Impact Of Missense Mutations Research Articles

Impact of Missense Mutations on Spike Protein Stability and Binding Affinity in the Omicron Variant.

The global effort to combat the COVID-19 pandemic faces ongoing uncertainty with the emergence of Variants of Concern featuring numerous mutations on the Spike (S) protein. In particular, the Omicron Variant is distinguished by 32 mutations, including 10 within its receptor-binding domain (RBD). These mutations significantly impact viral infectivity and the efficacy of vaccines and antibodies currently in use for therapeutic purposes. In our study, we employed structure-based computational saturation mutagenesis approaches to predict the effects of Omicron missense mutations on RBD stability and binding affinity, comparing them to the original Wuhan-Hu-1 strain. Our results predict that mutations such as G431W and P507W induce the most substantial destabilizations in the Wuhan-Hu-1-S/Omicron-S RBD. Notably, we postulate that mutations in the Omicron-S exhibit a higher percentage of enhancing binding affinity compared to Wuhan-S. We found that the mutations at residue positions G447, Y449, F456, F486, and S496 led to significant changes in binding affinity. In summary, our findings may shed light on the widespread prevalence of Omicron mutations in human populations. The Omicron mutations that potentially enhance their affinity for human receptors may facilitate increased viral binding and internalization in infected cells, thereby enhancing infectivity. This informs the development of new neutralizing antibodies capable of targeting Omicron's immune-evading mutations, potentially aiding in the ongoing battle against the COVID-19 pandemic.

Variant of uncertain significance Arg866Cys enhances disorderedness of h-BRCA1 (759-1064) region

High structural flexibility has been reported in the central region of BRCA1, which hinders the structural and functional evaluations of mutations identified in the domain. Additionally, the need to categorize variants of unknown significance (VUS) has increased due to the growth in the number of variants reported in clinical settings. Therefore, unraveling the disease-causing mechanism of VUS identified in different functional domains of BRCA1 is still challenging. The current study uses a multidisciplinary approach to assess the structural impact of BRCA1 Arg866Cys mutation discovered in the central domain of BRCA1. The structural alterations have been characterized using Circular-Dichroism spectroscopy, nano-DSF, and molecular-dynamics simulations. BRCA1 Arg866Cys mutant demonstrated more flexibility and lesser affinity to DNA than the wild-type protein. The BRCA1(759–1064) wild-type protein was shown to be a βII-rich protein with an induced D-O transition in the presence of DNA and 2,2,2-Trifluoroethanol (TFE). The protein's alpha-helical composition did not significantly change in the presence of TFE, besides an increase in β-turns and loops. Under Transmission Electron Microscopes (TEM), amyloid-like fibrils structure was detected for Arg866Cys mutant whereas the wild-type protein showed amorphous aggregates. An increased ThT fluorescence indicated β-rich composition and aggregation-prone behaviour for BRCA1 wild-type protein, while the fluorescence intensity was significantly quenched in the Arg866Cys mutant. Furthermore, increased conformational flexibility in the Arg866Cys variant was observed by principal component analysis. This work aims to comprehend the inherently disordered region of BRCA1 as well as the impact of missense mutations on folding patterns and binding to DNA for functional aspects.

In Silico Characterization of RNASEH2A Pathogenic Variants and Identification of Novel Splice Site Donor Variant c.549+1G>T in Indian Population.

