Identification of novel homozygous variants in FOXE3 and AP4M1 underlying congenital syndromic anophthalmia and microphthalmia.

BACKGROUND: Anophthalmia and microphthalmia are severe developmental ocular disorders that affect the size of the ocular globe and can be unilateral or bilateral. The disease is found in syndromic as well as non-syndromic forms. It is genetically caused by chromosomal aberrations, copy number variations and single gene mutations, along with non-genetic factors such as viral infections, deficiency of vitamin A and an exposure to alcohol or drugs during pregnancy. To date, more than 30 genes having different modes of inheritance patterns are identified as causing anophthalmia and microphthalmia. METHODS: In the present study, a clinical and genetic analysis was performed of six patients with anophthalmia and microphthalmia and/or additional phenotypes of intellectual disability, developmental delay and cerebral palsy from a large consanguineous Pakistani family. Whole exome sequencing followed by data analysis for variants prioritization and validation through Sanger sequencing was performed to identify the disease causing variant(s). American College of Medical Genetics and Genomics (ACMG) guidelines were applied to classify clinical interpretation of the prioritized variants. RESULTS: Clinical investigations revealed that the affected individuals are afflicted with anophthalmia. Three of the patients showed additional phenotype of intellectual disability, developmental delays and other neurological symptoms. Whole exome sequencing of the DNA samples of the affected members in the family identified a novel homozygous stop gain mutation (NM_012186: c.106G>T: p.Glu36*) in Forkhead Box E3 (FOXE3) gene shared by all affected individuals. Moreover, patients segregating additional phenotypes of spastic paraplegia, intellectual disability, hearing loss and microcephaly showed an additional homozygous sequence variant (NM_004722: c.953G>A: p.Arg318Gln) in AP4M1. Sanger sequencing validated the correct segregation of the identified variants in the affected family. ACMG guidelines predicted the variants to be pathogenic. CONCLUSIONS: We have investigated first case of syndromic anophthalmia caused by variants in the FOXE3 and AP4M1. The present findings are helpful for understanding pathological role of the mutations of the genes in syndromic forms of anophthalmia. Furthermore, the study signifies searching for the identification of second variant in families with patients exhibiting variable phenotypes. In addition, the findings will help clinical geneticists, genetic counselors and the affected family with respect to prenatal testing, family planning and genetic counseling.

Mutational spectrum of CFTR in cystic fibrosis patients with gastrointestinal and hepatobiliary manifestations.

BACKGROUND: Cystic fibrosis (CF) is a rare and debilitating autosomal recessive disorder. It hampers the normal function of various organs and causes severe damage to the lungs, and digestive system leading to recurring pneumonia. Cf also affects reproductive health eventually may cause infertility. The disease manifests due to genetic aberrations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This study aimed to screen for CFTR gene variants in Pakistani CF patients representing variable phenotypes. METHODS: Clinical exome and Sanger sequencing were performed after clinical characterization of 25 suspected cases of CF (CF1-CF25). ACMG guidelines were followed to interpret the clinical significance of the identified variants. RESULTS: Clinical investigations revealed common phenotypes such as pancreatic insufficiency, chest infections, chronic liver and lung diseases. Some patients also displayed symptoms like gastroesophageal reflux disease (GERD), neonatal cholestasis, acrodermatitis, diabetes mellitus, and abnormal malabsorptive stools. Genetic analysis of the 25 CF patients identified deleterious variants in the CFTR gene. Notably, 12% of patients showed compound heterozygous variants, while 88% had homozygous variants. The most prevalent variant was p. (Met1Thr or Met1?) at 24%, previously not reported in the Pakistani population. The second most common variant was p. (Phe508del) at 16%. Other variants, including p. (Leu218*), p. (Tyr569Asp), p. (Glu585Ter), and p. (Arg1162*) were also identified in the present study. Genetic analysis of one of the present patients showed a pathogenic variant in G6PD in addition to CFTR. CONCLUSION: The study reports novel and reported variants in the CFTR gene in CF patients in Pakistani population having distinct phenotypes. It also emphasizes screening suspected Pakistani CF patients for the p. (Met1Thr) variant because of its increased observance and prevalence in the study. Moreover, the findings also signify searching for additional pathogenic variants in the genome of CF patients, which may modify the phenotypes. The findings contribute valuable information for the diagnosis, genetic counseling, and potential therapeutic strategies for CF patients in Pakistan.

