Effects Of Missense Mutations Research Articles

Identification and mechanism determination of the efflux pump subunit amrB gene mutations linked to gentamicin susceptibility in clinical Burkholderia pseudomallei from Malaysian Borneo.

The bacterium Burkholderia pseudomallei is typically resistant to gentamicin but rare susceptible strains have been isolated in certain regions, such as Thailand and Sarawak, Malaysia. Recently, several amino acid substitutions have been reported in the amrB gene (a subunit of the amrAB-oprA efflux pump gene) that confer gentamicin susceptibility. However, information regarding the mechanism of the substitutions conferring the susceptibility is lacking. To understand the mechanism of amino acid substitution that confers susceptibility, this study identifies the corresponding mutations in clinical gentamicin-susceptible B. pseudomallei isolates from the Malaysian Borneo (n = 46; Sarawak: 5; Sabah: 41). Three phenotypically confirmed gentamicin-susceptible (GENs) strains from Sarawak, Malaysia, were screened for mutations in the amrB gene using gene sequences of gentamicin-resistant (GENr) strains (QEH 56, QEH 57, QEH20, and QEH26) and publicly available sequences (AF072887.1 and BX571965.1) as the comparator. The effect of missense mutations on the stability of the AmrB protein was determined by calculating the average energy change value (ΔΔG). Mutagenesis analysis identified a polymorphism-associated mutation, g.1056T > G, a possible susceptible-associated in-frame deletion, Delta V412, and a previously confirmed susceptible-associated amino acid substitution, T368R, in each of the three GENs isolates. The contribution of Delta V412 needs further confirmation by experimental mutagenesis analysis. The mechanism by which T368R confers susceptibility, as elucidated by in silico mutagenesis analysis using AmrB-modeled protein structures, is proposed to be due to the location of T368R in a highly conserved region, rather than destabilization of the AmrB protein structure.

5558131 GENETICS OF IRANIAN ALPHA THALASSEMIA PATIENTS; COMPREHENSIVE ORIGINAL STUDY

Background: Alpha thalassemia is the most prevalent monogenic gene disorder in the world and especially in Mediterranean countries. In the current hematological phenotype of patients with different genotypes, and also the effect of missense mutations on the protein function and stability were evaluated in a large cohort study. Methods: One thousand five hundred and and sixty subjects were participated in the study and divided into two groups; 259 normal subjects and 1301 alpha thalassemia carriers. Genomic DNA was extracted and analyzed using ARMS PCR, Multiplex Gap and direct sequencing. The effects of single nucleotide change on the protein function and stability were predicted by free available databases of human polymorphisms. Results: Sixty-three different genotypes were seen in the patients. The more prevalent was heterozygote form of -α3.7 (41.4%) followed by -α3.7 homozygote(11.6%) and - -MED(3.8%). The significant differences were seen in mean hemoglobin level [F=20.5, p<0.001] between the Alpha globin genotypes, when adjusted for gender. Moreover, 28 different mutations were found in our study. A significant relationship was seen between ethnicity and the alpha globin mutation frequency X2 (df;8) = 38.36, p <0.0001). Conclusion: Different genotypes could be displayed as different phenotypes. The mutation frequency distributions in our region is different from other parts of Iran. Significant differences are seen in the spectrum of mutation frequency between various ethnicity. Finally, some missense mutations maybe did not considerably effect on the proteins and could be a neutral mutations. Keywords: Alpha Thalassemia, Genotype, Phenotype, Southwest of Iran

Mitochondrial DNA Changes in Genes of Respiratory Complexes III, IV and V Could Be Related to Brain Tumours in Humans.