Background Aicardi-Goutieres syndrome (AGS) is a genetic disorder that has variable manifestations including neurological, immunological, and sometimes other system involvement in various combinations. Considering the high genetic and clinical diversity of AGS and the importance of RNASEH2 complex in the biological system, it is important to take a systematic approach to delineate the genetic diagnosis and impact of missense mutations. Methods Clinical targeted gene sequencing followed by Sanger validation was performed in an individual with the clinical features of AGS. Protein modeling studies of all the reported RNASEH2A missense variants till date were performed using freely available web servers BioGrid, ShinyGO. Protein structures were visualized using Pymol. Results and discussion We identified a novel homozygous splice site donor variant c.549+1G>T in RNASEH2A. Furthermore protein-interactome studies identifiedpotential genetic interactors that include RNASEH2A, RNASEH2B, TYMS, RNASEH2C, RPA1, ORC3, ORC2, CDC6, PCNA, LIG1, PRIM1, RFC2, DUT, GINS1, MCM7, FEN1, MCM4, GINS2, CDK4, and MCM5. Identified genes were mapped to specific pathways using SHINY GO. DNA replication and cell cycle, centrosome cycle, post-replication repair, nucleic acid and metabolic process, cellular response to stress, DNA metabolic process, nucleic acid phosphodiester bond hydrolysis, RNA phosphodiester bond hydrolysis, and DNA biosynthetic process were identified as the linked pathways with the prioritized genes. Conclusion In conclusion, a sophisticated genotype and phenotype correlation followed by linking the genes to the key biological pathways opens new avenues to understand disease pathology and plan for therapeutic interventions.

MO1010CLINICAL VARIABILITY OF ALPORT SYNDROME IN CHILDREN WITH MISSENSE COL4A5 VARIANTS

Abstract Background and Aims The phenotype-genotype relation is well established in patients (especially male) with X-linked Alport syndrome (XLAS). The aim of study was to define the spectrum of COL4A5 pathogenic variants and impact of missense mutation on disease progression. Method The NGS-based genetic testing of COL4A3, COL4A4, COL4A5 was performed in 186 children with suspected Alport syndrome. The comparison of clinical symptoms (frequency, age of presentation of macrohematuria, proteinuria, eGFR decrease, sensorineural deafness (SND), level of proteinuria (mg/m2/day) and eGFR (ml/min/1.73m2) at the last presentation) was carried out in male with XLAS due to different COL4A5 variants. Results The XLAS was diagnosed in 93 children (Ме 7,5[4;11], 51М) from 79 families. The following COL4A5 variants were revealed: missense (n=70, q=0.75), splice site (n=10 from 9 families, q=0.11), frame shift (n=6, q=0.06), nonsense (n=7, q=0.08). Missense variants were presented by COL4A5 c.1871G>A, p.(Gly624Asp) in 26% of cases (n=18). The mean age of ESRD was lower in male family member (35[24;35] years vs 48[40;55], p=0.023) with non COL4A5 p.(Gly624Asp). There was no difference in age on last presentation (13[9;14] vs 12[8;15], p=0.091) between pts with COL4A5 p.(Gly624Asp) and other missense variants. There was significant difference between the male with p.Gly624Asp and other missense variants in frequency of proteinuria (0.61 vs 0.25, p=0.043), SND (0.5 vs 0.08, p=0.03), age at onset (5[3;7] vs 14[8;16], p=0.02) and degree of proteinuria (441[78;1158] vs 66[22;160], p=0.023) and eGFR level (83[66;109] vs 96[91;104], p=0.048) at the last examination. Conclusion The XLAS is caused by missense variants in ¾ of cases in our cohort. We confirmed the other European studies results: the COL4A5 p.(Gly624Asp) is the most common variant of missense mutations characterized by mild phenotype of XLAS.

Vanishing white matter: Eukaryotic initiation factor 2B model and the impact of missense mutations.

