Identification of a mutation in TNRC18 in a patient with clinical features of Fazio-Londe disease.

Fazio-Londe disease and Brown-Vialetto-Van Laere syndrome are rare related neurological disorders. Although SLC52A3 and SLC52A2 that encode riboflavin transporters are their only known causative genes, many patients without mutations in these genes have been reported. Clinical and genetic data of a patient with features suggestive of Fazio-Londe disease are presented. Neurological examination revealed significant involvement of cranial nerves and weakness in the lower extremities. Pontobulbar presentations were prominent. EDX study suggested motor neuronopathy. Hearing was normal. She was diagnosed with FL disease. Response to riboflavin supplementation was not favorable. The patient's pedigree suggested recessive inheritance. SLC52A3 and SLC52A2 were screened and mutations were not observed. Results of exome sequencing and segregation analysis suggested that a mutation in TNRC18 is a candidate cause of disease in the patient. The three dimensional structure of the TNRC18 protein was predicted and it was noted that its two conserved domains (BAH and Tudor) interact and that the valine residue affected by the mutation is positioned close to both domains. A mutation in TNRC18 is cautiously reported as the possible cause of FL disease in the patient. The finding warrants further inquiries on TNRC18 about which little is presently known.

The construction of a testis transcriptional cell atlas from embryo to adult reveals various somatic cells and their molecular roles.

BACKGROUND: The testis is a complex organ that undergoes extensive developmental changes from the embryonic stage to adulthood. The development of germ cells, which give rise to spermatozoa, is tightly regulated by the surrounding somatic cells. METHODS: To better understand the dynamics of these changes, we constructed a transcriptional cell atlas of the testis, integrating single-cell RNA sequencing data from over 26,000 cells across five developmental stages: fetal germ cells, infants, childhood, peri-puberty, and adults. We employed various analytical techniques, including clustering, cell type assignments, identification of differentially expressed genes, pseudotime analysis, weighted gene co-expression network analysis, and evaluation of paracrine cell-cell communication, to comprehensively analyze this transcriptional cell atlas of the testis. RESULTS: Our analysis revealed remarkable heterogeneity in both somatic and germ cell populations, with the highest diversity observed in Sertoli and Myoid somatic cells, as well as in spermatogonia, spermatocyte, and spermatid germ cells. We also identified key somatic cell genes, including RPL39, RPL10, RPL13A, FTH1, RPS2, and RPL18A, which were highly influential in the weighted gene co-expression network of the testis transcriptional cell atlas and have been previously implicated in male infertility. Additionally, our analysis of paracrine cell-cell communication supported specific ligand-receptor interactions involved in neuroactive, cAMP, and estrogen signaling pathways, which support the crucial role of somatic cells in regulating germ cell development. CONCLUSIONS: Overall, our transcriptional atlas provides a comprehensive view of the cell-to-cell heterogeneity in the testis and identifies key somatic cell genes and pathways that play a central role in male fertility across developmental stages.

A novel missense variant in CDK5RAP2 associated with non-obstructive azoospermia.

OBJECTIVE: The most severe type of male infertility is non-obstructive azoospermia (NOA), where there is no sperm in the ejaculate due to failure of spermatogenesis, affecting 10%-20% of infertile men with azoospermia. Genetic studies have identified dozens of NOA genes. The main aim of the present study is to identify a novel monogenic mutation that may cause NOA. MATERIALS AND METHODS: We studied the pedigree of a consanguineous family with three NOA and one fertile brother by a family-based exome-sequencing, segregation analysis, insilico protein modeling and single-cell RNA sequencing data analysis. RESULTS: Bioinformatics analysis followed by sanger sequencing revealed that three NOA brothers were homozygous for a rare missense variant in Cyclin Dependent Kinase Regulatory Subunit Associated Protein 2 (Centrosomin) CDK5RAP2 (NM_018249:exon26:c.A4003T:p.R1335W, rs761196443). Protein modeling demonstrated that CDK5RAP2, Arg1335Trp resided nearby the Microtubule Associated Protein RP/EB Family Member 1 (EB1/MAPRE1) interaction site. As a consequence of the R1335W mutation, the positively charged Arginine was replaced by to the hydrophobic tryptophan residue, possibly leading to local instability in the structure and perturbation in the CDK5RAP2-MAPRE1 interaction. CONCLUSION: Our study reports a novel missense variant of CDK5RAP2 that segregates in homozygosity with male infertility and NOA in a consanguineous family. In silico structural predictions and gene expression data indicate a potential role of the CDK5RAP2 variant in causing defective centrosomic maturation during spermatogenesis.

