene, like SYTL4, is a protein-coding gene that belongs to a group of genes which host microRNA genes in their introns or exons. The novel Q236H amino acid variant in the TMEM187 in our patient is near the terminal end region of the protein which is represented by multiple sequence alignments and hidden Markov models, preventing comparative structural analysis of the variant harboring region. Like SYTL4, the TMEM187 gene is expressed in the brain and interacts with four known ASD genes, namely, HCFC1; TMLHE; MECP2; and GPHN. TMM187 is in linkage with MECP2, which is a well-known determinant of brain structure and size and is a well-known autism gene. Other members of the TMEM gene family, TMEM132E and TMEM132D genes are associated with bipolar and panic disorders, respectively, while TMEM231 is a known syndromic autism gene. Together, TMEM187 and SYTL4 genes directly interact with recognized important ASD genes, and their mRNAs are found in extracellular vesicles in the nervous system and stimulate target cells to translate into active protein. Our evidence shows that both these genes should be considered as candidate genes for autism. Additional biological testing is warranted to further determine the pathogenicity of these gene variants in the causation of autism.

Biallelic mutations in the LPAR6 gene causing autosomal recessive wooly hair/hypotrichosis phenotype in five Pakistani families.

Autosomal recessive wooly hair/hypotrichosis is an inherited disorder of hair characterized by less dense, short, and tightly curled hair on the scalp and sometimes less dense to complete absence of eyebrows and eyelashes. Autosomal recessive wooly hair/hypotrichosis phenotypes are mostly associated with pathogenic sequence variants in LIPH and LPAR6 genes. To find out the molecular basis of the disease, five families with autosomal recessive wooly hair/hypotrichosis were recruited for genetic analysis. Direct Sanger sequencing of LIPH and LPAR6 genes was carried out using BigDye chain termination chemistry. P2RY5 protein homology models were developed to study the effect of mutation on protein structure in a family having novel mutation. Sanger sequencing revealed a novel homozygous missense mutation (c.47A>T) in the LPAR6 gene in family A, while recurrent mutation (c.436G>A) was detected in the rest of the four families (B-E). Protein homology models for both native and mutant P2RY5 protein were developed to study the difference in subtle structural features because of Lys16Met (K16M) mutation. We observed that P2RY5K16M mutation results decrease in the number of ionic interactions detrimental to the protein stability. Protein modeling studies revealed that the novel mutation identified here decreased the number of ionic interactions by affecting physicochemical parameters of the protein, leading to an overall decrease in protein stability with no major secondary structural changes. The molecular analysis further confirms the frequent involvement of LPAR6 in autosomal recessive wooly hair/hypotrichosis, while the bioinformatic study revealed that the missense mutation destabilizes the overall structure of P2RY5 protein.

Heterozygous variants in MYBPC1 are associated with an expanded neuromuscular phenotype beyond arthrogryposis.

Encoding the slow skeletal muscle isoform of myosin binding protein-C, MYBPC1 is associated with autosomal dominant and recessive forms of arthrogryposis. The authors describe a novel association for MYBPC1 in four patients from three independent families with skeletal muscle weakness, myogenic tremors, and hypotonia with gradual clinical improvement. The patients carried one of two de novo heterozygous variants in MYBPC1, with the p.Leu263Arg variant seen in three individuals and the p.Leu259Pro variant in one individual. Both variants are absent from controls, well conserved across vertebrate species, predicted to be damaging, and located in the M-motif. Protein modeling studies suggested that the p.Leu263Arg variant affects the stability of the M-motif, whereas the p.Leu259Pro variant alters its structure. In vitro biochemical and kinetic studies demonstrated that the p.Leu263Arg variant results in decreased binding of the M-motif to myosin, which likely impairs the formation of actomyosin cross-bridges during muscle contraction. Collectively, our data substantiate that damaging variants in MYBPC1 are associated with a new form of an early-onset myopathy with tremor, which is a defining and consistent characteristic in all affected individuals, with no contractures. Recognition of this expanded myopathic phenotype can enable identification of individuals with MYBPC1 variants without arthrogryposis.

Protein modeling, molecular network and molecular dynamics study of newly sequenced interleukin-18 (IL-18) gene in Mus musculus.

Interleukin-18 (IL-18) belongs to the superfamily of IL-1 protein and exerts a pleiotropic pro-inflammatory effect on the body. Generally, this protein is significantly involved in immune defense during infection in cells, but sometimes its anomalous activities produce some inflammatory diseases like rheumatoid arthritis and Crohn's disease. In the present study, the IL-18 gene was isolated from mice and was subsequently cloned and sequenced. Further, the network analysis was carried out to explore the functional role of IL-18 protein in animals. The 3D protein structure of the IL-18 protein was generated and docked with appropriate 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate (CPS) ligand. Later the complex structure of the protein was subjected to molecular dynamics simulation (MDS) for 50 ns to determine the effect of ligand on protein. The network analysis explored the correlation of IL-18 protein with others proteins and their involvement in the different significant pathway to defend the cell from various diseases. As confirmed by MDS, the CPS:IL-18 complex was found to be highly stable. Our results further indicated that CPS ligand has the potential to act as a drug molecule, in future, for counteracting IL-18 activity. To date, no structural details were available for animal IL-18. Hence, the finding of this study will be useful in broadening the horizon towards a better understanding of the functional and structural aspects of IL-18 in animals.