BackgroundVanishing white matter (VWM) is a leukodystrophy, caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B)‐subunit genes (EIF2B1–EIF2B5); 80% are missense mutations. Clinical severity is highly variable, with a strong, unexplained genotype–phenotype correlation.Materials and MethodsWith information from a recent natural history study, we severity‐graded 97 missense mutations. Using in silico modeling, we created a new human eIF2B model structure, onto which we mapped the missense mutations. Mutated residues were assessed for location in subunits, eIF2B complex, and functional domains, and for information on biochemical activity.ResultsOver 50% of mutations have (ultra‐)severe phenotypic effects. About 60% affect the ε‐subunit, containing the catalytic domain, mostly with (ultra‐)severe effects. About 55% affect subunit cores, with variable clinical severity. About 36% affect subunit interfaces, mostly with severe effects. Very few mutations occur on the external eIf2B surface, perhaps because they have minor functional effects and are tolerated. One external surface mutation affects eIF2B‐substrate interaction and is associated with ultra‐severe phenotype.ConclusionMutations that lead to (ultra‐)severe disease mostly affect amino acids with pivotal roles in complex formation and function of eIF2B. Therapies for VWM are emerging and reliable mutation‐based phenotype prediction is required for propensity score matching for trials and in the future for individualized therapy decisions.

Impact of missense mutation (rs1990760) at interferon-induced helicase (1) gene on non-segmental vitiligo

ObjectiveTo investigate the role of IFIH1 gene polymorphism C/T (rs1990760) in non-segmental vitiligo and to correlate the results with the available clinical data. Patients and methodsA case-control study was conducted on a total number of 100 subjects and categorized into: 50 patients with non-segmental vitiligo and 50 age and gender matched healthy volunteers. Patients were recruited from the Dermatology Outpatient Clinic, Menoufia University hospitals. Control subjects were selected from the blood bank attendants Menoufia University Hospital. Every patient was subjected to history taking, complete general examination, detailed dermatological examination with determination of site and number of lesions, assessment of severity of vitiligo and assessment of disease activity. Results14 (28%) cases were with generalized vitiligo, 13 (26%) cases had acrofacial vitiligo, 12 (24%) cases were with focal vitiligo and 11 (22%) cases were with universal vitiligo. VASI score ranged from 3 to 50 with mean 16.78 ± 10.545 and VIDA score ranged from −1–4 with mean 2.08 ± 1.576. There was statistically highly significant difference between cases and controls regarding IFIH1 genotypes (P < 0.001), IFIH1 alleles (P < 0.001). TT genotype and T allele were significantly more frequent in non-segmental vitiligo patients when compared to healthy controls. There was statistically significant relationship between IFIH1 genotypes and onset of the disease, mucosal affection and type of non-segmental vitiligo (P = 0.005, 0.001, <0.001 respectively). There was statistically highly significant positive correlation between VASI score and duration of the disease in years (P < 0.001). ConclusionIFIH1 could be considered as a good candidate gene for vitiligo susceptibility.

Deciphering the impact of missense mutations on structure and dynamics of SMAD4 protein involved in pathogenesis of gall bladder cancer

Gall bladder cancer (GBC) is the most common malignancy of biliary tract cancer associated with high mortality rate and poor prognosis due to lack of suitable biomarkers. In this study, we explored the structural and functional effects of different missense mutations occurs in SMAD4 that was associated with the development of GBC. We utilized in silico methods to predict the harmful effects of nonsynonymous missense mutations and monitored the stability of protein. We found that all mutations (D351N, G352E, R361C, R361H, E526Q) associated with SMAD4 were deleterious in nature resulting in the formation of deformed or unstable protein structure. Molecular dynamics simulation studies revealed how these mutations affect protein stability, structure, conformation and function. We observed, different mutants increase the compactness and rigidity of SMAD4 protein, alter secondary structure composition, decrease the surface area and protein-ligand interaction and affect its conformation. Findings of current work indicated that the analyzed mutations might affect the structure of protein and its caliber to interact with other molecules, which probably related to functional impairment of SMAD4 upon D351N, G352E, R361C, R361H, E526Q mutations and their involvement in cancer. Hence, the present study has significance of rational drug design and further increase our understanding of GBC development. Communicated by Ramaswamy H. Sarma

The impact of missense mutation in PIGA associated to paroxysmal nocturnal hemoglobinuria and multiple congenital anomalies-hypotonia-seizures syndrome 2: A computational study