DrugRep-HeSiaGraph: when heterogenous siamese neural network meets knowledge graphs for drug repurposing.

BACKGROUND: Drug repurposing is an approach that holds promise for identifying new therapeutic uses for existing drugs. Recently, knowledge graphs have emerged as significant tools for addressing the challenges of drug repurposing. However, there are still major issues with constructing and embedding knowledge graphs. RESULTS: This study proposes a two-step method called DrugRep-HeSiaGraph to address these challenges. The method integrates the drug-disease knowledge graph with the application of a heterogeneous siamese neural network. In the first step, a drug-disease knowledge graph named DDKG-V1 is constructed by defining new relationship types, and then numerical vector representations for the nodes are created using the distributional learning method. In the second step, a heterogeneous siamese neural network called HeSiaNet is applied to enrich the embedding of drugs and diseases by bringing them closer in a new unified latent space. Then, it predicts potential drug candidates for diseases. DrugRep-HeSiaGraph achieves impressive performance metrics, including an AUC-ROC of 91.16%, an AUC-PR of 90.32%, an accuracy of 84.63%, a BS of 0.119, and an MCC of 69.31%. CONCLUSION: We demonstrate the effectiveness of the proposed method in identifying potential drugs for COVID-19 as a case study. In addition, this study shows the role of dipeptidyl peptidase 4 (DPP-4) as a potential receptor for SARS-CoV-2 and the effectiveness of DPP-4 inhibitors in facing COVID-19. This highlights the practical application of the model in addressing real-world challenges in the field of drug repurposing. The code and data for DrugRep-HeSiaGraph are publicly available at https://github.com/CBRC-lab/DrugRep-HeSiaGraph .

Intermediate-aided allostery mechanism for α-glucosidase by Xanthene-11v as an inhibitor using residue interaction network analysis.

Exploring allosteric inhibition and the discovery of new inhibitor binding sites are important studies in protein regulation mechanisms and drug discovery. Structural and network-based analyses of trajectories resulting from molecular dynamics (MD) simulations have been developed to discover protein dynamics, landscape, functions, and allosteric regions. Here, an experimentally suggested non-competitive inhibitor, xanthene-11v, was considered to explore its allosteric inhibition mechanism in α-glucosidase MAL12. Comparative structural and network analyses were applied to eight 250 ns independent MD simulations, four of which were performed in the free state and four of which were performed in ligand-bound forms. Projected two-dimensional free energy landscapes (FEL) were constructed from the probabilistic distribution of conformations along the first two principal components. The post-simulation analyses of the coordinates, side-chain torsion angles, non-covalent interaction networks, network communities, and their centralities were performed on α-glucosidase conformations and the intermediate sub-states. Important communities of residues have been found that connect the allosteric site to the active site. Some of these residues like Thr307, Arg312, TYR344, ILE345, Phe357, Asp406, Val407, Asp408, and Leu436 are the key messengers in the transition pathway between allosteric and active sites. Evaluating the probability distribution of distances between gate residues including Val407 in one community and Phe158, and Pro65 in another community depicted the closure of this gate due to the inhibitor binding. Six macro states of protein were deduced from the topology of FEL and analysis of conformational preference of free and ligand-bound systems to these macro states shows a combination of lock-and-key, conformational selection, and induced fit mechanisms are effective in ligand binding. All these results reveal structural states, allosteric mechanisms, and key players in the inhibition pathway of α-glucosidase by xanthene-11v.

DrugRep-KG: Toward Learning a Unified Latent Space for Drug Repurposing Using Knowledge Graphs.