Comprehensive in-silico analysis of deleterious SNPs in APOC2 and APOA5 and their differential expression in cancer and cardiovascular diseases conditions.

Genetic variations in APOC2 and APOA5 genes involve activating lipoprotein lipase (LPL), responsible for the hydrolysis of triglycerides (TG) in blood and whose impaired functions affect the TG metabolism and are associated with metabolic diseases. In this study, we investigate the biological significance of genetic variations at the DNA sequence and structural level using various computational tools. Subsequently, 8 (APOC2) and 17 (APOA5) non-synonymous SNPs (nsSNPs) were identified as high-confidence deleterious SNPs based on the effects of the mutations on protein conservation, stability, and solvent accessibility. Furthermore, based on our docking results, the interaction of native and mutant forms of the corresponding proteins with LPL depicts differences in root mean square deviation (RMSD), and binding affinities suggest that these mutations may affect their function. Furthermore, in vivo, and in vitro studies have shown that differential expression of these genes in disease conditions due to the influence of nsSNPs abundance may be associated with promoting the development of cancer and cardiovascular diseases. Preliminary screening using computational methods can be a helpful start in understanding the effects of mutations in APOC2 and APOA5 on lipid metabolism; however, further wet-lab experiments would further strengthen the conclusions drawn from the computational study.

A novel biallelic variant in the Popeye domain-containing protein 1 (POPDC1) underlies limb girdle muscle dystrophy type 25.

POPDC1 also known as BVES, is a highly conserved transmembrane protein, important for striated muscle function and homeostasis. Pathogenic variants in the POPDC1 gene are associated with limb-girdle muscular dystrophy type 25 (LGMDR25). In the present study, we performed trio-whole exome sequencing (WES) followed by Sanger sequencing on a single family having LGMD clinical features. Protein modeling of all POPDC1 missense variants (POPDC1Pro134Leu , POPDC1Ile193Ser , and POPDC1Ser201Phe ) associated with LGMDR25 were performed using Molecular Dynamics (MD) simulation. We identified a homozygous missense variant (c.401C>T; p.Pro134Leu) in the POPDC1 gene. Altered 3D structure, disruptive fluctuation, less compactness, and instability were observed in all the three variants of POPDC1 protein models. In comparison, POPDC1Ser201Phe protein dynamics were more unstable than other variants. Functional study of newly identified variant would add key answers to underlying mechanisms of the disease.

Molecular Dynamic Simulation Analysis of a Novel Missense Variant in CYB5R3 Gene in Patients with Methemoglobinemia.

Background and Objective: Mutations in the CYB5R3 gene cause reduced NADH-dependent cytochrome b5 reductase enzyme function and consequently lead to recessive congenital methemoglobinemia (RCM). RCM exists as RCM type I (RCM1) and RCM type II (RCM2). RCM1 leads to higher methemoglobin levels causing only cyanosis, while in RCM2, neurological complications are also present along with cyanosis. Materials and Methods: In the current study, a consanguineous Pakistani family with three individuals showing clinical manifestations of cyanosis, chest pain radiating to the left arm, dyspnea, orthopnea, and hemoptysis was studied. Following clinical assessment, a search for the causative gene was performed using whole exome sequencing (WES) and Sanger sequencing. Various variant effect prediction tools and ACMG criteria were applied to interpret the pathogenicity of the prioritized variants. Molecular dynamic simulation studies of wild and mutant systems were performed to determine the stability of the mutant CYB5R3 protein. Results: Data analysis of WES revealed a novel homozygous missense variant NM_001171660.2: c.670A > T: NP_001165131.1: p.(Ile224Phe) in exon 8 of the CYB5R3 gene located on chromosome 22q13.2. Sanger sequencing validated the segregation of the identified variant with the disease phenotype within the family. Bioinformatics prediction tools and ACMG guidelines predicted the identified variant p.(Ile224Phe) as disease-causing and likely pathogenic, respectively. Molecular dynamics study revealed that the variant p.(Ile224Phe) in the CYB5R3 resides in the NADH domain of the protein, the aberrant function of which is detrimental. Conclusions: The present study expanded the variant spectrum of the CYB5R3 gene. This will facilitate genetic counselling of the same and other similar families carrying mutations in the CYB5R3 gene.

