Clinical Characteristics and Genetic Analyses of Patients with Idiopathic Hypogonadotropic Hypogonadism

Objective: Idiopathic hypogonadotropic hypogonadism (IHH) is classified into two groups-Kalman syndrome and normosmic IHH (nIHH). Half of all cases can be explained by mutations in >50 genes. Targeted gene panel testing with nexrt generation sequencing (NGS) is required for patients without typical phenotypic findings. The aim was to determine the genetic etiologies of patients with IHH using NGS, including 54 IHH-associated genes, and to present protein homology modeling and protein stability analyzes of the detected variations. Methods: Clinical and demographic data of 16 patients (eight female), aged between 11.6-17.8 years, from different families were assessed. All patients were followed up for a diagnosis of nIHH, had normal cranial imaging, were without anterior pituitary hormone deficiency other than gonadotropins, had no sex chromosome anomaly, had no additional disease, and underwent genetic analysis with NGS between the years 2008-2021. Rare variants were classified according to the variant interpretation framework of the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology. Changes in protein structure caused by variations were modeled using RoseTTAFold and changes in protein stability resulting from variation were analyzed. Results: Half of the 16 had no detectable variation. Three (18.75%) had a homozygous (pathogenic) variant in the GNRHR gene, one (6.25%) had a compound heterozygous [likely pathogenic-variants of uncertain significance (VUS)] variant in PROK2 and four (25%) each had a heterozygous (VUS) variant in HESX1, FGF8, FLRT3 and DMXL2. Protein models showed that variants interpreted as VUS according to ACMG could account for the clinical IHH. Conclusion: The frequency of variation detection was similar to the literature. Modelling showed that the variant in five different genes, interpreted as VUS according to ACMG, could explain the clinical IHH.

Biallelic Novel USP53 Splicing Variant Disrupting the Gene Function that Causes Cholestasis Phenotype and Review of the Literature.

Introduction: Hereditary cholestasis is a heterogeneous group of liver diseases that mostly show autosomal recessive inheritance. The phenotype of cholestasis is highly variable. Molecular genetic testing offers an useful approach to differentiate different types of cholestasis because some symptoms and findings overlap. Biallelic variants in USP53 have recently been reported in cholestasis phenotype. Methods: In this study, we aimed to characterize clinical findings and biological insights on a novel USP53 splice variant causing cholestasis phenotype and provided a review of the literature. We performed whole-exome sequencing and then confirmed it with Sanger sequencing. In addition, as a result of in silico analyses and cDNA analysis, we showed that the USP53 protein in our patient was shortened. Results: We report a novel splice variant (NM_019050.2:c.238-1G>C) in the USP53 gene via whole-exome sequencing in a patient with cholestasis phenotype. This variant was confirmed by Sanger sequencing and was a result of family segregation analysis; it was found to be in a heterozygous state in the parents and the other healthy elder brother of our patient. According to in silico analyses, the change in the splice region resulted in an increase in the length of exon 2, whereas the stop codon after the additional 3 amino acids (VTF) caused the protein to terminate prematurely. Thus, the mature USP53 protein, consisting of 1,073 amino acids, has been reduced to a small protein of 82 amino acids. Conclusion: We propose a model for the tertiary structure of USP53 for the first time, and together with all these data, we support the association of biallelic variants of the USP53 gene with cholestasis phenotype. We also present a comparison of previously reported patients with USP53-associated cholestasis phenotype to contribute to the literature.

Investigation of changes in protein stability and substrate affinity of 3CL-protease of SARS-CoV-2 caused by mutations.

3CLpro of SARS-CoV-2 is one of the enzymes required for the replication process of the virus responsible for the COVID-19 pandemic. In this study, changes in protein stability and substrate affinity caused by mutations were investigated to stir the development of potent inhibitors. Sequence data of samples were obtained from the NCBI Virus database. Mutation analyses were performed with RDP4 and MegaX. 3CLpro tertiary models were created using Robetta. Molecular docking for peptidomimetic substrate and inhibitor ligand was done with Autodock v4.2 and Haddock v2.4. Protein stability analysis was performed using mCSM stability and DynaMut2. Twenty-four missense mutations in 3CLpro were identified in this study. Changes in the 3CLpro structure induced by the mutations Met49Thr, Leu167Ser, and Val202Ala resulted in significant levels of instability (-2.029,-2.612,-2.177 kcal.mol-1, respectively). The lowest interaction energy for substrate was -58.7 kcal.mol-1 and -62.6 kcal.mol-1 in wild-type and mutant, respectively. The lowest docking energy for ligand was -6.19 and -9.52 kcal.mol-1 for wild-type and mutant, respectively. This study reports for the first time that mutations cause increased substrate affinity of 3CLpro from SARS-CoV-2. This research provides important data for the development of potent peptidomimetic inhibitors for the treatment of COVID-19.