Mitochondrial DNA changes can contribute to both an increased and decreased likelihood of cancer. This process is complex and not fully understood. Polymorphisms and mutations, especially those of the missense type, can affect mitochondrial functions, particularly if the conservative domain of the protein is concerned. This study aimed to identify the possible relationships between brain gliomas and the occurrence of specific mitochondrial DNA polymorphisms and mutations in respiratory complexes III, IV and V. The investigated material included blood and tumour material collected from 30 Caucasian patients diagnosed with WHO grade II, III or IV glioma. The mitochondrial genetic variants were investigated across the mitochondrial genome using next-generation sequencing (MiSeq/FGx system—Illumina). The study investigated, in silico, the effects of missense mutations on the biochemical properties, structure and functioning of the encoded protein, as well as their potential harmfulness. The A14793G (MTCYB), A15758G, (MT-CYB), A15218G (MT-CYB), G7444A (MT-CO1) polymorphisms, and the T15663C (MT-CYB) and G8959A (ATP6) mutations were assessed in silico as harmful alterations that could be involved in oncogenesis. The G8959A (E145K) ATP6 missense mutation has not been described in the literature so far. In light of these results, further research into the role of mtDNA changes in brain tumours should be conducted.

Understanding and predicting the functional consequences of missense mutations in BRCA1 and BRCA2

BRCA1 and BRCA2 are tumour suppressor genes that play a critical role in maintaining genomic stability via the DNA repair mechanism. DNA repair defects caused by BRCA1 and BRCA2 missense variants increase the risk of developing breast and ovarian cancers. Accurate identification of these variants becomes clinically relevant, as means to guide personalized patient management and early detection. Next-generation sequencing efforts have significantly increased data availability but also the discovery of variants of uncertain significance that need interpretation. Experimental approaches used to measure the molecular consequences of these variants, however, are usually costly and time-consuming. Therefore, computational tools have emerged as faster alternatives for assisting in the interpretation of the clinical significance of newly discovered variants. To better understand and predict variant pathogenicity in BRCA1 and BRCA2, various machine learning algorithms have been proposed, however presented limited performance. Here we present BRCA1 and BRCA2 gene-specific models and a generic model for quantifying the functional impacts of single-point missense variants in these genes. Across tenfold cross-validation, our final models achieved a Matthew's Correlation Coefficient (MCC) of up to 0.98 and comparable performance of up to 0.89 across independent, non-redundant blind tests, outperforming alternative approaches. We believe our predictive tool will be a valuable resource for providing insights into understanding and interpreting the functional consequences of missense variants in these genes and as a tool for guiding the interpretation of newly discovered variants and prioritizing mutations for experimental validation.

Mutations affecting the N-terminal domains of SHANK3 point to different pathomechanisms in neurodevelopmental disorders

Shank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The effects of missense mutations on Shank3 function, and therefore the pathomechanisms are unclear. Several missense mutations in SHANK3 affect the N-terminal region, consisting of the Shank/ProSAP N-terminal (SPN) domain and a set of Ankyrin (Ank) repeats. Here we identify a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. We functionally analysed this and a series of other mutations, using biochemical and biophysical techniques. We observe two major effects: (1) a loss of binding to δ-catenin (e.g. in the p.L270M variant), and (2) interference with the intramolecular interaction between N-terminal SPN domain and the Ank repeats. This also interferes with binding to the α-subunit of the calcium-/calmodulin dependent kinase II (αCaMKII), and appears to be associated with a more severe neurodevelopmental pathology.

Mutational signatures in GATA3 transcription factor and its DNA binding domain that stimulate breast cancer and HDR syndrome

Transcription factors (TFs) play important roles in many biochemical processes. Many human genetic disorders have been associated with mutations in the genes encoding these transcription factors, and so those mutations became targets for medications and drug design. In parallel, since many transcription factors act either as tumor suppressors or oncogenes, their mutations are mostly associated with cancer. In this perspective, we studied the GATA3 transcription factor when bound to DNA in a crystal structure and assessed the effect of different mutations encountered in patients with different diseases and phenotypes. We generated all missense mutants of GATA3 protein and DNA within the adjacent and the opposite GATA3:DNA complex models. We mutated every amino acid and studied the new binding of the complex after each mutation. Similarly, we did for every DNA base. We applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations. After analyzing our data, we identified amino acids and DNA bases keys for binding. Furthermore, we validated those findings against experimental genetic data. Our results are the first to propose in silico modeling for GATA:DNA bound complexes that could be used to score effects of missense mutations in other classes of transcription factors involved in common and genetic diseases.