The mechanism of antigen-presentation of avian bone marrowed dendritic cells suppressed by infectious bronchitis virus.

Dendritic cells are first guard to defend avian infectious bronchitis virus (IBV) infection and invasion. While IBV always suppress dendritic cells and escape the degradation and presentation, which might help viruses to transfer and migrant. Initially, we compared two IBV's function in activating avian bone marrow dendritic cells (BMDCs) and found that both IBV (QX and M41) did not significantly increase surface marker of avian BMDCs. Moreover, a significant decrease of m6A modification level in mRNA, but an increased in the ut RNA were observed in avian BMDCs upon the prevalent IBV (QX) infection. Further study found that both non-structural protein 7 (NSP7) and NSP16 inhibited the maturation and cytokines secretion of BMDCs, as well as their antigen-presentation ability. Lastly, we found that gga-miR21, induced by both NSP7 and NSP16, inhibited the antigen presentation of avian BMDCs. Taken together, our results illustrated how IBV inhibited the antigen-presentation of avian DCs.

Biallelic loss-of-function variants in NEMF cause central nervous system impairment and axonal polyneuropathy.

We aimed to detect the causative gene in five unrelated families with recessive inheritance pattern neurological disorders involving the central nervous system, and the potential function of the NEMF gene in the central nervous system. Exome sequencing (ES) was applied to all families and linkage analysis was performed on family 1. A minigene assay was used to validate the splicing effect of the relevant discovered variants. Immunofluorescence (IF) experiment was performed to investigate the role of the causative gene in neuron development. The large consanguineous family confirms the phenotype-causative relationship with homozygous frameshift variant (NM_004713.6:c.2618del) as revealed by ES. Linkage analysis of the family showed a significant single-point LOD of 4.5 locus. Through collaboration in GeneMatcher, four additional unrelated families' likely pathogenic NEMF variants for a spectrum of central neurological disorders, two homozygous splice-site variants (NM_004713.6:c.574+1G>T and NM_004713.6:c.807-2A>C) and a homozygous frameshift variant (NM_004713.6: c.1234_1235insC) were subsequently identified and segregated with all affected individuals. We further revealed that knockdown (KD) of Nemf leads to impairment of axonal outgrowth and synapse development in cultured mouse primary cortical neurons. Our study demonstrates that disease-causing biallelic NEMF variants result in central nervous system impairment and other variable features. NEMF is an important player in mammalian neuron development.

Recalling the pathology of Parkinson's disease; lacking exact figure of prevalence and genetic evidence in Asia with an alarming outcome: A time to step-up.

Parkinson's disease (PD) is the second most common and progressive neurodegenerative disease globally, with major symptoms like bradykinesia, impaired posture, and tremor. Several genetic and environmental factors have been identified but elucidating the main factors have been challenging due to the disease's complex nature. Diagnosis, prognosis, and management of such diseases are challenging and require effective targeted attention in developing countries. Recently, PD is growing rapidly in many crowded Asian countries as an alarming threat with inadequate knowledge of its prevalence, genetic architecture, and geographic distribution. This study gave an in-depth overview of the prevalence, incidence and genomic/genetics studies published so far in the Asian population. To the best of our knowledge, PD has increased significantly in several Asian countries, including China, South Korea, Japan, Thailand, and Israel over the past few years, requiring a greater level of care and attention. Genetic screening of families with PD at national levels and establishing an official database of PD cases are essential to get a comprehensive and conclusive view of the exact prevalence and genetic diversity of PD in the Asian population to properly manage and treat the disease.

A comparison of intravenous midazolam and diazepam in management of status epilepticus in children.

