Evolutionary Conservation Analysis Research Articles

New biallelic mutations in WEE2: expanding the spectrum of mutations that cause fertilization failure or poor fertilization

To investigate the genetic cause of fertilization failure or poor fertilization. Genetic analysis. University-affiliated center. Twenty-four Chinese women who underwent assisted reproductive technology (ART) and had repeated fertilization failure or poor fertilization. None. Twenty-four affected patients were subjected to whole-exome sequencing and candidate mutations were validated by Sanger sequencing. Single-cell reverse transcription was used to analyze the functional characterization of the splice-site mutation invivo. Evolutionary conservation and molecular modeling analyses were used to predict the impact of missense mutations on secondary protein structure. Immunofluorescence was used to analyze the protein levels of WEE2 and phosphorylated CDC2. Biallelic mutations in WEE2 were identified in 5 of 24 (20.8%) Chinese patients with fertilization failure or poor fertilization. Among these individuals we found a novel splice-site mutation, two novel missense mutations, and a previously reported frame-shift mutation. Splicing mutation c.1136-2A>G of WEE2 caused an alteration of the reading frame and introduced a premature stop codon (p.Gly379Glufs*6/p.Asp380Leufs*39). The missense mutations c.585G>C (p.Lys195Asn) and c.1228C>T (p.Arg410Trp) produced obvious changes in secondary protein structures. Immunostaining indicated that mutated WEE2 resulted in the loss of phosphorylated CDC2. The phenotypes of women carrying WEE2 mutations exhibited slight variability, from total fertilization failure to poor fertilization. Novel mutations in the known causative gene WEE2 were identified in 5 of 24 women with fertilization failure or poor fertilization, indicating a high prevalence of WEE2 mutations in Chinese women experiencing fertilization failure or poor fertilization.

Large-scale validation of miRNAs by disease association, evolutionary conservation and pathway activity

ABSTRACTThe validation of microRNAs (miRNAs) identified by next generation sequencing involves amplification-free and hybridization-based detection of transcripts as criteria for confirming valid miRNAs. Since respective validation is frequently not performed, miRNA repositories likely still contain a substantial fraction of false positive candidates while true miRNAs are not stored in the repositories yet. Especially if downstream analyses are performed with these candidates (e.g. target or pathway prediction), the results may be misleading. In the present study, we evaluated 558 mature miRNAs from miRBase and 1,709 miRNA candidates from next generation sequencing experiments by amplification-free hybridization and investigated their distributions in patients with various disease conditions. Notably, the most significant miRNAs in diseases are often not contained in the miRBase. However, these candidates are evolutionary highly conserved. From the expression patterns, target gene and pathway analyses and evolutionary conservation analyses, we were able to shed light on the complexity of miRNAs in humans. Our data also highlight that a more thorough validation of miRNAs identified by next generation sequencing is required. The results are available in miRCarta (https://mircarta.cs.uni-saarland.de).

Mutation analysis of ZP1, ZP2, ZP3 and ZP4 genes in 152 Han Chinese samples with ovarian endometriosis