Selective functional inhibition of a tumor-derived p53 mutant by cytosolic chaperones identified using split-YFP in budding yeast.

Life requires the oligomerization of individual proteins into higher-order assemblies. In order to form functional oligomers, monomers must adopt appropriate 3D structures. Molecular chaperones transiently bind nascent or misfolded proteins to promote proper folding. Single missense mutations frequently cause disease by perturbing folding despite chaperone engagement. A misfolded mutant capable of oligomerizing with wild-type proteins can dominantly poison oligomer function. We previously found evidence that human-disease-linked mutations in Saccharomyces cerevisiae septin proteins slow folding and attract chaperones, resulting in a kinetic delay in oligomerization that prevents the mutant from interfering with wild-type function. Here, we build upon our septin studies to develop a new approach for identifying chaperone interactions in living cells, and use it to expand our understanding of chaperone involvement, kinetic folding delays, and oligomerization in the recessive behavior of tumor-derived mutants of the tumor suppressor p53. We find evidence of increased binding of several cytosolic chaperones to a recessive, misfolding-prone mutant, p53(V272M). Similar to our septin results, chaperone overexpression inhibits the function of p53(V272M) with minimal effect on the wild type. Unlike mutant septins, p53(V272M) is not kinetically delayed under conditions in which it is functional. Instead, it interacts with wild-type p53 but this interaction is temperature sensitive. At high temperatures or upon chaperone overexpression, p53(V272M) is excluded from the nucleus and cannot function or perturb wild-type function. Hsp90 inhibition liberates mutant p53 to enter the nucleus. These findings provide new insights into the effects of missense mutations.

MO035COMPUTATIONAL MODELING APPROACH FOR THE COMPREHENSIVE INTERPRETATION OF RARE TUBULOPATHIES

Abstract Background and Aims Kidney plays a central role on the maintenance of water homeostasis, acid-base and electrolytes balances through the activity of different types of ion channels/transporters expressed along the nephron segments. Mutations in genes encoding these transporters could subsequently lead to aberrant transporter activities, resulting in abnormal renal handling of electrolytes, thus represent monogenic form of rare kidney diseases. Accumulating number of mutations identified in genes responsible for such monogenic disorders demonstrated that eventual disease phenotypes may vary according to the type and localization of the mutation within the gene. Thus, careful evaluation of gene variation would be crucial prior to designing the strategy for the therapy in each case. Here we present various mutations from our patients, identified in genes including kcnj10, SLC12A1, SLC26A4 and clcn7 which are associated with rare tubulopathies EAST/SeSAME syndrome, Bartter’s syndrome, Pendred syndrome and Fanconi syndrome, respectively. In order to explore molecular mechanisms underlying the observed disease conditions in our patients, we have performed computational modeling analyses of these mutations in comparison with wild-type models. Method Three-dimensional homology models of Kir4.1, NKCC2, Pendrin and CLC-7 proteins were generated by Swiss-Model protein structure homology-modelling server (http://swissmodel. expasy.org) and I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/). Disease-related mutations including novel mutations identified from our patients were mapped onto the three-dimensional models and compared with wild-type models in terms of atomic interactions as well as secondary, tertiary and quaternary structures. Furthermore, we assessed possible effects of missense mutations on the function of ion channels/transporters using online bioinformatic prediction tools PolyPhen-2, Mutation taster, PROVEAN and SIFT. Results The three-dimensional model comparison between wild-type Kir4.1 and Ala167Val variant, which is related to EAST/SeSAME syndrome, revealed that Ala167Val located at the junction between transmembrane domain 2 (TM2) and C-terminus is predicted not to interrupt the sequence of the hydrogen bonds, thus not altering the TM2 alpha-helix structure. In addition, while PolyPhen-2 and Mutation taster evaluated Ala167Val as ‘probably damaging’, PROVEAN and SIFT predicted Ala167Val as ‘neutral’ and ‘tolerated’. These observations are in line with the clinical data demonstrating the milder phenotype in patients with Ala167Val mutation compared with the ones harboring frameshift mutations leading to truncated Kir4.1 channel (Figure. 1). Furthermore, computational modeling of wild-type NKCC2 and frameshift mutation Arg302Glyfs*3 variant clearly demonstrated that Arg302Glyfs*3 results in a loss of large part of the protein, indicating that NKCC2-Arg302Glyfs*3 is practically nonfunctional (Figure. 2). Conclusion Computational modeling of disease-related mutations in various ion channels/transporters represents a novel, powerful approach for comprehensive interpretation of the disease phenotypes observed in patients with rare tubulopathies. In addition, combination of in silico modeling and clinical data could provide us with further insight into molecular mechanisms underlying the renal transporter activities. Furthermore, this in silico computational modeling approach can be applicable and suggestive for novel pharmacological intervention as well as the visual disease severity assessment.