OBJECTIVE: To compare the efficacy of intravenous midazolam and diazepam in the management of status epilepticus seizures in children. METHODS: The comparative study was conducted in the paediatric neurological emergency unit of The Children's Hospital and the Institute of Child Health, Multan, Pakistan, from December 15, 2018, to May 14, 2019, and comprised paediatric patients of status epilepticus seizures which were divided into Diazepam and Midazolam groups. Data was analysed using Graph-Pad Prism 5. RESULTS: Of the 164 patients, 82(50%) were in each of the two groups. There was no significant difference between the groups in terms of weight, age, residence area of patients and mean duration of seizures (p>0.05). Status epilepticus seizures subsided after intravenous midazolam administration in 77(93.90%) cases, while success in the diazepam group 64(78.05%) (p<0.05). Mean time taken by midazolam to halt seizures was significantly shorter than diazepam (p<0.05) and less cases of treatment failure were observed with intravenous midazolam (p<0.05). Somnolence was observed after diazepam administration in 47(57.3%) cases (p=0.0001). CONCLUSION: Intravenous midazolam was found to be superior in efficacy than intravenous diazepam in controlling status epilepticus seizures.

Genetic polymorphisms in cell cycle regulatory genes CCND1 and CDK4 are associated with susceptibility to breast cancer.

PURPOSE: This study was performed to screen cyclin D1 (CCND1) and cyclin dependent kinases (CDK4) genes in order to evaluate their association with breast carcinogenesis. METHODS: The germline screening of these genes was carried out by combining polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP), followed by DNA sequence analysis. A total of 400 individuals (200 breast cancer patients and 200 healthy controls) were recruited prospectively for this study. RESULTS: Sequence analyses of the coding region of CCND1 revealed 12 mutations. When analyzed, a significant association was found between CCND1 mutations and breast cancer. It was observed that a 6-fold increased breast cancer risk (odds ratio/OR=5.75, 95% confidence interval/CI=1.26-26.33) was associated with Cys7Tyr in breast cancer patients when compared with healthy controls. In addition, a 5-fold increased breast cancer risk was associated with Trp63Stop mutation (OR=5.44, 95% CI=1.82-16.23), 10861C>A (OR=4.84; 95% CI=1.60-14.58) and 7720insTT (OR=5.32, 95% CI=1.98-14.23) in breast cancer patients compared to healthy controls. Concerning CDK4 gene, 5 mutations were identified and a significant association was observed between CDK4 gene mutations and breast cancer. It was observed that a 6-fold increased risk of breast cancer (OR=5.71, 95% CI=0.29-4.65) was associated with 5693 T>A. In addition, a 5-fold increased risk of breast cancer (OR=5.05, 95% CI=2.17-11.78) was associated with 5732 G>A in breast cancer patients compared to controls. CONCLUSION: In this study, our results showed that CCND1 and CDK4 mutations are associated with an increased risk of breast cancer, and may serve as biomarkers for early diagnosis and detection of breast cancer.

The Potential Roles of Host Cell miRNAs in Fine-Tuning Bovine Coronavirus (BCoV) Molecular Pathogenesis, Tissue Tropism, and Immune Regulation.