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal blood disorder that manifests with hemolytic anemia, thrombosis, and peripheral blood cytopenias. The disease is caused by the deficiency of two glycosylphosphatidylinositols (GPI)-anchored proteins (CD55 and CD59) in the hemopoietic stem cells. The deficiency of GPI-anchored proteins has been associated with the somatic mutations in phosphatidylinositol glycan class A (PIGA). However, the mutations that do not cause PNH is associated with the multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2). To best of our knowledge, no computational study has been performed to explore at an atomistic level the impact of PIGA missense mutations on the structure and dynamics of the protein. Therefore, we focused our study to provide molecular insights into the changes in protein structural dynamics upon mutation. In the initial step, screening for the most pathogenic mutations from the pool of publicly available mutations was performed. Further, to get a better understanding, pathogenic mutations were mapped to the modeled structure and the resulting protein was subjected to 100 ns molecular dynamics simulation. The residues close to C- and N-terminal regions of the protein were found to exhibit greater flexibility upon mutation. Our study suggests that four mutations are highly effective in altering the structural conformation and stability of the PIGA protein. Among them, mutant G48D was found to alter protein's structural dynamics to the greatest extent, both on a local and a global scale.

Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence

BackgroundNAC transcription factors contain five highly conserved subdomains which are required for protein dimerisation and DNA binding. Few residues within these subdomains have been identified as essential for protein function, and fewer still have been shown to be of biological relevance in planta. Here we use a positive regulator of senescence in wheat, NAM-A1, to test the impact of missense mutations at specific, highly conserved residues of the NAC domain on protein function.ResultsWe identified missense mutations in five highly conserved residues of the NAC domain of NAM-A1 in a tetraploid TILLING population. TILLING lines containing these mutations, alongside synonymous and non-conserved mutation controls, were grown under glasshouse conditions and scored for senescence. Four of the five mutations showed a significant and consistent delay in peduncle senescence but had no consistent effects on flag leaf senescence. All four mutant alleles with the delayed senescence phenotype also lost the ability to interact with the homoeolog NAM-B1 in a yeast two-hybrid assay. Two of these residues were previously shown to be involved in NAC domain function in Arabidopsis, suggesting conservation of residue function between species. Three of these four alleles led to an attenuated cell death response compared to wild-type NAM-A1 when transiently over-expressed in Nicotiana benthamiana. One of these mutations was further tested under field conditions, in which there was a significant and consistent delay in both peduncle and leaf senescence.ConclusionsWe combined field and glasshouse studies of a series of mutant alleles with biochemical analyses to identify four residues of the NAC domain which are required for NAM-A1 function and protein interaction. We show that mutations in these residues lead to a gradient of phenotypes, raising the possibility of developing allelic series of mutations for traits of agronomic importance. We also show that mutations in NAM-A1 more severely impact peduncle senescence, compared to the more commonly studied flag leaf senescence, highlighting this as an area deserving of further study. The results from this integrated approach provide strong evidence that conserved residues within the functional domains of NAC transcription factors have biological significance in planta.

The RUNX Transcriptional Coregulator, CBFβ, Suppresses Migration of ER+ Breast Cancer Cells by Repressing ERα-Mediated Expression of the Migratory Factor TFF1