Endometriosis is characterized by the ectopic implant of endometrial tissue outside the uterine cavity and found in ˜35–50% of subfertile women. Previous studies have found that endometriosis had frequent defects in zona pellucida (ZP), and mutations in ZP genes could lead to ZP defects, raising the possibility that mutations in ZP genes might exist in endometriosis. We analyzed a total of 152 Han Chinese samples with ovarian endometriosis for the presence of mutations in the ZP1, ZP2, ZP3 and ZP4 genes. Two novel nonsynonymous ZP4 mutations were identified in three out of 152 (2.0%) samples: a p.M1?/(c.3 G > C) mutation in a 27- and 35-year-old sample, respectively, and a p.A433 V (c.1298C > T) mutation in a 31-year-old patient. No mutations were detected in ZP1, ZP2 or ZP3 genes; furthermore, no mutations in ZP genes were identified in 85 female control samples without endometriosis. The p.M1?/(c.3 G > C) mutation could lead to the usage of a downstream translation initiation site, while the evolutionary conservation and protein structural modeling analyses suggested that the p.A433 V mutation might be functionally important. However, there were strikingly different fertility outcomes among the three samples with ZP4 mutations: the p.A433V-mutated sample had no problem in fertility; while the p.M1?-mutated samples presented with paradoxical effects on fertility: the 35-year-old patient had a child while the 27-year-old patient was infertile, who underwent two spontaneous abortions and an implantation failure after IVF treatment. These results suggested that the potential role of ZP4 mutations on human fertility might be more complex than we thought, and other genetic and environment factors might play a role. In conclusion, we identified two novel mutations in the ZP4 gene in 2.0% of Han Chinese patients with ovarian endometriosis for the first time, our results suggested that mutations in ZP4, but not ZP1, ZP2 and ZP3, might play active roles in the pathogenesis of ovarian endometriosis, despite the mutation-carriers present with complex fertility outcomes.

Towards a Central Role of ISL1 in the Bladder Exstrophy⁻Epispadias Complex (BEEC): Computational Characterization of Genetic Variants and Structural Modelling.

Genetic factors play a critical role in the development of human diseases. Recently, several molecular genetic studies have provided multiple lines of evidence for a critical role of genetic factors in the expression of human bladder exstrophy-epispadias complex (BEEC). At this point, ISL1 (ISL LIM homeobox 1) has emerged as the major susceptibility gene for classic bladder exstrophy (CBE), in a multifactorial disease model. Here, GWAS (Genome wide association studies) discovery and replication studies, as well as the re-sequencing of ISL1, identified sequence variants (rs9291768, rs6874700, c.137C > G (p.Ala46Gly)) associated with CBE. Here, we aimed to determine the molecular and functional consequences of these sequence variants and estimate the dependence of ISL1 protein on other predicted candidates. We used: (i) computational analysis of conserved sequence motifs to perform an evolutionary conservation analysis, based on a Bayesian algorithm, and (ii) computational 3D structural modeling. Furthermore, we looked into long non-coding RNAs (lncRNAs) residing within the ISL1 region, aiming to predict their targets. Our analysis suggests that the ISL1 protein specific N-terminal LIM domain (which harbors the variant c.137C > G), limits its transcriptional ability, and might interfere with ISL1-estrogen receptor α interactions. In conclusion, our analysis provides further useful insights about the ISL1 gene, which is involved in the formation of the BEEC, and in the development of the urinary bladder.

KEAP1/NRF2 signaling pathway mutations in cervical cancer.

The aim of the present study was to explore the potential involvement of mutations in the KEAP1/NRF2 signaling pathway in Chinese samples with cervical cancer. 236 Chinese patients with various types of cervical cancer were recruited, and the coding exons and the corresponding intron-exon boundaries of the KEAP1 and NRF2 genes were analyzed for the potential mutations in the KEAP1/NRF2 signaling pathway. A novel KEAP1 missense somatic mutation (c.1408C>T, p.R470C) and 5 NRF2 missense somatic mutations (c.72G>C, p.W24C; c.85G>T, p.D29Y; c.101G>A, p.R34Q; c.230A>C, p.D77A and c.242G>A p.G81D) were identified in 187 patients with cervical squamous cell carcinoma, respectively; no mutations were detected in other subtypes. All these mutations were heterozygous and predicted to be pathogenic by PolyPhen-2, MutationTaster programs, and evolutionary conservation analysis. Among these mutations, the KEAP1 (p.R470C) and 3 NRF2 mutations (p.D29Y, p.D77A, and p.G81D) were detected in cervical cancer for the first time. Also, no mutations were identified in our 21 adenosquamous carcinomas or 25 adenocarcinomas. We identified 6 potential diseases causing mutations in the KEAP1/NRF2 signaling pathway in 187 (3.2%) Chinese cases with cervical squamous cell carcinoma, implicating KEAP1/NRF2 signaling pathway might play an active role in the pathogenesis of this subtype of cervical cancer. Furthermore, among these detected mutations, the KEAP1 and 3 NRF2 mutations were reported in cervical cancer for the first time.