Drug repurposing or repositioning (DR) refers to finding new therapeutic applications for existing drugs. Current computational DR methods face data representation and negative data sampling challenges. Although retrospective studies attempt to operate various representations, it is a crucial step for an accurate prediction to aggregate these features and bring the associations between drugs and diseases into a unified latent space. In addition, the number of unknown associations between drugs and diseases, which is considered negative data, is much higher than the number of known associations, or positive data, leading to an imbalanced dataset. In this regard, we propose the DrugRep-KG method, which applies a knowledge graph embedding approach for representing drugs and diseases, to address these challenges. Despite the typical DR methods that consider all unknown drug-disease associations as negative data, we select a subset of unknown associations, provided the disease occurs because of an adverse reaction to a drug. DrugRep-KG has been evaluated based on different settings and achieves an AUC-ROC (area under the receiver operating characteristic curve) of 90.83% and an AUC-PR (area under the precision-recall curve) of 90.10%, which are higher than in previous works. Besides, we checked the performance of our framework in finding potential drugs for coronavirus infection and skin-related diseases: contact dermatitis and atopic eczema. DrugRep-KG predicted beclomethasone for contact dermatitis, and fluorometholone, clocortolone, fluocinonide, and beclomethasone for atopic eczema, all of which have previously been proven to be effective in other studies. Fluorometholone for contact dermatitis is a novel suggestion by DrugRep-KG that should be validated experimentally. DrugRep-KG also predicted the associations between COVID-19 and potential treatments suggested by DrugBank, in addition to new drug candidates provided with experimental evidence. The data and code underlying this article are available at https://github.com/CBRC-lab/DrugRep-KG.

Biomarker prediction in autism spectrum disorder using a network-based approach.

BACKGROUND: Autism is a neurodevelopmental disorder that is usually diagnosed in early childhood. Timely diagnosis and early initiation of treatments such as behavioral therapy are important in autistic people. Discovering critical genes and regulators in this disorder can lead to early diagnosis. Since the contribution of miRNAs along their targets can lead us to a better understanding of autism, we propose a framework containing two steps for gene and miRNA discovery. METHODS: The first step, called the FA_gene algorithm, finds a small set of genes involved in autism. This algorithm uses the WGCNA package to construct a co-expression network for control samples and seek modules of genes that are not reproducible in the corresponding co-expression network for autistic samples. Then, the protein-protein interaction network is constructed for genes in the non-reproducible modules and a small set of genes that may have potential roles in autism is selected based on this network. The second step, named the DMN_miRNA algorithm, detects the minimum number of miRNAs related to autism. To do this, DMN_miRNA defines an extended Set Cover algorithm over the mRNA-miRNA network, consisting of the selected genes and corresponding miRNA regulators. RESULTS: In the first step of the framework, the FA_gene algorithm finds a set of important genes; TP53, TNF, MAPK3, ACTB, TLR7, LCK, RAC2, EEF2, CAT, ZAP70, CD19, RPLP0, CDKN1A, CCL2, CDK4, CCL5, CTSD, CD4, RACK1, CD74; using co-expression and protein-protein interaction networks. In the second step, the DMN_miRNA algorithm extracts critical miRNAs, hsa-mir-155-5p, hsa-mir-17-5p, hsa-mir-181a-5p, hsa-mir-18a-5p, and hsa-mir-92a-1-5p, as signature regulators for autism using important genes and mRNA-miRNA network. The importance of these key genes and miRNAs is confirmed by previous studies and enrichment analysis. CONCLUSION: This study suggests FA_gene and DMN_miRNA algorithms for biomarker discovery, which lead us to a list of important players in ASD with potential roles in the nervous system or neurological disorders that can be experimentally investigated as candidates for ASD diagnostic tests.

Fluorescence labeling of anchor-modified Mart-1 peptide for increasing its affinity for HLA-A*0201: Hit two targets with one arrow.