Core binding factor β (CBFβ), the essential coregulator of RUNX transcription factors, is one of the most frequently mutated genes in estrogen receptor-positive (ER+) breast cancer. Many of these mutations are nonsense mutations and are predicted to result in loss of function, suggesting a tumor suppressor role for CBFβ. However, the impact of missense mutations and the loss of CBFβ in ER+ breast cancer cells have not been determined. Here we demonstrate that missense mutations in CBFβ accumulate near the Runt domain-binding region. These mutations inhibit the ability of CBFβ to form CBFβ-Runx-DNA complexes. We further show that deletion of CBFβ, using CRISPR-Cas9, in ER+ MCF7 cells results in an increase in cell migration. This increase in migration is dependent on the presence of ERα. Analysis of the potential mechanism revealed that the increase in migration is driven by the coregulation of Trefoil factor 1 (TFF1) by CBFβ and ERα. RUNX1-CBFβ acts to repress ERα-activated expression of TFF1. TFF1 is a motogen that stimulates migration and we show that knockdown of TFF1 in CBFβ-/- cells inhibits the migratory phenotype. Our findings reveal a new mechanism by which RUNX1-CBFβ and ERα combine to regulate gene expression and a new role for RUNX1-CBFβ in the prevention of cell migration by suppressing the expression of the motogen TFF1. IMPLICATIONS: Mutations in CBFβ contribute to the development of breast cancer by inducing a metastatic phenotype that is dependent on ER.

Impact of missense mutations in survival motor neuron protein (SMN1) leading to Spinal Muscular Atrophy (SMA): A computational approach.

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by the mutations in survival motor neuron 1 gene (SMN1). The molecular pathology of missense mutations in SMN1 is not thoroughly investigated so far. Therefore, we collected all missense mutations in the SMN1 protein, using all possible search terms, from three databases (PubMed, PMC and Google Scholar). All missense mutations were subjected to in silico pathogenicity, conservation, and stability analysis tools. We used statistical analysis as a QC measure for validating the specificity and accuracy of these tools. PolyPhen-2 demonstrated the highest specificity and accuracy. While PolyPhen-1 showed the highest sensitivity; overall, PolyPhen2 showed better measures in comparison to other in silico tools. Three mutations (D44V, Y272C, and Y277C) were identified as the most pathogenic and destabilizing. Further, we compared the physiochemical properties of the native and the mutant amino acids and observed loss of H-bonds and aromatic stacking upon the cysteine to tyrosine substitution, which led to the loss of aromatic rings and may reduce protein stability. The three mutations were further subjected to Molecular Dynamics Simulation (MDS) analysis using GROMACS to understand the structural changes. The Y272C and Y277C mutants exhibited maximum deviation pattern from the native protein as compared to D44V mutant. Further MDS analysis predicted changes in the stability that may have been contributed due to the loss of hydrogen bonds as observed in intramolecular hydrogen bond analysis and physiochemical analysis. A loss of function/structural impact was found to be severe in the case of Y272C and Y277C mutants in comparison to D44V mutation. Correlating the results from in silico predictions, physiochemical analysis, and MDS, we were able to observe a loss of stability in all the three mutants. This combinatorial approach could serve as a platform for variant interpretation and drug design for spinal muscular dystrophy resulting from missense mutations.

In silico analysis of Glanzmann variants of Calf-1 domain of \u03b1IIb\u03b23 integrin revealed dynamic allosteric effect

Integrin αIIbβ3 mediates platelet aggregation and thrombus formation. In a rare hereditary bleeding disorder, Glanzmann thrombasthenia (GT), αIIbβ3 expression / function are impaired. The impact of deleterious missense mutations on the complex structure remains unclear. Long independent molecular dynamics (MD) simulations were performed for 7 GT variants and reference structure of the Calf-1 domain of αIIb. Simulations were analysed using a structural alphabet to describe local protein conformations. Common and flexible regions as well as deformable zones were observed in all the structures. The most flexible region of Calf-1 (with highest B-factor) is rather a rigid region encompassed into two deformable zones. Each mutated structure barely showed any modifications at the mutation sites while distant conformational changes were observed. These unexpected results question the relationship between molecular dynamics and allostery; and the role of these long-range effects in the impaired αIIbβ3 expression. This method is aimed at studying all αIIbβ3 sub-domains and impact of missense mutations at local and global structural level.

Computational study of missense mutations in phenylalanine hydroxylase.

