Analysis Of Amino Acid Conservation Research Articles

Insight into the role of TXNRD2 in steroidogenesis through a novel homozygous TXNRD2 splice variant.

Adrenal cortisol production occurs through a biosynthetic pathway which depend on NADH and NADPH for energy supply. The mitochondrial respiratory chain and the reactive oxygen species (ROS) detoxification system are therefore important for steroidogenesis. Mitochondrial dysfunction leading to oxidative stress has been implicated in the pathogenesis of several adrenal conditions. Nonetheless, only very few patients with variants in one gene of the ROS detoxification system, Thioredoxin Reductase 2 (TXNRD2), have been described with variable phenotypes. Clinical, genetic, structural, and functional characterization of a novel, biallelic TXNRD2 splice variant. On human biomaterial, we performed whole exome sequencing to identify and RNA analysis to characterize the specific TXNRD2 splice variant. Amino acid conservation analysis and protein structure modeling were performed in silico. Using patient's fibroblast-derived human induced pluripotent stem cells, we generated adrenal-like cells (iALC) to study the impact of wild-type (WT) and mutant TXNRD2 on adrenal steroidogenesis and ROS production. The patient had a complex phenotype of primary adrenal insufficiency (PAI), combined with genital, ophthalmological, and neurological features. He carried a homozygous splice variant c.1348-1G > T in TXNRD2 which leads to a shorter protein lacking the C-terminus and thereby affecting homodimerization and flavin adenine dinucleotide binding. Patient-derived iALC showed a loss of cortisol production with overall diminished adrenal steroidogenesis, while ROS production was significantly increased. Lack of TXNRD2 activity for mitochondrial ROS detoxification affects adrenal steroidogenesis and predominantly cortisol production.

Enhancing Machine-Learning Prediction of Enzyme Catalytic Temperature Optima through Amino Acid Conservation Analysis.

Enzymes play a crucial role in various industrial production and pharmaceutical developments, serving as catalysts for numerous biochemical reactions. Determining the optimal catalytic temperature (Topt) of enzymes is crucial for optimizing reaction conditions, enhancing catalytic efficiency, and accelerating the industrial processes. However, due to the limited availability of experimentally determined Topt data and the insufficient accuracy of existing computational methods in predicting Topt, there is an urgent need for a computational approach to predict the Topt values of enzymes accurately. In this study, using phosphatase (EC 3.1.3.X) as an example, we constructed a machine learning model utilizing amino acid frequency and protein molecular weight information as features and employing the K-nearest neighbors regression algorithm to predict the Topt of enzymes. Usually, when conducting engineering for enzyme thermostability, researchers tend not to modify conserved amino acids. Therefore, we utilized this machine learning model to predict the Topt of phosphatase sequences after removing conserved amino acids. We found that the predictive model's mean coefficient of determination (R2) value increased from 0.599 to 0.755 compared to the model based on the complete sequences. Subsequently, experimental validation on 10 phosphatase enzymes with undetermined optimal catalytic temperatures shows that the predicted values of most phosphatase enzymes based on the sequence without conservative amino acids are closer to the experimental optimal catalytic temperature values. This study lays the foundation for the rapid selection of enzymes suitable for industrial conditions.

The role of SKA2 on affective disorder, post-traumatic stress disorder and suicide behavior: systematic review and in silico analysis.

Genes involved in the hypothalamic-pituitary-adrenal axis may be a robust biomarker of psychiatric disorders. Genetic polymorphisms of the SKA2 gene are associated with several behavioral disorders. In this study, we embarked on a systematic search of all possible reports of genetic association with SKA2 and affective disorder, post-traumatic stress disorder, and suicide behavior; the functional consequences of nsSNPs were explored through computational tools with an in silico analysis. Eight eligible articles were included. Our study identified that SKA2 did not show association with risk of Major Depression Disorder. Epigenetic variation at SKA2 mediates vulnerability to Post-Traumatic Stress Disorder. Studies provide strong preliminary evidence that alterations at the SKA2 gene covary with types of suicide behavior, including suicidal ideation, attempts, and completions. Results from in silico analysis predicted that I22S, I22G, I78T, A15L, D18R, R25L, N42I, Y21S, K14I, K14L, and L60R were the most structurally and functionally significant nsSNPs in SKA2. Amino acid conservation analysis revealed that the amino acids were highly conserved and some dissimilarities of mutant type amino acids from wild-type amino acids such as charge, size, and hydrophobicity were observed. In the future, SKA2 gene have the potential to be evaluated as prognostic biomarkers for diagnosis and research.