In silico analysis of nsSNPs in CYP19A1 gene affecting breast cancer associated aromatase enzyme

The human aromatase protein encoded by CYP19A1 gene is the principle enzyme involved in the biogenesis of oestrogen in adipose tissues. An excessive exposure to endogenous oestrogen is regarded as an important determinant in the risk of breast cancer. Thus, in the present study we have used multiple computational methods to identify the most deleterious nonsynonymous SNPs in CYP19A1 gene that caused probable genotypic-phenotypic alterations susceptible to breast cancer malignancy. In this study, a total of 338 nsSNPs were screened using 12 in silico tools including SIFT, PROVEAN, PolyPhene-2, SNAP2, I Mutant 3.0, MuPro, mCSM, PhD SNP, SNP&GO, P-Mut, Dr Cancer, and, CScape. Additionally the structural and functional consequences of missense mutations were validated using Consurf, ModPred, SOPMA, and, HOPE server tools. Of the 338 nsSNPs subjected to functional, protein stability, disease associated, and, cancer susceptible analysis, 14 variants were predicted to be highly deleterious mutants. Further, structural and molecular studies suggested 10 variants (R435H, Y77C, Y81C, E302K, E210K; and L451P, G49D, G131D, L204W and D309) to have various deformities and caused structural disturbances of the protein. Through the combination of multiple computational tools and strategized analysis, we report seven novel high risk nsSNPs of human aromatase enzyme in association with the pathogenesis of human breast cancer.

Prediction of Functional Consequences of Missense Mutations in ANO4 Gene.

The anoctamin (TMEM16) family of transmembrane protein consists of ten members in vertebrates, which act as Ca2+-dependent ion channels and/or Ca2+-dependent scramblases. ANO4 which is primarily expressed in the CNS and certain endocrine glands, has been associated with various neuronal disorders. Therefore, we focused our study on prioritizing missense mutations that are assumed to alter the structure and stability of ANO4 protein. We employed a wide array of evolution and structure based in silico prediction methods to identify potentially deleterious missense mutations in the ANO4 gene. Identified pathogenic mutations were then mapped to the modeled human ANO4 structure and the effects of missense mutations were studied on the atomic level using molecular dynamics simulations. Our data show that the G80A and A500T mutations significantly alter the stability of the mutant proteins, thus providing new perspective on the role of missense mutations in ANO4 gene. Results obtained in this study may help to identify disease associated mutations which affect ANO4 protein structure and function and might facilitate future functional characterization of ANO4.

Conserved missense variant in ALDH1A3 ortholog impairs fecundity in C. elegans.