Bovine coronavirus (BCoV) infection causes significant economic loss to the dairy and beef industries worldwide. BCoV exhibits dual tropism, infecting the respiratory and enteric tracts of cattle. The enteric BCoV isolates could also induce respiratory manifestations under certain circumstances. However, the mechanism of this dual tropism of BCoV infection has not yet been studied well. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and play a dual role in virus infection, mediating virus or modulating host immune regulatory genes through complex virus-host cell interactions. However, their role in BCoV infection remains unclear. This study aims to identify bovine miRNAs crucial for regulating virus-host interaction, influencing tissue tropism, and explore their potential as biomarkers and therapeutic agents against BCoV. We downloaded 18 full-length BCoV genomes (10 enteric and eight respiratory) from GenBank. We applied several bioinformatic tools to study the host miRNAs targeting various regions in the viral genome. We used the criteria of differential targeting between the enteric/respiratory isolates to identify some critical miRNAs as biological markers for BCoV infection. Using various online bioinformatic tools, we also searched for host miRNA target genes involved in BCoV infection, immune evasion, and regulation. Our results show that four bovine miRNAs (miR-2375, miR-193a-3p, miR-12059, and miR-494) potentially target the BCoV spike protein at multiple sites. These miRNAs also regulate the host immune suppressor pathways, which negatively impacts BCoV replication. Furthermore, we found that bta-(miR-2338, miR-6535, miR-2392, and miR-12054) also target the BCoV genome at certain regions but are involved in regulating host immune signal transduction pathways, i.e., type I interferon (IFN) and retinoic acid-inducible gene I (RIG-I) pathways. Moreover, both miR-2338 and miR-2392 also target host transcriptional factors RORA, YY1, and HLF, which are potential diagnostic markers for BCoV infection. Therefore, miR-2338, miR-6535, miR-2392, and miR-12054 have the potential to fine-tune BCoV tropism and immune evasion and enhance viral pathogenesis. Our results indicate that host miRNAs play essential roles in the BCoV tissue tropism, pathogenesis, and immune regulation. Four bovine miRNAs (miR-2375, bta-miR-193a-3p, bta-miR-12059, and bta-miR-494) target BCoV-S glycoprotein and are potentially involved in several immune suppression pathways during the viral infection. These miRNA candidates could serve as good genetic markers for BCoV infection. However, further studies are urgently needed to validate these identified miRNAs and their target genes in the context of BCoV infection and dual tropism and as genetic markers.

Machine learning tools used for mapping some immunogenic epitopes within the major structural proteins of the bovine coronavirus (BCoV) and for the in silico design of the multiepitope-based vaccines.

Introduction: BCoV is one of the significant causes of enteritis in young calves; it may also be responsible for many respiratory outbreaks in young calves. BCoV participates in the development of bovine respiratory disease complex in association with other bacterial pathogens. Our study aimed (1) to map the immunogenic epitopes (B and T cells) within the major BCoV structural proteins. These epitopes are believed to induce a robust immune response through the interaction with major histocompatibility complex (MHC class II) molecules (2) to design some novel BCoV multiepitope-based vaccines. Materials and Methods: The goal is achieved through several integrated in silico prediction computational tools to map these epitopes within the major BCoV structural proteins. The final vaccine was constructed in conjugation with the Choleratoxin B toxin as an adjuvant. The tertiary structure of each vaccine construct was modeled through the AlphaFold2 tools. The constructed vaccine was linked to some immunostimulants such as Toll-like receptors (TLR2 and TLR4). We also predicted the affinity binding of these vaccines with this targeted protein using molecular docking. The stability and purity of each vaccine construct were assessed using the Ramachandran plot and the Z-score values. We created the in silico cloning vaccine constructs using various expression vectors through vector builder and Snap gene. Results and discussion: The average range of major BCoV structural proteins was detected within the range of 0.4 to 0.5, which confirmed their antigen and allergic properties. The binding energy values were detected between -7.9 and -9.4 eV and also confirmed their best interaction between our vaccine construct and Toll-like receptors. Our in silico cloning method expedited the creation of vaccine constructs and established a strong basis for upcoming clinical trials and experimental validations. Conclusion: Our designed multiepitope vaccine candidates per each BCoV structural protein showed high antigenicity, immunogenicity, non-allergic, non-toxic, and high-water solubility. Further studies are highly encouraged to validate the efficacy of these novel BCoV vaccines in the natural host.

The deleterious variants of N-acetylgalactosamine-6-sulfatase (GalN6S) enzyme trigger Morquio a syndrome by disrupting protein foldings.