Hyperphenylalaninemia (HPA) is one of the most common metabolic disorders. HPA, which is transmitted by an autosomal recessive mode of inheritance, is caused by mutations of the phenylalanine hydroxylase gene. Most mutations are missense and lead to reduced protein stability and/or impaired catalytic function. The impact of such mutations varies, ranging from classical phenylketonuria (PKU), mild PKU, to non-PKU HPA phenotypes. Despite the fact that HPA is a monogenic disease, clinical data show that one PKU genotype can be associated with more in vivo phenotypes, which indicates the role of other (still unknown) factors. To better understand the phenotype-genotype relationships, we analyzed computationally the impact of missense mutations in homozygotes stored in the BIOPKU database. A total of 34 selected homozygous genotypes was divided into two main groups according to their phenotypes: (A) genotypes leading to non-PKU HPA or combined phenotype non-PKU HPA/mild PKU and (B) genotypes leading to classical PKU, mild PKU or combined phenotype mild PKU/classical PKU. Combining in silico analysis and molecular dynamics simulations (in total 3μs) we described the structural impact of the mutations, which allowed us to separate 32 out of 34 mutations between groups A and B. Testing the simulation conditions revealed that the outcome of mutant simulations can be modulated by the ionic strength. We also employed programs SNPs3D, Polyphen-2, and SIFT but based on the predictions performed we were not able to discriminate mutations with mild and severe PKU phenotypes.

Abstract 5148: CanDrA: Cancer-specific driver missense mutation annotation with optimized features.

Abstract Driver mutations are somatic mutations that provide growth advantage to tumor cells, while passenger mutations are those that are not functionally associated with oncogenesis. Distinguishing drivers from passengers is challenging because drivers occur much less frequently than passengers, they turn to have low prevalence, and their functions are multifactorial. Missense mutations are excellent candidates for driver identification, as they occur more frequently and are potentially easier to target than other types of mutations. Although several methods have been developed for predicting the functional impact of missense mutations, only a few are specifically designed for identifying driver mutations. As more mutations are being discovered, more accurate predictive models can be developed using machine learning approaches that systematically characterize the commonality and peculiarity of somatic mutations under specific cancer background. Here, we present such a Cancer Driver Annotation (CanDrA) tool that assesses the driver potential of missense somatic mutations based on a set of 95 structural and evolutionary features computed by over 10 functional prediction algorithms from SNVBOX, VEP, ANNOVAR and Mutation Assessor. Through feature optimization and supervised training, CanDrA outperforms existing tools in analysing the glioblastoma multiforme and the ovarian carcinoma data in The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia project. Citation Format: Yong Mao, Han Chen, Han Liang, Funda Meric-Bernstam, Gordon Mills, Ken Chen. CanDrA: Cancer-specific driver missense mutation annotation with optimized features. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5148. doi:10.1158/1538-7445.AM2013-5148