The choice of sequence homologs included in multiple sequence alignments has a dramatic impact on evolutionary conservation analysis.

The analysis of sequence conservation patterns has been widely utilized to identify functionally important (catalytic and ligand-binding) protein residues for over a half-century. Despite decades of development, on average state-of-the-art non-template-based functional residue prediction methods must predict ∼25% of a protein's total residues to correctly identify half of the protein's functional site residues. The overwhelming proportion of false positives results in reported 'F-Scores' of ∼0.3. We investigated the limits of current approaches, focusing on the so-far neglected impact of the specific choice of homologs included in multiple sequence alignments (MSAs). The limits of conservation-based functional residue prediction were explored by surveying the binding sites of 1023 proteins. A straightforward conservation analysis of MSAs composed of randomly selected homologs sampled from a PSI-BLAST search achieves average F-Scores of ∼0.3, a performance matching that reported by state-of-the-art methods, which often consider additional features for the prediction in a machine learning setting. Interestingly, we found that a simple combinatorial MSA sampling algorithm will in almost every case produce an MSA with an optimal set of homologs whose conservation analysis reaches average F-Scores of ∼0.6, doubling state-of-the-art performance. We also show that this is nearly at the theoretical limit of possible performance given the agreement between different binding site definitions. Additionally, we showcase the progress in this direction made by Selection of Alignment by Maximal Mutual Information (SAMMI), an information-theory-based approach to identifying biologically informative MSAs. This work highlights the importance and the unused potential of optimally composed MSAs for conservation analysis. Supplementary data are available at Bioinformatics online.

Novel CTCF mutations in Chinese patients with ovarian endometriosis.

Endometriosis is a common gynecological disease characterized by the outgrowth of the endometrium, however, the detailed molecular etiology remains largely uncharacterized. Recent studies have implicated that endometriosis is potentially a precancerous lesion, and that CCCTC‑binding factor (CTCF) mutations may be involved in the pathogenesis of this disorder. However, the detailed CTCF mutation spectrum in Chinese patients with ovarian endometriosis remains largely unknown. In the present study, a cohort of 92patients with ovarian endometriosis were analyzed for the presence of CTCF mutations by sequencing the entire coding regions. In addition, 67 healthy eutopic endometrial tissues and 46healthy ovarian tissues from control samples (without endometriosis) were also analyzed. In total, two CTCF missense mutations, p.K206E (c.616A>G) and p.H373L (c.1118A>T), were identified in 2/92 (2.2%) endometriotic lesions. The patient with the p.K206E mutation was 26years old and diagnosed with primary infertility, whereas the patient with the p.H373L mutation was 37years old and concurrently diagnosed with uterine leiomyoma. The p.H373L mutation was previously identified in endometrial cancer samples with low frequency, while the p.K206E mutation was novel. In addition, no CTCF mutations were detected in the 67healthy eutopic endometrial and 46 healthy ovarian tissue samples. Insilico prediction and evolutionary conservation analysis suggested that these CTCF mutations may be pathogenic. In summary, the present study identified 2potential pathogenic CTCF mutations in endometriotic lesions from 2/92patients with ovarian endometriosis. These results, together with a prior exome‑sequencing based study, suggest that CTCF mutations may be involved in the development of ovarian endometriosis.

Analysis of CARD10 and CARD11 somatic mutations in patients with ovarian endometriosis.