Lysosomal enzymes degrade cellular macromolecules, while their inactivation causes human hereditary metabolic disorders. Mucopolysaccharidosis IVA (MPS IVA; Moquio A syndrome) is one of the lysosomal storage disorders caused by a defective Galactosamine-6-sulfatase (GalN6S) enzyme. In several populations, disease incidence is elevated due to missense mutations brought on by non-synonymous allelic variation in the GalN6S enzyme. Here, we studied the effect of non-synonymous single nucleotide polymorphism (nsSNPs) on the structural dynamics of the GalN6S enzyme and its binding with N-acetylgalactosamine (GalNAc) using all-atom molecular dynamics simulation and an essential dynamics approach. Consequently, in this study, we have identified three functionally disruptive mutations in domain-I and domain-II, that is, S80L, R90W, and S162F, which presumably contribute to post-translational modifications. The study delineated that both domains work cooperatively, and alteration in domain II (S80L, R90W) leads to conformational changes in the catalytic site in domain-I, while mutation S162F mainly provokes higher residual flexibility of domain II. These results show that these mutations impair the hydrophobic core, implying that Morquio A syndrome is caused by misfolding of the GalN6S enzyme. The results also show the instability of the GalN6S-GalNAc complex upon substitution. Overall, the structural dynamics resulting from point mutations give the molecular rationale for Moquio A syndrome and, more importantly, the Mucopolysaccharidoses (MPS) family of diseases, re-establishing MPS IVA as a protein-folding disease.Communicated by Ramaswamy H. Sarma.

Effectiveness of Autoinoculation in Patients with Multiple Warts Presenting at a Tertiary Care Hospital.

OBJECTIVE: To determine the effectiveness of autoinoculation in patients with multiple skin warts. STUDY DESIGN: A Quasi-experimental study. PLACE AND DURATION OF STUDY: Dermatology department of PNS Shifa Hospital, from April to October 2021. METHODOLOGY: Ninety-six patients with multiple warts were enrolled in this study after informed consent. Under strict aseptic measures and local anaesthesia, wart tissue was removed and crushed on a glass slide with the scalpel. Autoinoculation was done on flexor aspects of bilateral forearms after making a subcutaneous pocket which was then stitched. Patients were assessed at monthly intervals for 03 months and after 01 month of the last autoinoculation to see sustained response. Effectiveness was recorded according to ordinal scale; worsening of lesions/no response at <50% resolution/partial response at >50%- <100% resolution and complete response at 100% resolution. RESULTS: Complete response was observed in 88 (91.66%) of the cases, no response was observed in 2 (2.1%) cases where as 6 (6.3%) showed worsening. All patients were compliant with the follow-up. No new eruptions were observed. CONCLUSION: Autoinoculation is a minimally invasive and cost-effective procedure with excellent response to wart. It also decreases recurrence by generating viral-specific immunity. KEY WORDS: Viral warts, Autoinoculation, Multiple, Immunotherapy, Verrucous, Treatment, HPV, Resistant.

Structural and dynamics insights into the GBA variants associated with Parkinson's disease.

The GBA1 gene encodes for the lysosomal enzyme glucocerebrosidase (GCase), which maintains glycosphingolipid homeostasis and regulates the autophagy process. Genomic variants of GBA1 are associated with Goucher disease; however, several heterozygous variants of GBA (E326K, T369M, N370S, L444P) are frequent high-risk factors for Parkinson's disease (PD). The underlying mechanism of these variants has been revealed through functional and patient-centered research, but the structural and dynamical aspects of these variants have not yet been thoroughly investigated. In the current study, we used a thorough computational method to pinpoint the structural changes that GBA underwent because of genomic variants and drug binding mechanisms. According to our findings, PD-linked nsSNP variants of GBA showed structural variation and abnormal dynamics when compared to wild-typ. The docking analysis demonstrated that the mutants E326K, N370S, and L444P have higher binding affinities for Ambroxol. Root means square deviation (RMSD), Root mean square fluctuation analysis (RMSF), and MM-GBSA analysis confirmed that the Ambroxol are more stable in the binding site of N370S and L444P, and that their binding affinities are stronger as compared to the wild-type and T369M variants of GBA. The evaluation of hydrogen bonds and the calculation of the free binding energy provided additional evidence in favor of this conclusion. When docked with Ambroxol, GBA demonstrated an increase in binding affinity and catalytic activity. Understanding the therapeutic efficacy and potential against the aforementioned changes in the GBA will be beneficial in order to use more efficient methods for developing novel drugs.Communicated by Ramaswamy H. Sarma.

Truncated DNM1 variant underlines developmental delay and epileptic encephalopathy.