One of the most successful strategies in designing peptide-based cancer vaccines is modifying natural epitope peptides to increase their binding strength to human leukocyte antigens (HLAs). Anchor-modified Mart-1 peptide (ELAGIGILTV) is among the artificial epitope peptides with the highest binding affinity for HLA-A*0201. In this study, by fluorescence labeling of its either C- or N-terminus with Nε -(5-carboxyfluorescein)-l-lysine, we not only made it traceable but also drastically increased its binding strength to HLA-A*0201. HLA streptamer, for the first time, is introduced for measuring the binding constants (Ka ) of the labeled peptides. The affinity of the labeled peptides for the HLA-A*201 of the MCF-7 cells was extraordinarily high and co-incubating them with the highest possible amount of the unlabeled peptide, as a competitor, did not significantly prohibit them from binding to the HLA. The reproducibility of the obtained results was confirmed by using the T2 cell line. The HLA-deficient K562 cell line was used as the negative control. With in silico simulations, we found two hydrophobic pockets on both sides of HLA-A*0201 for anchoring the C- or N-terminal 5-carboxyfluorescein probe, which can explain the extraordinary affinity of the labeled peptides for the HLA-A*0201.

Integrated Bioinformatic Analysis of Differentially Expressed Genes Associated with Wound Healing.

OBJECTIVE: Wound healing is a complex process involving the coordinated interaction of various genes and molecular
pathways. The study aimed to uncover novel therapeutic targets, biomarkers and candidate genes for drug development
to improve successful wound repair interventions.
Materials and Methods: This study is a network-meta analysis study. Nine wound healing microarray datasets obtained
from the Gene Expression Omnibus (GEO) database were used for this study. Differentially expressed genes (DEGs)
were described using the Limma package and shared genes were used as input for weighted gene co-expression
network analysis. The Gene Ontology analysis was performed using the EnrichR web server, and construction of a
protein-protein interaction (PPI) network was achieved by the STRING and Cytoscape.
Results: A total of 424 DEGs were determined. A co-expression network was constructed using 7692 shared genes
between nine data sets, resulting in the identification of seven modules. Among these modules, those with the top 20
genes of up and down-regulation were selected. The top down-regulated genes, including TJP1, SEC61A1, PLEK,
ATP5B, PDIA6, PIK3R1, SRGN, SDC2, and RBBP7, and the top up-regulated genes including RPS27A, EEF1A1,
HNRNPA1, CTNNB1, POLR2A, CFL1, CSNk1E, HSPD1, FN1, and AURKB, which can potentially serve as therapeutic
targets were identified. The KEGG pathway analysis found that the majority of the genes are enriched in the "Wnt
signaling pathway".
Conclusion: In our study of nine wound healing microarray datasets, we identified DEGs and co-expressed modules
using WGCNA. These genes are involved in important cellular processes such as transcription, translation, and posttranslational
modifications. We found nine down-regulated genes and ten up-regulated genes, which could serve as
potential therapeutic targets for further experimental validation. Targeting pathways related to protein synthesis and cell
adhesion and migration may enhance wound healing, but additional experimental validation is needed to confirm the
effectiveness and safety of targeted interventions.

Identification of UBA1 as the causative gene of an X-linked non-Kennedy spinal-bulbar muscular atrophy.

BACKGROUND AND PURPOSE: Spinal-bulbar muscular atrophy (SBMA) (Kennedy's disease) is a motor neuron disease. Kennedy's disease is nearly exclusively caused by mutations in the androgen receptor encoding gene (AR). The results of studies aimed at identification of the genetic cause of a disease that best approximates SBMA in a pedigree (four patients) without mutations in AR are reported. METHODS: Clinical investigations included thorough neurological and non-neurological examinations and testing. Genetic analysis was performed by exome sequencing using standard protocols. UBA1 mutations were modeled on the crystal structure of UBA1. RESULTS: The clinical features of the patients are described in detail. A missense mutation in UBA1 (c.T1499C; p.Ile500Thr) was identified as the probable cause of the non-Kennedy SBMA in the pedigree. Like AR, UBA1 is positioned on chromosome X. UBA1 is a highly conserved gene. It encodes ubiquitin-like modifier activating enzyme 1 (UBA1) which is the major E1 enzyme of the ubiquitin-proteasome system. Interestingly, UBA1 mutations can also cause infantile-onset X-linked spinal muscular atrophy (XL-SMA). The mutation identified here and the XL-SMA causative mutations were shown to affect amino acids positioned in the vicinity of UBA1's ATP binding site and to cause structural changes. CONCLUSION: UBA1 was identified as a novel SBMA causative gene. The gene affects protein homeostasis which is one of most important components of the pathology of neurodegeneration. The contribution of this same gene to the etiology of XL-SMA is discussed.