PREDICTION OF HIGH-RISK NSSNPS ASSOCIATED WITH WISP3 GENE EXPRESSION: AN IN SILICO STUDY

Objective: The primary aim of this investigation is to comprehensively examine the detrimental effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on the WISP3 gene. This objective will be accomplished through intricate evaluations encompassing protein stability prediction, amino acid conservation analysis, investigation of protein-protein interactions (PPI), scrutiny of post-translational modifications (PTM), and the utilization of bioinformatics tools to forecast the potential association between nsSNPs and various diseases. By implementing these sophisticated methodologies, we aim to unveil the intricate mechanisms by which harmful nsSNPs influence the functionality and pathological implications of the WISP3 gene. Methods: Retrieved rsIDs of SNPs from the dbSNP database and filtered using 5 in silico programs. Selected nsSNPs were subjected to further analysis i.e., protein stability and conservation analysis, solvent accessibility analysis, PPI and PTM analysis, prediction and evaluation of both native and mutant protein, and identification of cancer association and gene expression analysis. Results: The study found that seven (C122Y, C145Y, C52Y, C78R, C75G, N233K, and R245I) of the nsSNPs are potentially vulnerable due to their higher conservancy and ability to reduce protein stability. Two (D271N and Q56H) of the nsSNPs from the initial screening were found to be associated with colon adenocarcinoma. Conclusion: The study's findings could help researchers design experiments to validate the predictions and develop potential treatments for diseases associated with the WISP3 gene.

Phenotypic characteristics of taurodontism and a novel WNT10A variant in non-syndromic oligodontia family

ObjectiveVariants in wingless-type MMTV integration site family member 10A (WNT10A) have been proposed to be the most common cause of non-syndromic oligodontia (NSO). The goal of the present study was to identify the novel WNT10A variants in Chinese families with NSO. DesignClinical data were collected from 39 families with oligodontia admitted to the Hospital of Stomatology Hebei Medical University (China) from 2016 to 2022. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify WNT10A variants in three families with non-syndromic oligodontia. Amino acid conservation analysis and protein conformational analysis were conducted for the WNT10A variant. Genotype-phenotype analysis was performed on the previously reported WNT10A variants related to NSO. ResultsWe found a novel heterozygous WNT10A variant c.1127 G>A (p.Cys376Tyr) and two reported heterozygous variants c.460 C>A (p.Leu154Met) and c.511 C>T (p.Arg171Cys). Structural modeling showed that the novel WNT10A variant was located in a highly conserved domain, which led to structural damage of WNT10A protein. In addition, we found that the phenotype of the WNT10A variants affected the maxillary second premolars, followed by the mandibular second premolars, and rarely affected the maxillary central incisor. Herein, it is the first time to report that NSO patients with WNT10A monoallele mutation carry taurodontism phenotype and 6.1% prevalence of taurodontism in WNT10A-related NSO patients. ConclusionsOur results demonstrated that the novel variant c.1127 G>A (p.Cys376Tyr) of WNT10A causes NSO. The present study expanded the known variation spectrum of WNT10A and provided valuable information for genetic counseling of families.

In Silico Analysis Identified Putative Pathogenic Missense nsSNPs in Human SLITRK1 Gene.

Human DNA contains several variations, which can affect the structure and normal functioning of a protein. These variations could be single nucleotide polymorphisms (SNPs) or insertion-deletions (InDels). SNPs, as opposed to InDels, are more commonly present in DNA and may cause genetic disorders. In the current study, several bioinformatic tools were used to prioritize the pathogenic variants in the SLITRK1 gene. Out of all of the variants, 16 were commonly predicted to be pathogenic by these tools. All the variants had very low frequency, i.e., <0.0001 in the global population. The secondary structure of all filtered variants was predicted, but no structural change was observed at the site of variation in any variant. Protein stability analysis of these variants was then performed, which determined a decrease in protein stability of 10 of the variants. Amino acid conservation analysis revealed that all the amino acids were highly conserved, indicating their structural and functional importance. Protein 3D structure of wildtype SLITRK1 and all of its variants was predicted using I-TASSER, and the effect of variation on 3D structure of the protein was observed using the Missense3D tool, which presented the probable structural loss in three variants, i.e., Asn529Lys, Leu496Pro and Leu94Phe. The wildtype SLITRK1 protein and these three variants were independently docked with their close interactor protein PTPRD, and remarkable differences were observed in the docking sites of normal and variants, which will ultimately affect the functional activity of the SLITRK1 protein. Previous studies have shown that mutations in SLITRK1 are involved in Tourette syndrome. The present study may assist a molecular geneticist in interpreting the variant pathogenicity in research as well as diagnostic setup.