Background: Developmental and epileptic encephalopathies (DEEs) signify a group of heterogeneous neurodevelopmental disorder associated with early-onset seizures accompanied by developmental delay, hypotonia, mild to severe intellectual disability, and developmental regression. Variants in the DNM1 gene have been associated with autosomal dominant DEE type 31A and autosomal recessive DEE type 31B. Methods: In the current study, a consanguineous Pakistani family consisting of a proband (IV-2) was clinically evaluated and genetically analyzed manifesting in severe neurodevelopmental phenotypes. WES followed by Sanger sequencing was performed to identify the disease-causing variant. Furthermore, 3D protein modeling and dynamic simulation of wild-type and mutant proteins along with reverse transcriptase (RT)-based mRNA expression were checked using standard methods. Results: Data analysis of WES revealed a novel homozygous non-sense variant (c.1402G>T; p. Glu468*) in exon 11 of the DNM1 gene that was predicted as pathogenic class I. Variants in the DNM1 gene have been associated with DEE types 31A and B. Different bioinformatics prediction tools and American College of Medical Genetics guidelines were used to verify the identified variant. Sanger sequencing was used to validate the disease-causing variant. Our approach validated the pathogenesis of the variant as a cause of heterogeneous neurodevelopmental disorders. In addition, 3D protein modeling showed that the mutant protein would lose most of the amino acids and might not perform the proper function if the surveillance non-sense-mediated decay mechanism was skipped. Molecular dynamics analysis showed varied trajectories of wild-type and mutant DNM1 proteins in terms of root mean square deviation, root mean square fluctuation and radius of gyration. Similarly, RT-qPCR revealed a substantial reduction of the DNM1 gene in the index patient. Conclusion: Our finding further confirms the association of homozygous, loss-of-function variants in DNM1 associated with DEE type 31B. The study expands the genotypic and phenotypic spectrum of pathogenic DNM1 variants related to DNM1-associated pathogenesis.

Commonly expressed key transcriptomic profiles of sepsis in the human circulation and brain via integrated analysis.

BACKGROUND: Sepsis is the leading cause of death in intensive care units and is characterized by multiple organ failure, including dysfuction of the immune system and brain. This study aims to determine the differential effect of sepsis on specific circulating immune cell subsets compared with brain transcriptome and identify the genes co-expressed by them, so as to identify key genes and regulatory factors involved in the pathogenesis of sepsis induced brain injury and identify novel therapeutic targets. METHODS: The GSE133822 and GSE135838 datasets were obtained from the Gene Expression Omnibus (GEO) database and utilized for bioinformatics analyses. Functional enrichment analysis was used to identify commonly expressed genes that were differentially expressed between sepsis patients and non-sepsis patients with critical illness; protein-protein interaction (PPI) networks were also generated. Then, key transcriptomic biomarkers were further validated in an external dataset from the GEO. We also investigated the expression of key mRNAs in peripheral blood mononuclear cells (PBMCs) from sepsis patients by quantitative PCR (qPCR) and an in-vitro model stimulated by lipopolysaccharide (LPS) was generated in brain cell lines. RESULTS: The transcriptomic profiles of brain tissue were relatively similar as those of specific immune cells. In addition, our validation showed that these key genes were up regulated both in PBMCs in sepsis patients and LPS-treated brain cells. CONCLUSION: Brain injury in sepsis was correlated with circulating immune responses, and the expression of DEFA3, MMP8, MMP9 and LCN2 might be potential diagnostic biomarkers as well as therapeutic target in septic brain dysfunction.

Construction of a Novel Infectious Clone of Recombinant Herpesvirus of Turkey Fc-126 Expressing VP2 of IBDV.