Accumulating evidence demonstrates that mutations in ALDH1A3 (the aldehyde dehydrogenase 1 family, member A3) are associated with developmental defects. The ALDH1A3 enzyme catalyzes retinoic acid biosynthesis and is essential to patterning and neuronal differentiation in the development of embryonic nervous system. Several missense mutations in ALDH1A3 have been identified in family studies of autosomal recessive microphthalmia, autism spectrum disorder, and other neurological disorders. However, there has been no evidence from animal models that verify the functional consequence of missense mutations in ALDH1A3. Here, we introduced the equivalent of the ALDH1A3 C174Y variant into the Caenorhabditis elegans ortholog, alh-1, at the corresponding locus. Mutant animals with this missense mutation exhibited decreased fecundity by 50% compared to wild-type animals, indicating disrupted protein function. To our knowledge, this is the first ALDH1A3 C174Y missense model, which might be used to elucidate the effects of ALDH1A3 C174Y missense mutation in the retinoic acid signaling pathway during development.

Ligand-specific pharmacogenetic effects of nonsynonymous mutations.

In pharmacogenomics, variable receptor phenotypes, resulting from genetic polymorphisms, are often described as a change in protein function or regulation observed upon exposure to a drug. However, in some instances, phenotypes are defined using a class of medications rather than individual drugs. This paradigm assumes that a variation associated with a drug response phenotype will retain the magnitude and direction of the effect for other drugs with the same mechanism of action. However, nonsynonymous polymorphisms may have ligand-specific effects. The purpose of this study was to investigate the potential for point mutations to asymmetrically affect the binding of different drugs to a common target. Ligand binding data from site-directed mutagenesis studies on five G-protein coupled receptors (beta-1 and -2 adrenergic, dopamine D2, angiotensin II and mu-opioid receptor) were collected and analyzed. Binding data from 81 studies for 253 ligands with 447 mutant proteins, including 10 naturally occurring human variants, were analyzed, yielding 1989 mutation-ligand pairs. Fold change in binding affinity for mutant proteins, relative to the wild-type, for different drugs was examined for ligand-specific effects, with a fold-change difference of one or more orders of magnitude between agents considered significant. Of the mutations examined, 49% were associated with ligand-specific effects. One human variant (T164I, beta-2 adrenergic receptor) showed ligand-specific effects for antiasthmatic agents. These results indicate that ligand-specific changes in binding are a possible consequence of missense mutations. This implies that caution needs to be exercised when grouping drugs together during design or interpretation of genotype-phenotype association studies.

Abstract 2160: Immune escape mutations in HLA Class I, B2M and CASP8 genes shape tumor evolution