Endometriosis is a complex and heterogeneous pre-malignant inflammatory disease harboring multiple gene mutations. Previous studies have suggested that caspase recruitment domain family member (CARD)10 and CARD11 mutations may exist in endometriosis. In the present study, a collection of endometriotic lesions and paired peripheral blood from 101 patients with ovarian endometriosis were obtained, and the entire coding sequences of the CARD10 and CARD11 genes were sequenced. Evolutionary conservation analysis and online prediction programs were applied to analyze the disease-causing potential of the identified mutations. A total of 4 novel somatic mutations were identified in 4 out of the 101 (4.0%) samples: 2 in-frame deletions in CARD10 (c.785_790delAGGAGA, p.K272_E273delKE; c.785_802delAGGAGAAGGAGAAGGAGA, p.K272_V277delKEPDNV) and 2 heterozygous missense mutations in CARD11 (c.49G>T, p.D17Y; c.160G>C, p.E54Q). The sample with CARD10 p.K272_E273delKE deletion was obtained from a 47-year-old patient who was also diagnosed with uterine leiomyoma, while the CARD10 p.K272_V277delKEPDNV-mutated sample was from a 43-year-old patient exhibiting a decreased blood eosinophil granulocyte ratio (0.3%) and an elevated serum creatine kinase level (314 U/l). The patient with the CARD11 p.D17Y mutation was 38 years old and exhibited an increased level of cancer antigen 125 (45.4 U/ml), while the patient with the CARD11 p.E54Q mutation was 46 years old and exhibited no other gynecological conditions. Evolutionary conservation analysis and online prediction programs suggested that these mutations may be disease-causing. In summary, 4 novel somatic mutations in the CARD10 and CARD11 genes were identified from amongst 101 cases of ovarian endometriosis for the first time, these mutations may serve active roles in the development of ovarian endometriosis.

Bioinformatic and Functional Analysis of a Key Determinant Underlying the Substrate Selectivity of the Al Transporter, Nrat1.

Nrat1 is a member of the natural resistance-associated macrophage protein (Nramp) family of metal ion transporters in all organisms. Different from other Nramp members capable of transporting divalent metals, Nrat1 specifically transports trivalent aluminum (Al) ion. However, molecular mechanism underlying the Al transport selectivity of Nrat1 remains unknown. Here, we performed structure-function analyses of Nrat1 and other Nramp members to gain insights into the determinants of ion selectivity. A phylogenetic analysis showed that plant Nramp transporters could be divided into five groups. OsNrat1 was found in one of the individual clades and clustered with SbNrat1 and ZmNrat1 on the evolutionary tree. Structural modeling revealed that Nrat1 transporters adopted a common LeuT fold shared by many Nramp-family transporters that likely employed an identical transport mechanism. Sequence alignment and evolutionary conservation analysis of amino acids identified a metal-permeation pathway of Nrat1 centered at the metal binding site. The metal binding site of Nrat1 was characterized by two conserved sequence motifs, i.e., the Asp-Pro-Ser-Asn motif (motif A) and the Ala-Ile-Ile-Thr motif (motif B). Replacement of the Ala-Met-Val-Met motif B of the OsNramp3 manganese (Mn) transporter to that of Nrat1 resulted in a partial gain of Al transport activity and a total loss of Mn in yeast. Conversely, substitution of the motif B of OsNrat1 with that of OsNramp3 altered the Al transport activity. These observations indicated the metal binding site, particularly the motif B, as a key determinant of Al selectivity of Nrat1.

Novel TRERF1 mutations in Chinese patients with ovarian endometriosis.