RAB33B and PCNT variants in two Pakistani families with skeletal dysplasia and short stature

BackgroundSkeletal dysplasia is a heterogeneous group of disorders resulting from different genetic variants in humans. The current study was designed to identify the genetic causes of skeletal dysplasia and short stature in two consanguineous families from Pakistan, both comprised of multiple affected individuals. Patients in one family had proportionate short stature with reduced head circumference while affected individuals in the other family had disproportionate short stature.MethodsClinical data were obtained and radiological examinations of the index patients were completed. Whole genome sequencing for probands from both families were performed followed by Sanger sequencing to confirm segregation of identified variants in the respective families. In-silico pathogenicity score prediction for identified variant and amino acid conservation analysis was completed.ResultsWhole Genome Sequencing identified a known biallelic variant c.6176_6189delGTCAGCTGCCGAAG; p.(Gln2060ArgfsTer48) in PCNT gene and a novel biallelic variant c.174delC; p.(Asp60ThrfsTer7) in RAB33B gene respectively in affected members of the two families. Clinical imaging revealed platyspondyly and varus deformity in the legs of the affected members in the first family. Radiographs indicated severe platyspondyly, genu valgus deformity of legs and pectus carinatum for the patients in the second family.ConclusionIn this study we report the phenotypes and genetic variants in two unrelated families with two distinct forms of skeletal dysplasia. This study strengthens the previous findings that patients harboring PCNT variants are phenotypically homogeneous and also extends the genotypic spectrum of RAB33B variants.

Novel pathogenic variant of MYBPC3 responsible for hypertrophic cardiomyopathy.

This study aims to investigate the pathogenic gene variant in a family with hypertrophic cardiomyopathy by using whole-exome sequencing and to explore the relationship between the gene variant and clinical phenotype. Peripheral blood was collected from a family with hypertrophic cardiomyopathy, and deoxyribonucleic acid was extracted. The possible pathogenic genes were detected by whole-exome sequencing, and the variant was verified by Sanger sequencing. Functional change in the variant was predicted by bioinformatics software. Clinical data of the family members are analysed simultaneously. The proband carries a novel heterozygous nonsense variant of MYBPC3:c.2731G > T (p.E911X). The analysis of amino acid conservation suggests that the variation is highly conserved. The three-dimensional protein structure shows that the variant in MYBPC3 results in the incompleteness of the fibronectintype-III2 (p872-967) domain and deletion of Ig-like C2-type 6 (p971-1065) and fibronectin type-III 3 and Ig-like C2-type 7 (p1181-1274) domains, in which p1253-1268 is predicted to have a transmembrane helix structure. Clinical data indicate that the phenotypes of variant carriers with hypertrophic cardiomyopathy are diverse, suggesting the functional damages to the protein of MYBPC3. The phenotypes of variant carriers with hypertrophic cardiomyopathy caused by the novel variant in MYBPC3: c.2731G > T (p.E911X) exhibit variable severity and clinical manifestations. Whole-exome sequencing can be used to comprehensive screen hypertrophic cardiomyopathy genes and provide a strong basis for early screening and accurate diagnosis and treatment of hypertrophic cardiomyopathy in children.

Genome-wide analysis, identification, evolution and genomic organization of dehydration responsive element-binding (DREB) gene family in Solanum tuberosum.