Thermal stability enhancement: Fundamental concepts of protein engineering strategies to manipulate the flexible structure.

Increasing the temperature by just a few degrees may lead to structural perturbation or unfolding of the protein and consequent loss of function. The concepts of flexibility and rigidity are fundamental for understanding the relationships between function, structure and stability. Protein unfolding can often be triggered by thermal fluctuations with flexible residues usually on the protein surface. Therefore, identification and knowledge of the effect of modification to flexible regions in protein structures are required for efficient protein engineering and the rational design of thermally stable proteins. The most flexible regions in protein are loops, hence their rigidification is one of the effective strategies for increasing thermal stability. Directed evolution or rational design by computational prediction can also lead to the generation of thermally stable proteins. Computational protein design has been improved significantly in recent years and has successfully produced de novo stable backbone structures with optimized sequences and functions. This review discusses intramolecular and intermolecular interactions that determine the protein structure, and the strategies utilized in the mutagenesis of mesophilic proteins to stabilize and improve the functional characteristics of biocatalysts by describing efficient techniques and strategies to rigidify flexible loops at appropriate positions in the structure of the protein.

A rare frameshift mutation in SYCP1 is associated with human male infertility.

Proper assembly of the synaptonemal complex is essential for successful meiosis, and impairments in the process lead to infertility. Meiotic transverse filament proteins encoded by the SYCP1 (synaptonemal complex protein 1) gene are one of the main components of the synaptonemal complex and play an important role in correct synapsis and recombination. Family-based whole-exome sequencing revealed a rare homozygous SYCP1 frameshift mutation (c.2892delA: p.K967Nfs*1) in two men with severe oligozoospermia, followed by validation and segregation through Sanger sequencing. This single nucleotide deletion not only changes lysine 967 (K) into asparagine (N) but also causes a premature stop codon, which leads to deletion of 968-976 residues from the end of the C-tail region of the SYCP1 protein. Although, sycp1 knockout male mice are reported to be sterile with a complete lack of spermatids and spermatozoa, to date no SYCP1 variant has been associated with human oligozoospermia. HADDOCK analysis indicated that this mutation decreases the ability of the truncated SYCP1 protein to bind DNA. Immunodetection of ϒH2AX signals in SYCP1 mutant semen cells, and a 40% DNA fragmentation index might indicate that a small number of DNA double-strand breaks, which require SYCP1 and/or synapsis to be repaired, are not efficiently repaired, resulting in defects in differentiation of germline cells and appearance of the oligozoospermia phenotype. To our knowledge, this is the first report of a homozygous SYCP1 mutation that decreases sperm count. Further studies are required to determine the function of the SYCP1 mutation, which is potentially associated with human oligozoospermia.

A Novel Missense Variant in Actin Binding Domain of MYH7 Is Associated With Left Ventricular Noncompaction.

Cardiomyopathies are a group of common heart disorders that affect numerous people worldwide. Left ventricular non-compaction (LVNC) is a structural disorder of the ventricular wall, categorized as a type of cardiomyopathy that mostly caused by genetic disorders. Genetic variations are underlying causes of developmental deformation of the heart wall and the resultant contractile insufficiency. Here, we investigated a family with several affected members exhibiting LVNC phenotype. By whole-exome sequencing (WES) of three affected members, we identified a novel heterozygous missense variant (c.1963C>A:p.Leu655Met) in the gene encoding myosin heavy chain 7 (MYH7). This gene is evolutionary conserved among different organisms. We identified MYH7 as a highly enriched myosin, compared to other types of myosin heavy chains, in skeletal and cardiac muscles. Furthermore, MYH7 was among a few classes of MYH in mouse heart that highly expresses from early embryonic to adult stages. In silico predictions showed an altered actin-myosin binding, resulting in weaker binding energy that can cause LVNC. Moreover, CRISPR/Cas9 mediated MYH7 knockout in zebrafish caused impaired cardiovascular development. Altogether, these findings provide the first evidence for involvement of p.Leu655Met missense variant in the incidence of LVNC, most probably through actin-myosin binding defects during ventricular wall morphogenesis.