New concepts for CML clonality

BCR-ABL1 compound mutations, defined as ≥2 mutations within the same BCR-ABL1 allele, can confer high-level resistance to imatinib and other ABL1 tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). Even the third generation ABL1 TKI, ponatinib, which is uniformly effective against BCR-ABL1 point mutants, remains vulnerable to certain BCR-ABL1 compound mutants [1, 7]. Therapy escape attributable to compound mutations may become more common with greater clinical use of ponatinib in refractory CML. To determine the frequency of compound mutations among CML patients on ABL1 TKI therapy, we examined cDNA samples (N=47) with clear Sanger direct sequencing evidence of two BCR-ABL1 kinase domain mutations. Using an amplicon cloning and sequencing method, we confirmed that a high proportion of patients (70%; 33/47) with double mutations harbored compound mutations. In contrast to prevailing stepwise models, we found sequential, branching, and parallel routes to compound mutations (Figure ​(Figure1A).1A). The frequency of clones harboring compound mutations with >2 missense mutations was low (10%). At the same time, the likelihood of silent mutations increased disproportionately with the total number of mutations per clone, suggesting a limited tolerance for missense mutations in the BCR-ABL1 kinase domain [4]. Silent mutations are not subject to selection pressure and provide a convenient measure of mutation rate. Their increased frequency in clones with multiple mutations suggests clones that instead acquire additional missense mutations are not competitive, possibly because of impaired kinase function. Figure 1 A. Model depicting two mutations in a compound mutant clone and also in parallel as two independent clones. A subclone of the mutant α clonal pool might acquire mutation β, resulting in co-existence of α and α/β ... In 39% of patients with compound mutations, subclones representing each of the component mutations co-existed with the compound mutations (Figure ​(Figure1A).1A). This observation dictates that at least one of the two component mutations arose independently in single and compound mutant subclones and opens the possibility that the same mutation detected at two time points might be of different clonal origin. Thus, disease recurrence with the ‘same’ mutation after a transient response does not definitively establish a clonal relationship. This might also explain why a patient with a certain mutation may show a different level of sensitivity to the same TKI during the course of treatment (Figure ​(Figure1B).1B). Alternatively, a clone may acquire additional non BCR-ABL1 kinase mutation-based resistance mechanisms. Given the large number of kinase domain mutations identified in patients with resistance, independent acquisition of the very same mutation is puzzling and strongly suggests a tendency for nonrandom acquisition of certain mutations over others. Various DNA repair mechanisms are defective in CML cells [2], but this does not explain the preferential and repetitive acquisition of identical mutations at certain residues. It will be interesting to examine whether the point mutation rate in other regions of the CML genome is elevated and whether there are recurrent mutational hotspots. Such a study would shed light on whether the ABL1 kinase domain is located in a part of the genome that is unusually predisposed to mutation acquisition [6]. Cloning and sequencing of the BCR-ABL1 kinase domain in our study of CML samples also revealed many mutations that were not detected by conventional Sanger direct sequencing [4]. The relatively high frequency of these low-level mutations might be due to DNA errors induced by the BCR-ABL1 oncoprotein in CML cells [3]. Models to explain mutational complexity in cancer have invoked a sequential, orderly hierarchy in which the progression of events can be deduced retrospectively. Our study in CML challenges this simplistic framework [4]. Clinical observations are consistent with acquisition of the second mutation by the pre-existing single mutant clone [5, 8], suggesting that introduction of mutations in BCR-ABL1 kinase domain happens constantly in certain CML patients [9] and that either: 1) not all observed compound mutations predate TKI therapy or 2) the observed compound mutations predate TKI therapy but existing detection methods are not sufficiently sensitive for identifying low-level mutations in pre-therapeutic samples. The latter, if proven, suggests that mutation acquisition is sharply curtailed on TKI therapy. Our study demonstrates that compound mutations are common in patients with two BCR-ABL1 mutations and frequently reflect a highly complex clonal network whose evolution may be limited by the negative impact of missense mutations on kinase function.

Bernoulli mixture models in application to the evaluation of algorithms estimating functionality of missense mutations

Background Whole genome and whole exome sequencing projects yield thousands of missense mutations with unknown functionality. Direct estimation of the sensitivity and specificity of bioinformatic algorithms predicting the impact of missense mutations on protein function requires a ‘gold standard’ or set of mutations with known functionality. In the absence of a gold standard, additional statistical methods are needed to estimate the accuracy of these algorithms. It has been shown informative predictions depend on the algorithm and sequence alignment employed and often algorithms disagree as to which mutations are predicted deleterious or neutral [1].