BackgroundThe dehydration responsive element-binding (DREB) gene family plays a crucial role as transcription regulators and enhances plant tolerance to abiotic stresses. Although the DREB gene family has been identified and characterized in many plants, knowledge about it in Solanum tuberosum (Potato) is limited.ResultsIn the present study, StDREB gene family was comprehensively analyzed using bioinformatics approaches. We identified 66 StDREB genes through genome wide screening of the Potato genome based on the AP2 domain architecture and amino acid conservation analysis (Valine at position 14th). Phylogenetic analysis divided them into six distinct subgroups (A1–A6). The categorization of StDREB genes into six subgroups was further supported by gene structure and conserved motif analysis. Potato DREB genes were found to be distributed unevenly across 12 chromosomes. Gene duplication proved that StDREB genes experienced tandem and segmental duplication events which led to the expansion of the gene family. The Ka/Ks ratios of the orthologous pairs also demonstrated the StDREB genes were under strong purification selection in the course of evolution. Interspecies synteny analysis revealed 45 and 36 StDREB genes were orthologous to Arabidopsis and Solanum lycopersicum, respectively. Moreover, subcellular localization indicated that StDREB genes were predominantly located within the nucleus and the StDREB family’s major function was DNA binding according to gene ontology (GO) annotation.ConclusionsThis study provides a comprehensive and systematic understanding of precise molecular mechanism and functional characterization of StDREB genes in abiotic stress responses and will lead to improvement in Solanum tuberosum.

Cytochrome P450 Monooxygenase CYP139 Family Involved in the Synthesis of Secondary Metabolites in 824 Mycobacterial Species.

Tuberculosis (TB) is one of the top infectious diseases causing numerous human deaths in the world. Despite enormous efforts, the physiology of the causative agent, Mycobacterium tuberculosis, is poorly understood. To contribute to better understanding the physiological capacity of these microbes, we have carried out extensive in silico analyses of the 1111 mycobacterial species genomes focusing on revealing the role of the orphan cytochrome P450 monooxygenase (CYP) CYP139 family. We have found that CYP139 members are present in 894 species belonging to three mycobacterial groups: M. tuberculosis complex (850-species), Mycobacterium avium complex (34-species), and non-tuberculosis mycobacteria (10-species), with all CYP139 members belonging to the subfamily “A”. CYP139 members have unique amino acid patterns at the CXG motif. Amino acid conservation analysis placed this family in the 8th among CYP families belonging to different biological domains and kingdoms. Biosynthetic gene cluster analyses have revealed that 92% of CYP139As might be associated with producing different secondary metabolites. Such enhanced secondary metabolic potentials with the involvement of CYP139A members might have provided mycobacterial species with advantageous traits in diverse niches competing with other microbial or viral agents, and might help these microbes infect hosts by interfering with the hosts’ metabolism and immune system.

DREB Genes from Common Bean (Phaseolus vulgaris L.) Show Broad to Specific Abiotic Stress Responses and Distinct Levels of Nucleotide Diversity.

We analyzed the nucleotide variability and the expression profile of DREB genes from common bean, a crop of high economic and nutritional value throughout the world but constantly affected by abiotic stresses in cultivation areas. As DREB genes have been constantly associated with abiotic stress tolerance, we systematically categorized 54 putative PvDREB genes distributed in the common bean genome. It involved from AP2 domain location and amino acid conservation analysis (valine at the 14th position) to the identification of conserved motifs within peptide sequences representing six subgroups (A-1 to A-6) of PvDREB proteins. Four genes (PvDREB1F, PvDREB2A, PvDREB5A, and PvDREB6B) were cloned and analyzed for their expression profiles under abiotic stresses and their nucleotide and amino acid diversity in genotypes of Andean and Mesoamerican origin, showing distinct patterns of expression and nucleotide variability. PvDREB1F and PvDREB5A showed high relative inducibilities when genotypes of common bean were submitted to stresses by drought, salt, cold, and ABA. PvDREB2A inducibility was predominantly localized to the stem under drought. PvDREB6B was previously described as an A-2 (DREB2) gene, but a detailed phylogenetic analysis and its expression profile clearly indicated it belongs to group A-6. PvDREB6B was found as a cold- and dehydration-responsive gene, mainly in leaves. Interestingly, PvDREB6B also showed a high nucleotide and amino acid diversity within its coding region, in comparison to the others, implicating in several nonsynonymous amino acid substitutions between Andean and Mesoamerican genotypes. The expression patterns and nucleotide diversity of each DREB found in this study revealed fundamental characteristics for further research aimed at understanding the molecular mechanisms associated with drought, salt, and cold tolerance in common bean, which could be performed based on association mapping and functional analyses.