Novel STAG3 variant associated with primary ovarian insufficiency and non-obstructive azoospermia in an Iranian consanguineous family.

Non-obstructive azoospermia (NOA) and primary ovarian insufficiency (POI) present the most severe forms of male and female infertility. In the last decade, the increasing use of whole exome sequencing (WES) in genomics studies of these conditions has led to the introduction of a number of novel genes and variants especially in meiotic genes with restricted expression to gonads. In this study, exome sequencing of a consanguineous Iranian family with one POI and two NOA cases in three siblings showed that all three patients were double homozygous for a novel in-frame deletion and a novel missense variant in STAG3 (NM_001282717.1:c.1942G > A: p.Ala648Thr; NM_001282717.1:c.1951_1953del: p. Leu652del). Both variants occur within a short proximity of each other affecting the relatively conserved armadillo-type fold superfamily feature. STAG3 is a specific meiotic cohesin complex component that interacts with the α-kleisin subunit through this feature. Protein homology modeling indicated that the in-frame deletion destabilizes kleisin biding by STAG3. Although the missense variant did not seem to affect the binding significantly, protein homology modeling suggests that it further destabilizes kleisin binding when in double homozygous state with the deletion. Our findings are in line with several other studies having associated deleterious variants affecting this region with male and female infertility in humans and mouse models. This is the first report associating an in-frame STAG3 variant with NOA and POI in a single family. SUMMARY SENTENCE: A patient with primary ovarian failure and her two brothers with non-obstructive azoospermia were double homozygous for a novel in-frame deletion and a novel missense variant in STAG3 that potentially disrupt the protein's meiotic functions.

Integration and gene co-expression network analysis of scRNA-seq transcriptomes reveal heterogeneity and key functional genes in human spermatogenesis.

Spermatogenesis is a complex process of cellular division and differentiation that begins with spermatogonia stem cells and leads to functional spermatozoa production. However, many of the molecular mechanisms underlying this process remain unclear. Single-cell RNA sequencing (scRNA-seq) is used to sequence the entire transcriptome at the single-cell level to assess cell-to-cell variability. In this study, more than 33,000 testicular cells from different scRNA-seq datasets with normal spermatogenesis were integrated to identify single-cell heterogeneity on a more comprehensive scale. Clustering, cell type assignments, differential expressed genes and pseudotime analysis characterized 5 spermatogonia, 4 spermatocyte, and 4 spermatid cell types during the spermatogenesis process. The UTF1 and ID4 genes were introduced as the most specific markers that can differentiate two undifferentiated spermatogonia stem cell sub-cellules. The C7orf61 and TNP can differentiate two round spermatid sub-cellules. The topological analysis of the weighted gene co-expression network along with the integrated scRNA-seq data revealed some bridge genes between spermatogenesis's main stages such as DNAJC5B, C1orf194, HSP90AB1, BST2, EEF1A1, CRISP2, PTMS, NFKBIA, CDKN3, and HLA-DRA. The importance of these key genes is confirmed by their role in male infertility in previous studies. It can be stated that, this integrated scRNA-seq of spermatogenic cells offers novel insights into cell-to-cell heterogeneity and suggests a list of key players with a pivotal role in male infertility from the fertile spermatogenesis datasets. These key functional genes can be introduced as candidates for filtering and prioritizing genotype-to-phenotype association in male infertility.

The penicillin binding protein 1A of Helicobacter pylori, its amoxicillin binding site and access routes.