Abstract The immune system has several mechanisms to recognize tumor cells as foreign and eliminate them. Somatically altered genes can create peptides, or neoantigens, that class I HLA (HLA-I) molecules can present to cytotoxic T-cells. As a result, people with suppressed immune systems commonly have a higher rate of cancer. PanCancer data of 9423 tumor exomes from the Cancer Genome Atlas project identified 299 frequently mutated cancer genes, 4 of which (HLA-A, HLA-B, beta-2-microglobulin (B2M), and caspase-8 (CASP8) are associated with the neoantigen presentation system. By mutating genes essential to immune recognition and cytotoxicity, tumors can escape the immune system. Tumors with a high mutation burden (MB) and thus more neoantigens experience even greater pressure to mutate HLA-I genes to escape detection. Therefore, the immune system shapes tumor evolution. Tumors with a higher mutation burden respond more effectively to immune checkpoint inhibitors (ICI) such as anti-PD-L1 and anti-CTLA-4 drugs. However, there are many PD-L1 positive patients who fail to respond. These patients may have immune escape (IE) mutations inhibiting neoantigen presentation to T-cells, and a database of immune escape mutations would help inform cancer treatment and improve response to immunotherapy. From the TCGA Pan-Cancer dataset we identified samples with mutations in HLA-A, HLA-B, HLA-C and mutations or loss of B2M or CASP8. We also identified samples with microsatellite instability (MSI) and mutations in DNA Polymerase E (POLE), both of which are associated with high mutation burden. We predicted individual HLA genotypes using multiple programs and aligned tumor and normal reads to these alleles, and called mutations. We used the Integrative Genome Viewer, to validate the mutations and used multiple programs to identify potential functional effects of missense mutations. Finally, we used chi-squared tests to find associations between genes and phenotypes. There were substantially more mutations in HLA-A and HLA-B genes than in HLA-C. Loss-of-function (LoF) mutations accounted for over 50% of the mutations in HLA-A and 54% in HLA-B, but are significantly suppressed (25%) in HLA-C (X2=21; P=3 × 10-5). Since all HLA-C alleles are ligands for natural killer (NK) cell receptors (KIR) this indicates that NK cell recognition of tumor cells is important. Over 90% of HLA-I missense variants were predicted to be functional (likely to impact the stability, membrane localization, B2M binding, peptide binding of the protein, the signal peptide or T cell recognition). Cervical, head and neck, stomach, colon and lung cancer have the highest level of IE mutations (9-20%). Immune escape mutations were significantly higher in MSI (45%) and POLE mutated (72%) tumors as compared to MSI- and POLE wild type tumors (3.9%, X2=470 P&amp;lt;&amp;lt;0.0001). An increased knowledge of these IE mutations can be useful in identifying tumor or patient subsets that may respond better to ICI therapies. Citation Format: Megan Ren, Mathias Viard, Arman Bashirova, Meredith Yeager, Lisa Mirabello, Mary Carrington, Michael Dean. Immune escape mutations in HLA Class I, B2M and CASP8 genes shape tumor evolution [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2160.

Computational analysis of hereditary spastic paraplegia mutations in the kinesin motor domains of KIF1A and KIF5A

Hereditary spastic paraplegias (HSPs) are a genetically heterogeneous collection of neurodegenerative disorders categorized by progressive lower-limb spasticity and frailty. The complex HSP forms are characterized by various neurological features including progressive spastic weakness, urinary sphincter dysfunction, extra pyramidal signs and intellectual disability (ID). The kinesin superfamily proteins (KIFs) are microtubule-dependent molecular motors involved in intracellular transport. Kinesins directionally transport membrane vesicles, protein complexes, and mRNAs along neurites, thus playing important roles in neuronal development and function. Recent genetic studies have identified kinesin mutations in patients with HSPs. In this study, we used the computational approaches to investigate the 40 missense mutations associated with HSP and ID in KIF1A and KIF5A. We performed homology modeling to construct the structures of kinesin–microtubule binding domain and kinesin–tubulin complex. We applied structure-based energy calculation methods to determine the effects of missense mutations on protein stability and protein–protein interaction. The results revealed that the most of disease-causing mutations could change the folding free energy of kinesin motor domain and the binding free energy of kinesin–tubulin complex. We found that E253K associated with ID in KIF1A decrease the protein stability of kinesin motor domains. We showed that the HSP mutations located in kinesin–tubulin complex interface, such as K253N and R280C in KIF5A, can destabilize the kinesin–tubulin complex. The computational analysis provides useful information for understanding the roles of kinesin mutations in the development of ID and HSPs.