Endometriosis is an estrogen-dependent precancerous lesion exhibiting frequently perturbed level of steroid hormones and transcriptional‑regulating factor1 (TRERF1) has a crucial role in the production of steroid hormones including estrogen. Endometriosis has previously been revealed to be a precancerous lesion that harbors somatic mutations in cancer‑associated genes. Therefore, the authors of the present study hypothesize that TRERF1 aberrations may be involved in the development of endometriosis. In the present study, endometriotic lesions and paired blood samples from 92individuals with ovarian endometriosis were analyzed for the potential presence of TRERF1 mutations by sequencing the entire coding region and the corresponding intron‑exon boundaries of the TRERF1 gene. Two heterozygous missense somatic mutations [c.3166A>C (p.K1056Q) and c.3187 G>A (p.G1063R)] in the TRERF1 gene were identified in two out of 92 ectopic endometria (2.2%), to the best of our knowledge, these mutations have not been previously reported. From the two samples with TRERF1 mutations, one sample was from a 42‑year‑old patient also diagnosed with uterine leiomyoma and the other mutation was identified in a 36‑year‑old woman exhibiting no other apparent gynecological conditions. The evolutionary conservation analysis and insilico prediction of these TRERF1 mutations suggested that they may be pathogenic. To the best of our knowledge, the present study was the first to identify 2novel, potentially 'disease‑causing' TRERF1 somatic mutations in the endometriotic lesions in 2out of 92patients with ovarian endometriosis; therefore, TRERF1 mutations may be involved in the pathogenesis of ovarian endometriosis.

Analysis of viral diversity for vaccine target discovery

BackgroundViral vaccine target discovery requires understanding the diversity of both the virus and the human immune system. The readily available and rapidly growing pool of viral sequence data in the public domain enable the identification and characterization of immune targets relevant to adaptive immunity. A systematic bioinformatics approach is necessary to facilitate the analysis of such large datasets for selection of potential candidate vaccine targets.ResultsThis work describes a computational methodology to achieve this analysis, with data of dengue, West Nile, hepatitis A, HIV-1, and influenza A viruses as examples. Our methodology has been implemented as an analytical pipeline that brings significant advancement to the field of reverse vaccinology, enabling systematic screening of known sequence data in nature for identification of vaccine targets. This includes key steps (i) comprehensive and extensive collection of sequence data of viral proteomes (the virome), (ii) data cleaning, (iii) large-scale sequence alignments, (iv) peptide entropy analysis, (v) intra- and inter-species variation analysis of conserved sequences, including human homology analysis, and (vi) functional and immunological relevance analysis.ConclusionThese steps are combined into the pipeline ensuring that a more refined process, as compared to a simple evolutionary conservation analysis, will facilitate a better selection of vaccine targets and their prioritization for subsequent experimental validation.

Associations of ERAP1 coding variants and domain specific interaction with HLA-C∗06 in the early onset psoriasis patients of India

Interferon-γ-induced aminopeptidase ERAP1 trims peptides within the endoplasmic reticulum so that they can be loaded onto MHC class I and presented to the CD8+ T-cells. ERAP1 association and its interaction with HLA-C∗06 is controversial across different populations. We have investigated the association and possible functional role of non-synonymous SNPs at different exons of ERAP1 (rs26653: Arg127Pro, rs30187: Lys528Arg and rs27044: Gln730Glu) and their interactions with HLA-C∗06 in psoriasis. Significant associations of HLA-C∗06 (OR=5.47, P<2.2×10−16), rs30187 (OR 1.35, P=7.4×10−4) and rs27044 (OR=1.24, P=5.8×10−3) were observed. All three ERAP1 SNPs showed significant association only for HLA-C∗06 positive patients, while rs30187 and rs27044 showed significant association only for early onset patients (rs30187: OR=1.47, P=9.6×10−5; rs27044: OR=1.36, P=3.3×10−4). No differential expression of ERAP1 was observed either between paired uninvolved and involved skin tissues of psoriasis patients or between non-risk and risk variants in the involved skin. Significant epistatic interaction was observed between HLA-C∗06 and the SNP (rs27044) located at the peptide-binding cavity of ERAP1. Evolutionary conservation analysis among mammals showed confinement of Lys528 and Gln730 within highly conserved regions of ERAP1 and suggested the possible detrimental effect of this allele in ERAP1 regulation.