Mutations in Main Protease of SARS CoV-2 Decreased Boceprevir Affinity

Abstract The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global health emergency. The main protease (Mpro) is crucial for the life cycle of coronaviruses. Boceprevir is a potential inhibitor and drug candidate for the Mpro of SARS-CoV-2. In this study, changes in the protein structure of the Mpro due to mutations in SARS-CoV-2 and the effects of these changes on boceprevir affinity, an important potential therapeutic agent, were investigated. The mutations were analyzed with RDP4 and MegaX. A three-dimensional model of mutant Mpro was generated by ProMod3. Qualitative Model Energy Analysis, ProSA, and MolProbity tools were used for structural validation and modeling of the wild-type and mutant Mpro proteins. Topological differences of the wild-type and mutant Mpro were calculated with the i-Tasser TM-Score. Molecular docking was performed using AutoDock 4.2. Functional dynamic structure models were created with DynOmics. Seven mutations (L89F, K90R, P108S, A191V, T224A, A234V and S254F) were detected in the Mpro of SARS-CoV-2. The mutations caused a decrease in the affinity of boceprevir, a potential protease inhibitor. The boceprevir was docked to the active site of Mpro, and the binding energies were −10.34 and −9.41 kcal.mol-1 for the wild-type and the mutant, respectively. The Debye-Waller factors calculated by elastic network model analysis were 0.58 and 0.64 Å2 for the wild-type Mpro and mutant Mpro, respectively. Mutations in structures that are important drug targets for SARS-CoV-2 may render existing therapeutics ineffective in its treatment.

Comprehensive structural analysis of the open and closed conformations of Theileria annulata enolase by molecular modelling and docking.

Theileria annulata is an apicomplexan parasite which is responsible for tropical theileriosis in cattle. Due to resistance of T. annulata against commonly used antitheilerial drug, new drug candidates should be identified urgently. Enolase might be a druggable protein candidate which has an important role in glycolysis, and could also be related to several cellular functions as a moonlight protein. In this study; we have described three-dimensional models of open and closed conformations of T. annulata enolase by homology modeling method for the first time with the comprehensive domain, active site and docking analyses. Our results show that the enolase has similar folding patterns within enolase superfamily with conserved catalytic loops and active site residues. We have described specific insertions, possible plasminogen binding sites, electrostatic potential surfaces and positively charged pockets as druggable regions in T. annulata enolase.

[Comparative analysis at the nucleotide level of the genes encoding the lactate dehydrogenase enzyme of Plasmodium vivax and Plasmodium falciparum.]. / Plasmodium vivax ve Plasmodium falciparum'un laktat dehidrogenaz enzimini kodlayan genlerinin nükleotid düzeyinde karsilastirmali analizi.

In this study, the nucleotide sequence of the enzyme lactate dehydrogenase from Plasmodium vivax has been compared to the same enzyme from another malaria parasite Plasmodium falciparum. It was found that the identity between two sequences was 74.8%. The percentage of the GC value was found to be higher in the Plasmodium vivax lactate dehydrogenase (46.6%) than in that of Plasmodium falciparum (33%). The nucleotide sequence that corresponds to the 5 amino acid insertion in Plasmodium lactate dehydrogenase is also present in Plasmodium vivax. This site will be targeted in the design of novel antimalarials for Plasmodium vivax as has been for Plasmodium falciparum.

Cloning, sequence and expression of the lactate dehydrogenase gene from the human malaria parasite, Plasmodium vivax.

Increased drug resistance to anti-malarials highlights the need for the development of new therapeutics for the treatment of malaria. To this end, the lactate dehydrogenase (LDH) gene was cloned and sequenced from genomic DNA of Plasmodium vivax ( PvLDH) Belem strain. The 316 amino acid protein-coding region of the PvLDH gene was inserted into the prokaryotic expression vector pKK223-3 and a 34 kDa protein with LDH activity was expressed in E. coli. Structural differences between human LDHs and PfLDH make the latter an attractive target for inhibitors leading to novel anti-malarial drugs. The sequence similarity between PvLDH and PfLDH (90% residue identity and no insertions or deletions) indicate that the same approach could be applied to Plasmodium vivax, the most common human malaria parasite in the world.