Evaluation of cancer related missense mutations in CENPH

Objective: CENPH, centromere protein H, is one of the constitutive kinetochore proteins. High expression of CENPH has been shown in various forms of cancers; however, studies searching the effect of CENPH mutations in cancers are limited. Therefore, the aim of this study is to investigate the potential effects of the missense mutations of CENPH that have been identified in different cancers.&#x0D; Materials and Methods: Missense CENPH mutations, which have been observed in cancers, were downloaded from the COSMIC v89. The effect of missense mutations was predicted by using PredictSNP1.0. The protein structure of the CENPH protein was generated with I-TASSER and missense mutations were visualized on CENPH protein with UCSF Chimera. Structural effects of selected mutations were assessed with HOPE.&#x0D; Results: 34 missense mutations were observed in human cancers. Of the 34 missense mutations 18 mutations were predicted as deleterious and 16 mutations were predicted as neutral with ranging expected accuracies. Predicted missense mutations showed a scattered pattern on 3D CENPH protein. Two of the predicted deleterious missense mutations with higher expected accuracy were further analyzed and assessed according to amino acid properties.&#x0D; Conclusion: This study provided a systematic analysis and evaluation of missense mutations on a CENPH protein that have been observed in different cancers.

PB2401 GENETICS OF IRANIAN ALPHA THALASSEMIA PATIENTS; COMPREHENSIVE ORIGINAL STUDY

Please indicate where the abstract has been published before: The full paper was published in Biochemical genetics journalBackground:Alpha thalassemia is the most prevalent monogenic gene disorder in the world and especially in Mediterranean countries. In the current hematological phenotype of patients with different genotypes, and also the effect of missense mutations on the protein function and stability were evaluated in a large cohort study.Aims:To evaluate the genetics of large populations of Iranian alpha thalassemia patientsMethods:One thousand five hundred and sixty subjects were participated in the study and divided into two groups; 259 normal subjects and 1301 alpha thalassemia carriers. Genomic DNA was extracted and analyzed using ARMS PCR, Multiplex Gap, and direct sequencing. The effects of single nucleotide change on the protein function and stability were predicted by free available databases of human polymorphisms.Results:Sixty‐three different genotypes were seen in the patients. The more prevalent was heterozygote form of ‐α3.7 (41.4%) followed by ‐α3.7 homozygote(11.6%) and ‐ ‐MED(3.8%). The significant differences were seen in mean hemoglobin level [F = 20.5, p &lt; 0.001] between the Alpha globin genotypes when adjusted for gender. Moreover, 28 different mutations were found in our study. A significant relationship was seen between ethnicity and the alpha globin mutation frequency X2 (df;8) = 38.36, p &lt;0.0001).Summary/Conclusion:Different genotypes could be displayed as different phenotypes. The mutation frequency distributions in our region are different from other parts of Iran. Significant differences are seen in the spectrum of mutation frequency between various ethnicity. Finally, some missense mutations maybe did not considerably effect on the proteins and could be a neutral mutation.image

Effects of genetic variants in the TSPO gene on protein structure and stability.

The 18 kDa translocator protein (TSPO) is an evolutionary conserved cholesterol binding protein localized in the outer mitochondrial membrane. Expression of TSPO is upregulated in activated microglia in various neuroinflammatory, neurodegenerative, and neoplastic disorders. Therefore, TSPO radioligands are used as biomarkers in positron emission tomography (PET) studies. In particular, a common A147T polymorphism in the TSPO gene affects binding of several high affinity TSPO radioligands. Given the relevance of TSPO as a diagnostic biomarker in disease processes, we systematically searched for mutations in the human TSPO gene by a wide array of evolution and structure based bioinformatics tools and identified potentially deleterious missense mutations. The two most frequently observed missense mutations A147T and R162H were further analysed in structural models of human wildtype and mutant TSPO proteins. The effects of missense mutations were studied on the atomic level using molecular dynamics simulations. To analyse putative effects of A147T and R162H variants on protein stability we established primary dermal fibroblast cultures from wt and homozygous A147T and R162H donors. Stability of endogenous TSPO protein, which is abundantly expressed in fibroblasts, was studied using cycloheximide protein degradation assay. Our data show that the A147T mutation significantly alters the flexibility and stability of the mutant protein. Furthermore both A147T and R162H mutations decreased the half-life of the mutant proteins by about 25 percent, which could in part explain its effect on reduced pregnenolone production and susceptibility to neuropsychiatric disorders. The present study is the first comprehensive bioinformatic analysis of genetic variants in the TSPO gene, thereby extending the knowledge about the clinical relevance of TSPO nsSNPs.