Halophytic NHXs confer salt tolerance by altering cytosolic and vacuolar K+ and Na+ in Arabidopsis root cell

While the role of the vacuolar NHX Na+/H+ exchangers in plant salt tolerance has been demonstrated on numerous occasions, their control over cytosolic ionic relations has never been functionally analysed in the context of subcellular Na+ and K+ homeostasis. In this work, PutNHX1 and SeNHX1 were cloned from halophytes Puccinellia tenuiflora and Salicornia europaea and transiently expressed in Arabidopsis wild type Col-0 and the nhx1 mutant. Phylogentic analysis, topological prediction, analysis of evolutionary conservation, the topology structure and analysis of hydrophobic or polar regions of PutNHX1 and SeNHX1 indicated that they are unique tonoplast Na+/H+ antiporters with characteristics for salt tolerance. As a part of the functional assessment, cytosolic and vacuolar Na+ and K+ in different root tissues and ion fluxes from root mature zone of Col-0, nhx1 and their transgenic lines were measured. Transgenic lines sequestered large quantity of Na+ into root cell vacuoles and also promoted high cytosolic and vacuolar K+ accumulation. Expression of PutNHX1 and SeNHX1 led to significant transient root Na+ uptake in the four transgenic lines upon recovery from salt treatment. In contrast, the nhx1 mutant maintained a prolonged Na+ efflux and the nhx1:PutNHX1 and nhx1:SeNHX1 lines started to actively pump Na+ out of the cell. Overall, our findings suggest that PutNHX1 and SeNHX1 improve Na+ sequestration in the vacuole and K+ retention in the cytosol and vacuole of root cells of Arabidopsis, and that they interact with other regulatory mechanisms to provide a highly orchestrated regulation of ionic relations among intracellular cell compartments.

GNAQ mutation R183Q as a potential cause of familial Sturge-Weber syndrome: A case report.

Sturge-Weber syndrome (SWS) is a rare neurocutaneous disorder whose etiology remains unclear. To investigate the genetic contribution underlying this disease, the genetic variants of a 4-generation family with a history of SWS was analyzed in the present study. SWS was diagnosed in 3 of the family members (II-1, III-11 and IV-6). Sanger sequencing was performed to identify mutations in G protein subunit αq (GNAQ) and RAS p21 protein activator 1 exons in the 3 patients with SWS and other unaffected family members. Notably, a non-synonymous single-nucleotide variant at codon 183 on exon 4 of the GNAQ gene was identified as the only pathogenic site. This variant generated a substitution of arginine (R) with glutamine and resulted in a change of function of the encoded protein. Evolutionary conservation analysis revealed that the mutated residue 183 (R) of GNAQ is highly conserved across several vertebrate species. Furthermore, an immunofluorescence staining assay demonstrated that the substitution of arginine with glutamine resulted in a change in the sub-cellular localization of the GNAQ recombinant protein in vitro. These findings may aid in the development of novel diagnostic markers and/or therapeutic targets for the treatment of patients with familial SWS.

Bacterial Polysaccharide Specificity of the Pattern Recognition Receptor Langerin Is Highly Species-dependent

The recognition of pathogen surface polysaccharides by glycan-binding proteins is a cornerstone of innate host defense. Many members of the C-type lectin receptor family serve as pattern recognition receptors facilitating pathogen uptake, antigen processing, and immunomodulation. Despite the high evolutionary pressure in host-pathogen interactions, it is still widely assumed that genetic homology conveys similar specificities. Here, we investigate the ligand specificities of the human and murine forms of the myeloid C-type lectin receptor langerin for simple and complex ligands augmented by structural insight into murine langerin. Although the two homologs share the same three-dimensional structure and recognize simple ligands identically, a screening of more than 300 bacterial polysaccharides revealed highly diverging avidity and selectivity for larger and more complex glycans. Structural and evolutionary conservation analysis identified a highly variable surface adjacent to the canonic binding site, potentially forming a secondary site of interaction for large glycans.

Positive selection in cathelicidin host defense peptides: adaptation to exogenous pathogens or endogenous receptors?