The increased virulence of infectious bursal disease virus (IBDV) is a threat to the chicken industry. The construction of novel herpesvirus of turkey-vectored (HVT) vaccines expressing VP2 of virulent IBDV may be a promising vaccine candidate for controlling this serious disease in chickens. We generated a novel infectious clone of HVT Fc-126 by inserting mini-F sequences in lieu of the glycoprotein C (gC) gene. Based on this bacterial artificial chromosome (BAC), a VP2 expression cassette containing the pMCMV IE promoter and a VP2 sequence from the virulent IBDV NJ09 strain was inserted into the noncoding area between the UL55 and UL56 genes to generate the HVT vector VP2 recombinant, named HVT-VP2-09. The recovered vectored mutant HVT-VP2-09 exhibited higher titers (p = 0.0202 at 36 h) or similar growth kinetics to the parental virus HVT Fc-126 (p = 0.1181 at 48 h and p = 0.1296 at 64 h). The high reactivation ability and strong expression of VP2 by HVT-VP2-09 in chicken embryo fibroblasts (CEFs) were confirmed by indirect immunofluorescence (IFA) and Western blotting. The AGP antibodies against IBDV were detected beginning at 3 weeks post-inoculation (P.I.) of HVT-VP2-09 in 1-day-old SPF chickens. Seven of ten chickens immunized with HVT-VP2-09 were protected post-challenge (P.C.) with the virulent IBDV NJ09 strain. In contrast, all chickens in the challenge control group showed typical IBD lesions in bursals, and eight of ten died P.C. In this study, we demonstrated that (i) a novel HVT BAC with the whole genome of the Fc-126 strain was obtained with the insertion of mini-F sequences in lieu of the gC gene; (ii) HVT-VP2-09 harboring the VP2 expression cassette from virulent IBDV exhibited in vitro growth properties similar to those of the parental HVT virus in CEF cells; and (iii) HVT-VP2-09 can provide efficient protection against the IBDV NJ09 strain.

Proteomics approaches: A review regarding an importance of proteome analyses in understanding the pathogens and diseases.

Proteomics is playing an increasingly important role in identifying pathogens, emerging and re-emerging infectious agents, understanding pathogenesis, and diagnosis of diseases. Recently, more advanced and sophisticated proteomics technologies have transformed disease diagnostics and vaccines development. The detection of pathogens is made possible by more accurate and time-constrained technologies, resulting in an early diagnosis. More detailed and comprehensive information regarding the proteome of any noxious agent is made possible by combining mass spectrometry with various gel-based or short-gun proteomics approaches recently. MALDI-ToF has been proved quite useful in identifying and distinguishing bacterial pathogens. Other quantitative approaches are doing their best to investigate bacterial virulent factors, diagnostic markers and vaccine candidates. Proteomics is also helping in the identification of secreted proteins and their virulence-related functions. This review aims to highlight the role of cutting-edge proteomics approaches in better understanding the functional genomics of pathogens. This also underlines the limitations of proteomics in bacterial secretome research.

Molecular Insights into the Role of Pathogenic nsSNPs in GRIN2B Gene Provoking Neurodevelopmental Disorders.

The GluN2B subunit of N-methyl-D-aspartate receptors plays an important role in the physiology of different neurodevelopmental diseases. Genetic variations in the GluN2B coding gene (GRIN2B) have consistently been linked to West syndrome, intellectual impairment with focal epilepsy, developmental delay, macrocephaly, corticogenesis, brain plasticity, as well as infantile spasms and Lennox-Gastaut syndrome. It is unknown, however, how GRIN2B genetic variation impacts protein function. We determined the cumulative pathogenic impact of GRIN2B variations on healthy participants using a computational approach. We looked at all of the known mutations and calculated the impact of single nucleotide polymorphisms on GRIN2B, which encodes the GluN2B protein. The pathogenic effect, functional impact, conservation analysis, post-translation alterations, their driving residues, and dynamic behaviors of deleterious nsSNPs on protein models were then examined. Four polymorphisms were identified as phylogenetically conserved PTM drivers and were related to structural and functional impact: rs869312669 (p.Thr685Pro), rs387906636 (p.Arg682Cys), rs672601377 (p.Asn615Ile), and rs1131691702 (p.Ser526Pro). The combined impact of protein function is accounted for by the calculated stability, compactness, and total globularity score. GluN2B hydrogen occupancy was positively associated with protein stability, and solvent-accessible surface area was positively related to globularity. Furthermore, there was a link between GluN2B protein folding, movement, and function, indicating that both putative high and low local movements were linked to protein function. Multiple GRIN2B genetic variations are linked to gene expression, phylogenetic conservation, PTMs, and protein instability behavior in neurodevelopmental diseases. These findings suggest the relevance of GRIN2B genetic variations in neurodevelopmental problems.