Genetics of Iranian Alpha-Thalassemia Patients: A Comprehensive Original Study.

Alpha thalassemia is the most prevalent monogenic gene disorder in the world, especially in Mediterranean countries. In the current hematological phenotype of patients with different genotypes, the effects of missense mutations on the protein function and also stability were evaluated in a large cohort study. A total of 1,560 subjects were enrolled in the study and divided into two groups: 259 normal subjects; and 1301 alpha-thalassemia carriers. Genomic DNA was extracted and analyzed using ARMS PCR, Multiplex Gap, and direct sequencing. The effects of single nucleotide change on the protein function and stability were predicted by freely available databases of human polymorphisms. Sixty-three different genotypes were seen in the patients. The more prevalent was heterozygote form of -α3.7 (41.4%) followed by -α3.7 homozygote (11.6%) and -MED (3.8%). The significant differences were seen in mean hemoglobin level [F = 20.5, p < 0.001] between the Alpha-globin genotypes, when adjusted for gender. Moreover, 28 different mutations were found in our study. A significant relationship was seen between ethnicity and the alpha-globin mutation frequency χ2 (df;8) = 38.36, p < 0.0001). Different genotypes could display as different phenotypes. The mutation frequency distributions in our region are different from those of other parts of Iran. Significant differences are seen in the spectrum of mutation frequency among various ethnicities. Finally, some missense mutations might not have considerable effect on the proteins, and they could be neutral mutations.

Performance of in silico prediction tools for the classification of rare BRCA1/2 missense variants in clinical diagnostics

BackgroundThe use of next-generation sequencing approaches in clinical diagnostics has led to a tremendous increase in data and a vast number of variants of uncertain significance that require interpretation. Therefore, prediction of the effects of missense mutations using in silico tools has become a frequently used approach. Aim of this study was to assess the reliability of in silico prediction as a basis for clinical decision making in the context of hereditary breast and/or ovarian cancer.MethodsWe tested the performance of four prediction tools (Align-GVGD, SIFT, PolyPhen-2, MutationTaster2) using a set of 236 BRCA1/2 missense variants that had previously been classified by expert committees. However, a major pitfall in the creation of a reliable evaluation set for our purpose is the generally accepted classification of BRCA1/2 missense variants using the multifactorial likelihood model, which is partially based on Align-GVGD results. To overcome this drawback we identified 161 variants whose classification is independent of any previous in silico prediction. In addition to the performance as stand-alone tools we examined the sensitivity, specificity, accuracy and Matthews correlation coefficient (MCC) of combined approaches.ResultsPolyPhen-2 achieved the lowest sensitivity (0.67), specificity (0.67), accuracy (0.67) and MCC (0.39). Align-GVGD achieved the highest values of specificity (0.92), accuracy (0.92) and MCC (0.73), but was outperformed regarding its sensitivity (0.90) by SIFT (1.00) and MutationTaster2 (1.00). All tools suffered from poor specificities, resulting in an unacceptable proportion of false positive results in a clinical setting. This shortcoming could not be bypassed by combination of these tools. In the best case scenario, 138 families would be affected by the misclassification of neutral variants within the cohort of patients of the German Consortium for Hereditary Breast and Ovarian Cancer.ConclusionWe show that due to low specificities state-of-the-art in silico prediction tools are not suitable to predict pathogenicity of variants of uncertain significance in BRCA1/2. Thus, clinical consequences should never be based solely on in silico forecasts. However, our data suggests that SIFT and MutationTaster2 could be suitable to predict benignity, as both tools did not result in false negative predictions in our analysis.

Pharmacogenomics of cytochrome P450: Effects of missense mutations on drug metabolism

Pharmacogenomics of cytochrome P450: Effects of missense mutations on drug metabolism