BACKGROUND: Amoxicillin-resistant H. pylori strains are increasing worldwide. To explore the potential resistance mechanisms involved, the 3D structure modeling and access tunnel prediction for penicillin-binding proteins (PBP1A) was performed, based on the Streptococcus pneumoniae, PBP 3D structure. Molecular covalent docking was used to determine the interactions between amoxicillin (AMX) and PBP1A. RESULTS: The AMX-Ser368 covalent complex interacts with the binding site residues (Gly367, Ala369, ILE370, Lys371, Tyr416, Ser433, Thr541, Thr556, Gly557, Thr558, and Asn560) of PBP1A, non-covalently. Six tunnel-like structures, accessing the PBP1A binding site, were characterized, using the CAVER algorithm. Tunnel-1 was the ultimate access route, leading to the drug catalytic binding residue (Ser368). This tunnel comprises of eighteen amino acid residues, 8 of which are shared with the drug binding site. Subsequently, to screen the presence of PBP1A mutations, in the binding site and tunnel residues, in our clinical strains, in vitro assays were performed. H. pylori strains, isolated under gastroscopy, underwent AMX susceptibility testing by E-test. Of the 100 clinical strains tested, 4 were AMX-resistant. The transpeptidase domain of the pbp1a gene of these resistant, plus 10 randomly selected AMX-susceptible strains, were amplified and sequenced. Of the amino acids lining the tunnel-1 and binding site residues, three (Ser414Arg, Val469Met and Thr556Ser) substitutions, were detected in 2 of the 4 resistant and none of the sequenced susceptible strains, respectively. CONCLUSIONS: We hypothesize that mutations in amino acid residues lining the binding site and/or tunnel-1, resulting in conformational/spatial changes, may block drug binding to PBP1A and cause AMX resistance.

Rare missense variant in MSH4 associated with primary gonadal failure in both 46, XX and 46, XY individuals.

STUDY QUESTION: Can whole-exome sequencing (WES) reveal a shared pathogenic variant responsible for primary gonadal failure in both male and female patients from a consanguineous family? SUMMARY ANSWER: Patients with primary ovarian insufficiency (POI) and non-obstructive azoospermia (NOA) were homozygous for the rare missense variant p. S754L located in the highly conserved MSH4 MutS signature motif of the ATPase domain. An oligozoospermic patient was heterozygous for the variant. WHAT IS KNOWN ALREADY: MSH4 is a meiosis-specific protein expressed at a certain level in the testes and ovaries. Along with its heterodimer partner MSH5, it is responsible for double-strand Holliday junction recognition and stabilization, to ensure accurate chromosome segregation during meiosis. Knockout male and female mice for Msh4 and Msh5 are reportedly infertile due to meiotic arrest. In humans, MSH4 is associated with male and female gonadal failure, with distinct variations in the MutS domain V. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of a consanguineous family with multiple cases of gonadal failure in both genders. The subject family was recruited in Iran, in 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: The proband who is affected by POI, an NOA brother, a fertile sister and their parents were subjected to WES. The discovered variant was validated in these individuals, and the rest of the family was also genotyped by Sanger sequencing. The variant was not detected in 800 healthy Iranian individuals from the Iranome database nor in 30 sporadic NOA and 30 sporadic POI patients. Suggested effect in aberrant splicing was studied by RT-PCR. Moreover, protein homology modeling was used to further investigate the amino acid substitution in silico. MAIN RESULTS AND THE ROLE OF CHANCE: The discovered variant is very rare and has never been reported in the homozygous state. It occurs in the ATPase domain at Serine 754, the first residue within the highly conserved MutS signature motif, substituting it with a Leucine. All variant effect prediction tools indicated this variant as deleterious. Since the substitution occurs immediately before the Walker B motif at position 755, further investigations based on protein homology were conducted. Considering the modeling results, the nature of the substituted amino acid residue and the distances between p. S754L variation and the residues of the Walker B motif suggested the possibility of conformational changes affecting the ATPase activity of the protein. LARGE SCALE DATA: We have submitted dbSNP entry rs377712900 to ClinVar under SCV001169709, SCV001169708 and SCV001142647 for oligozoospermia, NOA and POI, respectively. LIMITATIONS, REASONS FOR CAUTION: Studies in model organisms can shed more light on the role of this variant as our results were obtained by variant effect prediction tools and protein homology modeling. WIDER IMPLICATIONS OF THE FINDINGS: Identification of variants in meiotic genes should improve genetic counseling for both male and female infertility. Also, as two of our NOA patients underwent testicular sperm extraction (TESE) with no success, ruling out the existence of pathogenic variants in meiotic genes in such patients prior to TESE could prove useful. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by Royan Institute in Tehran, Iran, and Institut Pasteur in Paris, France. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Biallelic variant in cyclin B3 is associated with failure of maternal meiosis II and recurrent digynic triploidy.