In silico and in vitro Studies of Human 5α‐reductase Type II Reveal New Loss of Function Variants

BackgroundAndrogens are steroid hormones necessary for human sex development. Testosterone (T) and the more potent dihydrotestosterone (DHT) are maybe the best‐known androgens, which exert their effect by binding and activating the androgen receptor. The 5α steroid reductases (SRD5As) catalyze the conversion of T to DHT in the classical androgen production pathway, or from 17‐hydroxyprogesterone to 17OH‐dihydroprogesterone, and androstenedione to androstanedione in alternate pathways leading to DHT. There are two enzymes with differential expression, of which SRD5A2 is expressed in reproductive organs and liver, and catalyzes the reaction of T to DHT more efficiently than the SRD5A1. In humans, SRD5A2 loss‐of‐function mutations are known, and cause severe 46XY undervirilization, while gain‐of‐function variants have been suggested in androgen excess syndromes such as premature adrenarche, the polycystic ovary syndrome or prostate tumors, but these have not been identified so far.AimWe aimed to search for potential gain‐of‐function mutations in the human SRD5A2 gene.MethodsWe searched sequence databases for genetic variants and performed bioinformatic and functional tests on a set of selected variants. After the amino acid conservation analysis of SRD5A2, a novel 3D protein model was constructed to identify the exact position of amino acids in the tertiary structure and predict their effect on protein function and substrate interaction. We then collected 116 coding SNPs in SRD5A2 from OMIM, dbSNP, Pubmed, Clinvar, HGMD, and UniProt databases. These SNPs were ranked according to their association with phenotypes, physical location in our 3D model, and molecular dynamics simulation studies. Finally, we selected nine coding SNPs for in vitro studies. These SNPs were located within or close to highly conserved areas that form the binding cavities for substrates or the cofactor NADPH. The SRD5A2 variants were expressed in HEK293 cells, and enzyme activity was assessed by conversion of testosterone (T), progesterone (Prog) and androstenedione (Δ4A) to their 5α‐reduced metabolites.ResultVariants R50A and P173S showed partial activity with substrates T (34% and 28%) and Δ4A (37% and 22%). With Prog as substrate variants P106L, P106A, L167S, and R168C, showed partial activity (15% to 64%). Functional testing of all other variants showed loss‐of‐function. As predicted in our in silico analysis, all coding SNPs affected enzyme activity. However, none of them showed gain‐of‐functionConclusionWe provide a novel protein model for studies of SRD5A2. We have characterized seven human SRD5A2 variants, which although did not show any gain of function, might be of clinical relevance for their loss of enzyme activity. It is possible that individuals carrying these SNPs show a minor, yet unidentified phenotype.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Amyotrophic Lateral Sclerosis Type 20 - In Silico Analysis and Molecular Dynamics Simulation of hnRNPA1.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that affects the upper and lower motor neurons. 5–10% of cases are genetically inherited, including ALS type 20, which is caused by mutations in the hnRNPA1 gene. The goals of this work are to analyze the effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on hnRNPA1 protein function, to model the complete tridimensional structure of the protein using computational methods and to assess structural and functional differences between the wild type and its variants through Molecular Dynamics simulations. nsSNP, PhD-SNP, Polyphen2, SIFT, SNAP, SNPs&GO, SNPeffect and PROVEAN were used to predict the functional effects of nsSNPs. Ab initio modeling of hnRNPA1 was made using Rosetta and refined using KoBaMIN. The structure was validated by PROCHECK, Rampage, ERRAT, Verify3D, ProSA and Qmean. TM-align was used for the structural alignment. FoldIndex, DICHOT, ELM, D2P2, Disopred and DisEMBL were used to predict disordered regions within the protein. Amino acid conservation analysis was assessed by Consurf, and the molecular dynamics simulations were performed using GROMACS. Mutations D314V and D314N were predicted to increase amyloid propensity, and predicted as deleterious by at least three algorithms, while mutation N73S was predicted as neutral by all the algorithms. D314N and D314V occur in a highly conserved amino acid. The Molecular Dynamics results indicate that all mutations increase protein stability when compared to the wild type. Mutants D314N and N319S showed higher overall dimensions and accessible surface when compared to the wild type. The flexibility level of the C-terminal residues of hnRNPA1 is affected by all mutations, which may affect protein function, especially regarding the protein ability to interact with other proteins.