The cause of adaptive protein evolution includes internal (for example, co-evolution of ligand-receptor pairs) and external (for example, adaptation to different ecological niches) mechanisms. Host defense peptides (HDPs) are a class of vertebrate-specific cationic antimicrobial peptides evolving under positive selection. Besides their antibiotic activity, HDPs also exert an effect on multiple host immune cells, thus providing an ideal model to study selective agents driving their evolution. On the basis of a combination of computational and experimental approaches, we studied the evolution of LL-37-type HDPs in mammals, the mature peptide of cathelicidin CAP18 (herein termed CAP18-MP) and investigated the driving force behind the evolution. Using codon-substitution maximum likelihood models, we analyzed CAP18-MPs in 40 species belonging to nine mammalian Orders and identified four positively selected sites (PSSs) that all are located on two terminal unordered regions of CAP18-MPs. Grafting the two positively selected regions of human or whale CAP18-MP to the α-helical scaffold of a rabbit homolog (substituting its corresponding parts) led to no alterations in antibacterial activity, spectrum and action mode. Likewise, further deletion of the two terminal regions did not alter its functional features. Evolutionary conservation analysis of mammalian FPR2, a receptor known to interact with the C-terminal positively selected region of LL-37, revealed high evolutionary variability in its ligand-binding extracellular loop domains, matching sequence diversity of the unordered regions in CAP18-MPs. This is the first report describing that the signature of positive selection of cathelicidins is not associated with their direct bactericidal activity, but rather with the evolutionary variability of their endogenous receptors.

Evolution of the Exon-Intron Structure in Ciliate Genomes.

A typical eukaryotic gene is comprised of alternating stretches of regions, exons and introns, retained in and spliced out a mature mRNA, respectively. Although the length of introns may vary substantially among organisms, a large fraction of genes contains short introns in many species. Notably, some Ciliates (Paramecium and Nyctotherus) possess only ultra-short introns, around 25 bp long. In Paramecium, ultra-short introns with length divisible by three (3n) are under strong evolutionary pressure and have a high frequency of in-frame stop codons, which, in the case of intron retention, cause premature termination of mRNA translation and consequent degradation of the mis-spliced mRNA by the nonsense-mediated decay mechanism. Here, we analyzed introns in five genera of Ciliates, Paramecium, Tetrahymena, Ichthyophthirius, Oxytricha, and Stylonychia. Introns can be classified into two length classes in Tetrahymena and Ichthyophthirius (with means 48 bp, 69 bp, and 55 bp, 64 bp, respectively), but, surprisingly, comprise three distinct length classes in Oxytricha and Stylonychia (with means 33–35 bp, 47–51 bp, and 78–80 bp). In most ranges of the intron lengths, 3n introns are underrepresented and have a high frequency of in-frame stop codons in all studied species. Introns of Paramecium, Tetrahymena, and Ichthyophthirius are preferentially located at the 5' and 3' ends of genes, whereas introns of Oxytricha and Stylonychia are strongly skewed towards the 5' end. Analysis of evolutionary conservation shows that, in each studied genome, a significant fraction of intron positions is conserved between the orthologs, but intron lengths are not correlated between the species. In summary, our study provides a detailed characterization of introns in several genera of Ciliates and highlights some of their distinctive properties, which, together, indicate that splicing spellchecking is a universal and evolutionarily conserved process in the biogenesis of short introns in various representatives of Ciliates.

The OPA1 Gene Mutations Are Frequent in Han Chinese Patients with Suspected Optic Neuropathy.