BACKGROUND: Triploidy is one of the most common chromosome abnormalities affecting human gestation and accounts for an important fraction of first-trimester miscarriages. Triploidy has been demonstrated in a few cases of recurrent pregnancy loss (RPL) but its molecular mechanisms are unknown. This study aims to identify the genetic cause of RPL associated with fetus triploidy. METHODS: We investigated genomic imprinting, genotyped sequence-tagged site (STS) markers and performed exome sequencing in a family including two sisters with RPL. Moreover, we evaluated oocyte maturation in vivo and in vitro and effect of the candidate protein variant in silico. RESULTS: While features of hydatidiform mole were excluded, the presence of triploidy of maternal origin was demonstrated in the fetuses. Oocyte maturation was deficient and all the maternally inherited pericentromeric STS alleles were homozygous in the fetuses. A deleterious missense variant (p.V1251D) of the cyclin B3 gene (CCNB3) affecting a residue conserved in placental mammals and located in a region that can interact with the cyclin-dependent kinase 1 or cyclin-dependent kinase 2 cosegregated in homozygosity with RPL. CONCLUSION: Here, we report a family in which a damaging variant in cyclin B3 is associated with the failure of oocyte meiosis II and recurrent fetus triploidy, implicating a rationale for CCNB3 testing in RPL.

The outward shift of clarithromycin binding to the ribosome in mutant Helicobacter pylori strains.

OBJECTIVES: Disruption of protein synthesis, by drug-mediated restriction of the ribosomal nascent peptide exit tunnel (NPET), may inhibit bacterial growth. Here, we have studied the secondary and tertiary structures of domain V of the 23S rRNA in the wild-type and mutant (resistant) H. pylori strains and their mechanisms of interaction with clarithromycin (CLA). METHODS: H pylori strains, isolated from cultured gastric biopsies, underwent CLA susceptibility testing by E test, followed by PCR amplification and sequencing of domain V of 23S rRNA. The homology model of this domain in H pylori, in complex with L4 and L22 accessory proteins, was determined based on the E. coli ribosome 3D structure. The interactions between CLA and 23S rRNA complex were determined by molecular docking studies. RESULTS: Of the 70 H pylori strains, isolated from 200 dyspeptic patients, 11 (16%) were CLA-resistant. DNA sequencing identified categories with no (A), A2142G (B), and A2143G (C) mutations. Docking studies of our homology model of 23S rRNA complex with CLA showed deviated positions for categories B and C, in reference to category A, with 12.19 Å and 7.92 Å RMSD values, respectively. In both mutant categories, CLA lost its interactions at positions 2142 and 2587 and gained two new bonds with the L4 accessory protein. CONCLUSION: Our data suggest that, in mutant H pylori strains, once the nucleotides at positions 2142 and 2587 are detached from the drug, CLA interacts with and is peeled back by the L4 accessory protein, removing the drug-imposed spatial restriction of the NPET.

Profiling of Initial Available SARS-CoV-2 Sequences from Iranian Related COVID-19 Patients.

The etiologic agent SARS-CoV-2 has caused the outbreak of COVID-19 which is spread widely around the world. It is vital to uncover and investigate the full genome sequence of SARS-CoV-2 throughout the world to track changes in this virus. To this purpose, SARS-CoV-2 full genome sequence profiling of 20 patients in Iran and different countries that already had a travel history to Iran or contacts with Iranian cases were provided from the GISAID database. The bioinformatics analysis showed 44 different nucleotide mutations that caused 26 nonsynonymous mutations in protein sequences with regard to the reference full genome of the SARS-CoV-2 sequence (NC_045512.2). R207C, V378I, M2796I, L3606F, and A6407V in ORF1ab were common mutations in these sequences. Also, some of the detected mutations only were found in Iranian data in comparison with all the available sequences of SARS-CoV-2. The position of S protein mutations showed they were far from the binding site of this protein with angiotensin-converting enzyme-2 (ACE2) as the host cell receptor. These results can be helpful to design specific diagnostic tests, trace the SARS-CoV-2 sequence changes in Iran, and explore therapeutic drugs and vaccines.