Revisiting the Interaction between the Chaperone Skp and Lipopolysaccharide

The bacterial outer membrane comprises two main classes of components, lipids and membrane proteins. These nonsoluble compounds are conveyed across the aqueous periplasm along specific molecular transport routes: the lipid lipopolysaccharide (LPS) is shuttled by the Lpt system, whereas outer membrane proteins (Omps) are transported by chaperones, including the periplasmic Skp. In this study, we revisit the specificity of the chaperone-lipid interaction of Skp and LPS. High-resolution NMR spectroscopy measurements indicate that LPS interacts with Skp nonspecifically, accompanied by destabilization of the Skp trimer and similar to denaturation by the nonnatural detergent lauryldimethylamine-N-oxide (LDAO). Bioinformatic analysis of amino acid conservation, structural analysis of LPS-binding proteins, and MD simulations further confirm the absence of a specific LPS binding site on Skp, making a biological relevance of the interaction unlikely. Instead, our analysis reveals a highly conserved salt-bridge network, which likely has a role for Skp function.

Crystal structure of the FRP and identification of the active site for modulation of OCP-mediated photoprotection in cyanobacteria

Photosynthetic reaction centers are sensitive to high light conditions, which can cause damage because of the formation of reactive oxygen species. To prevent high-light induced damage, cyanobacteria have developed photoprotective mechanisms. One involves a photoactive carotenoid protein that decreases the transfer of excess energy to the reaction centers. This protein, the orange carotenoid protein (OCP), is present in most cyanobacterial strains; it is activated by high light conditions and able to dissipate excess energy at the site of the light-harvesting antennae, the phycobilisomes. Restoration of normal antenna capacity involves the fluorescence recovery protein (FRP). The FRP acts to dissociate the OCP from the phycobilisomes by accelerating the conversion of the active red OCP to the inactive orange form. We have determined the 3D crystal structure of the FRP at 2.5 Å resolution. Remarkably, the FRP is found in two very different conformational and oligomeric states in the same crystal. Based on amino acid conservation analysis, activity assays of FRP mutants, FRP:OCP docking simulations, and coimmunoprecipitation experiments, we conclude that the dimer is the active form. The second form, a tetramer, may be an inactive form of FRP. In addition, we have identified a surface patch of highly conserved residues and shown that those residues are essential to FRP activity.

Regulation of ATG6/Beclin-1 homologs by abiotic stresses and hormones in rice (Oryza sativa L.)

Autophagy, a complex and conserved mechanism, serving as a defense response in all eukaryotic organisms, is regulated by several proteins, among which ATG proteins are the most important due to their involvement in autophagosome formation. ATG6/Beclin-1 proteins, reported to be essential for autophagosome formation and assigned as a conserved domain, were subjected to database searches. We found three homologs in the rice (Oryza sativa) genome. A phylogeny tree was constructed to establish their across species relationship, which divided them into three distinct groups; two for plants, i.e., monocots and dicots, and one for animals. Evolutionary study of this family by critical amino acid conservation analysis revealed significant functional divergence. The finding of important stress-related cis-acting elements in the promoter region of rice ATG6 genes demonstrated their involvement in abiotic stress responses. Furthermore, expression profiling of rice ATG6 genes based on microarray data, as well as by semi-quantitative RT-PCR showed differential expression when subjected to different stresses suggesting the involvement of OsATG6 genes in abiotic stresses (heat, cold and drought) and hormone (abscisic acid) responses. Analysis of co-expressed genes showed that most of them annotated to DNA repair pathways and proteolysis, etc. Collectively, these results suggest the involvement of OsATG6 genes in different stresses, and provide a basis for further functional studies to investigate the biological mechanism of action of these genes under abiotic stresses.

Rare mtDNA variants in Leber hereditary optic neuropathy families with recurrence of myoclonus