While many patients with hereditary optic neuropathies are caused by mitochondrial DNA (mtDNA) mutations of Leber's hereditary optic neuropathy (LHON), a significant proportion of them does not have mtDNA mutation and is caused by mutations in genes of the nuclear genome. In this study, we investigated whether the OPA1 gene, which is a pathogenic gene for autosomal dominant optic atrophy (ADOA), is frequently mutated in these patients. We sequenced all 29 exons of the OPA1 gene in 105 Han Chinese patients with suspected LHON. mtDNA copy number was quantified in blood samples from patients with and without OPA1 mutation and compared to healthy controls. In silico program-affiliated prediction, evolutionary conservation analysis, and in vitro cellular assays were performed to show the potential pathogenicity of the mutations. We identified nine OPA1 mutations in eight patients; six of them are located in exons and three are located in splicing sites. Mutation c.1172T > G has not been reported before. When we combined our data with 193 reported Han Chinese patients with optic neuropathy and compared to the available data of 4327 East Asians by the Exome Aggregation Consortium (ExAC), we found a significant enrichment of potentially pathogenic OPA1 mutations in Chinese patients. Cellular assays for OPA1 mutants c.869G > A and c.2708_2711del showed abnormalities in OPA1 isoforms, mitochondrial morphology, and cellular reactive oxygen species (ROS) level. Our results indicated that screening OPA1 mutation is needed for clinical diagnosis of patients with suspected optic neuropathy.

Probing the Intermolecular Interfaces between Claudin Protomers

Tight junctions (TJ) are structurally defined by networks of linear sealing strands between adjoining epithelial cells; this structure regulates the ion and solute permeation through the intercellular space. Claudins, a large family of transmembrane proteins, are the main components of the TJ strand network. A crystal structure of mclaudin-15 monomer was reported recently; however, the crucial domains facilitating polymerization into highly ordered, yet flexible, strands still remain unknown. In this study, we aimed to identify the crucial claudin protomer interfaces required for polymerization using both in silico and in vitro methods. First, we use computational methods including evolutionary couplings, conservation analysis, and solvent accessible surface area evaluation to identify potential key residues on the oligomerization interface. We then perform mutagenesis analysis, confocal imaging of TJ formation in transfected cultured cells, and freeze fracture EM to test the in silico data. We further use molecular docking to construct claudin oligomeric models and molecular dynamics to study intermolecular interactions at the atomic level. Two distinct intermolecular interfaces were obtained after an extensive screening of 1200 interfaces during in silico docking using in silico and in vitro data as constraints. Specifically, hydrophobic residues in extracellular loop1 and loop2 form a “head-to-head” (-trans) interface. In addition, intermolecular hydrogen bonds between hydrophilic residues on the extracellular helix and charged residues on the β-strand (β5) form an axial (-cis) interface. Mutations of these residues eliminated, reduced, or weakened the TJ strands. Combining the data obtained from these different approaches, we further propose an octameric extracellular pore model, which is consistent with the selectivity, size (∼10 A), and evidence for pore-lining residues reported previously. Our study provides mechanistic insight into TJ polymerization, paracellular molecular flow through TJ, and potential targets for TJ modulation.

Mitochondrial tRNALeu(CUN) A12307G variant may not be associated pancreatic cancer.

Mitochondrial DNA mutations that lead to mitochondrial dysfunction have long been proposed to play important roles in the development of pancreatic cancer. Of these, alterations to mitochondrial tRNA genes constitute the largest group. Most recently, a variation at position 12307 in the gene encoding tRNA(Leu(CUN)) has been reported to be associated with this disease. However, the molecular mechanism underlying this relationship remains poorly understood. To assess this association, we evaluated this variant by evolutionary conservation analysis, measurements of allelic frequencies among control subjects, and use of several bioinformatic tools to estimate potential structural and functional alterations. We found this residue to have a high conservation index; however, the presence of the A12307G variation in control subjects revealed by a literature search suggested it to be common in human populations. Moreover, RNAfold results showed that this variant did not alter the secondary structure of tRNA(Leu(CUN)). Through the application of a pathogenicity scoring system, this variant was determined to be a "neutral polymorphism," with a score of only 4 points based on current data. Thus, the contribution of the A12307G variant to pancreatic cancer needs to be addressed in further experimental studies.