The impact of missense mutations on human behavior

The impact of missense mutations on human behavior

Predictions on impact of missense mutations on structure function relationship of PAX6 and its alternatively spliced isoform PAX6(5a)

The PAX6 contains two DNA-binding domains, paired domain (PD), homeodomain (HD), and a transactivation domain (TD). Only the crystal structure of PD and the solution structure of HD of PAX6 are known. Mutations in PAX6 show variable penetrance, and expressivity of ocular and neural diseases, but the mechanism is poorly understood. Its alternatively spliced isoform PAX6(5a), is also required in a specific ratio for optimal functions. To understand impact of missense mutations on stability, and conformation of PAX6, whose functional analyses are described in PAX6 allelic variant database, were considered. Representative mutations like PAX6-L46R, -C52R, -V126D, -R128C, -R242T, -P375Q, -Q422R, -V256E, and -S259P from PD, HD, and TD of PAX6 were explored. The secondary structures were analyzed through PSIPRED, and relative solvent accessibilities (RSA) of the mutant and the wild type amino acid residues were compared through SABLE. The change in the contact residues and calculations of energy level were studied through SVMcon, MUpro, and FoldX, respectively. The 3D modeling was performed with the help of MODELLER and models were visualized in Chimera. Predictions suggest mutation induced alterations in local conformation or misfolding in DNA-binding domains of PAX6 and PAX6(5a). The predicted impact of mutations via secondary structure, changes in free energy, stability, conformation, and experimental reports on DNA-binding and transactivation, necessarily provides a strong background to explain structure-function relationship of PAX6 and PAX6(5a). However, because of their predictive nature, these findings need to be validated with other experimental evidences when structure of full length PAX6 is available.

The impact of CFNS-causing EFNB1 mutations on ephrin-B1 function

BackgroundMutations of EFNB1 cause the X-linked malformation syndrome craniofrontonasal syndrome (CFNS). CFNS is characterized by an unusual phenotypic pattern of inheritance, because it affects heterozygous females more severely than hemizygous males. This sex-dependent inheritance has been explained by random X-inactivation in heterozygous females and the consequences of cellular interference of wild type and mutant EFNB1-expressing cell populations. EFNB1 encodes the transmembrane protein ephrin-B1, that forms bi-directional signalling complexes with Eph receptor tyrosine kinases expressed on complementary cells. Here, we studied the effects of patient-derived EFNB1 mutations predicted to give rise to truncated ephrin-B1 protein or to disturb Eph/ephrin-B1 reverse ephrin-B1 signalling. Five mutations are investigated in this work: nonsense mutation c.196C > T/p.R66X, frameshift mutation c.614_615delCT, splice-site mutation c.406 + 2T > C and two missense mutations p.P54L and p.T111I. Both missense mutations are located in the extracellular ephrin domain involved in Eph-ephrin-B1 recognition and higher order complex formation.MethodsNonsense mutation c.196C > T/p.R66X, frameshift mutation c.614_615delCT and splice-site mutation c.406+2T > C were detected in the primary patient fibroblasts by direct sequencing of the DNA and were further analysed by RT-PCR and Western blot analyses.The impact of missense mutations p.P54L and p.T111I on cell behaviour and reverse ephrin-B1 cell signalling was analysed in a cell culture model using NIH 3T3 fibroblasts. These cells were transfected with the constructs generated by in vitro site-directed mutagenesis. Investigation of missense mutations was performed using the Western blot analysis and time-lapse microscopy.Results and DiscussionNonsense mutation c.196C > T/p.R66X and frameshift mutation c.614_615delCT escape nonsense-mediated RNA decay (NMD), splice-site mutation c.406+2T > C results in either retention of intron 2 or activation of a cryptic splice site in exon 2. However, c.614_615delCT and c.406+2T > C mutations were found to be not compatible with production of a soluble ephrin-B1 protein. Protein expression of the p.R66X mutation was predicted unlikely but has not been investigated.Ectopic expression of p.P54L ephrin-B1 resists Eph-receptor mediated cell cluster formation in tissue culture and intracellular ephrin-B1 Tyr324 and Tyr329 phosphorylation. Cells expressing p.T111I protein show similar responses as wild type expressing cells, however, phosphorylation of Tyr324 and Tyr329 is reduced.ConclusionsPathogenic mechanisms in CFNS manifestation include impaired ephrin-B1 signalling combined with cellular interference.