To investigate the mechanisms underlying myoclonus in Leber hereditary optic neuropathy (LHON). Five patients and one unaffected carrier from two Italian families bearing the homoplasmic 11778/ND4 and 3460/ND1 mutations underwent a uniform investigation including neurophysiologic studies, muscle biopsy, serum lactic acid after exercise, and muscle ((31)P) and cerebral ((1)H) magnetic resonance spectroscopy (MRS). Biochemical investigations on fibroblasts and complete mitochondrial DNA (mtDNA) sequences of both families were also performed. All six individuals had myoclonus. In spite of a normal EEG background and the absence of giant SEPs and C reflex, EEG-EMG back-averaging showed a preceding jerk-locked EEG potential, consistent with a cortical generator of the myoclonus. Specific comorbidities in the 11778/ND4 family included muscular cramps and psychiatric disorders, whereas features common to both families were migraine and cardiologic abnormalities. Signs of mitochondrial proliferation were seen in muscle biopsies and lactic acid elevation was observed in four of six patients. (31)P-MRS was abnormal in five of six patients and (1)H-MRS showed ventricular accumulation of lactic acid in three of six patients. Fibroblast ATP depletion was evident at 48 hours incubation with galactose in LHON/myoclonus patients. Sequence analysis revealed haplogroup T2 (11778/ND4 family) and U4a (3460/ND1 family) mtDNAs. A functional role for the non-synonymous 4136A>G/ND1, 9139G>A/ATPase6, and 15773G>A/cyt b variants was supported by amino acid conservation analysis. Myoclonus and other comorbidities characterized our Leber hereditary optic neuropathy (LHON) families. Functional investigations disclosed a bioenergetic impairment in all individuals. Our sequence analysis suggests that the LHON plus phenotype in our cases may relate to the synergic role of mtDNA variants.

Hereditary haemorrhagic telangiectasia: mutation detection, test sensitivity and novel mutations

Background: Hereditary haemorrhagic telangiectasia (HHT) is a genetic disorder present in 1 in 8000 people and associated with arteriovenous malformations. Genetic testing can identify individuals at risk of developing the...

Structural basis for mammalian vitamin B 12 transport by transcobalamin

Cobalamin (Cbl, vitamin B(12)) serves for two essential cofactors in mammals. The pathway for its intestinal absorption, plasma transport, and cellular uptake uses cell surface receptors and three Cbl-transporting proteins, haptocorrin, intrinsic factor, and transcobalamin (TC). We present the structure determination of a member of the mammalian Cbl-transporter family. The crystal structures of recombinant human and bovine holo-TCs reveal a two-domain architecture, with an N-terminal alpha(6)-alpha(6) barrel and a smaller C-terminal domain. One Cbl molecule in base-on conformation is buried inside the domain interface. Structural data combined with previous binding assays indicate a domain motion in the first step of Cbl binding. In a second step, the weakly coordinated ligand H(2)O at the upper axial side of added H(2)O-Cbl is displaced by a histidine residue of the alpha(6)-alpha(6) barrel. Analysis of amino acid conservation on TC's surface in orthologous proteins suggests the location of the TC-receptor-recognition site in an extended region on the alpha(6)-alpha(6) barrel. The TC structure allows for the mapping of sites of amino acid variation due to polymorphisms of the human TC gene. Structural information is used to predict the overall fold of haptocorrin and intrinsic factor and permits a rational approach to the design of new Cbl-based bioconjugates for diagnostic or therapeutic drug delivery.

Analysis of missense variants in the PKHD1-gene in patients with autosomal recessive polycystic kidney disease (ARPKD)

Autosomal recessive polycystic kidney disease (ARPKD) is a severe form of polycystic kidney disease characterized by enlarged kidneys and congenital hepatic fibrosis. Given the poor prognosis for the majority of children with the severe perinatal ARPKD phenotype, there is a regular request for prenatal testing. ARPKD is caused by mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, which consists of 86 exons that are variably assembled into a number of alternatively spliced transcripts. The longest transcript, comprising 67 exons, encodes the protein fibrocystin/polyductin. We have set up mutation analysis by direct sequencing of these 67 exons. In 39 mainly Dutch families we identified: 11 nonsense mutations, 15 deletions/insertions, 5 splice site mutations, and 39 missense mutations. To classify missense variants we combined evolutionary conservation, using the human, chimpanzee, dog, mouse, chicken and frog Pkhd1 sequences, with the Grantham score for chemical differences. Thirty-three missense mutations were considered pathogenic and seven were classified as rare, probably pathogenic variants. In addition to sequence analysis, multiplex ligation-dependent probe amplification (MLPA) was used to identify multiple exon deletions. However, no large deletions in the PKHD1 gene were identified. In 31 index patients two mutations were found, in 6 patients one mutation was found, leading to a mutation detection rate of 87%. The analysis of amino acid conservation as well as applying the Grantham score for chemical differences allowed us to determine the pathogeneity for nearly all new missense mutations and thus proved to be useful tools to classify missense variants.