Uncharacterised Mutations Research Articles

Disruptor: Computational identification of oncogenic mutants disrupting protein-protein and protein-DNA interactions

We report an Osprey-based computational protocol to prospectively identify oncogenic mutations that act via disruption of molecular interactions. It is applicable to analyse both protein-protein and protein-DNA interfaces and it is validated on a dataset of clinically relevant mutations. In addition, it is used to predict previously uncharacterised patient mutations in CDK6 and p16 genes, which are experimentally confirmed to impair complex formation.

Operationalising targeted next-generation sequencing for routine diagnosis of drug-resistant TB.

Phenotypic drug susceptibility testing (pDST) for Mycobacterium tuberculosis can take up to 8 weeks, while conventional molecular tests identify a limited set of resistance mutations. Targeted next-generation sequencing (tNGS) offers rapid results for predicting comprehensive drug resistance, and this study sought to explore its operational feasibility within a public health laboratory in Mumbai, India. Pulmonary samples from consenting patients testing Xpert MTB-positive were tested for drug resistance by conventional methods and using tNGS. Laboratory operational and logistical implementation experiences from study team members are shared below. Of the total number of patients tested, 70% (113/161) had no history of previous TB or treatment; however, 88.2% (n = 142) had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB). There was a high concordance between resistance predictions of tNGS and pDST for most drugs, with tNGS more accurately identifying resistance overall. tNGS was integrated and adapted into the laboratory workflow; however, batching samples caused significantly longer result turnaround time, fastest at 24 days. Manual DNA extraction caused inefficiencies; thus protocol optimisations were performed. Technical expertise was required for analysis of uncharacterised mutations and interpretation of report templates. tNGS cost per sample was US$230, while for pDST this was US$119. Implementation of tNGS is feasible in reference laboratories. It can rapidly identify drug resistance and should be considered as a potential alternative to pDST.

Identifying novel oncogenic RET mutations and characterising their sensitivity to RET-specific inhibitors

BackgroundRearranged during transfection (RET) is a well-known proto-oncogene. Multiple RET oncogenic alterations have been identified, including fusions and mutations. Although RET fusions have been reported in multiple cancers, RET mutations...

High-Throughput Mutational Screen Identifies Phenotypically Relevant Categories of Mutations in Fumarate Hydratase

Fumarate Hydratase (FH) is an enzyme of the citric acid (TCA) cycle that is responsible for reversibly catalysing the conversion between fumarate and malate. Deficiency in FH is linked to numerous syndromes in humans, but notably the buildup of the oncometabolite fumarate caused by mutational inactivation is a major cause of clear cell renal carcinoma. We sought to explore the mutational landscape of FH in silico, to predict the functional effects of many detected mutations, and categorise detected but un-characterised mutations in human populations. Through the use of homology modelling and mutational energy predicting tools such as Rosetta and Foldx we can accurately detect mutations and mutational hotspots with high disruptive capability. Furthermore, through performing molecular dynamics simulations we show that hinge regions of the protein can be stabilized or destabilized by mutations, with consequences on the binding affinity of the enzyme for its substrates. We can additionally categorise a large majority of mutations and potential mutations into functional groups, and predict which detected mutations in the human population are likely to be loss-of-function, and therefore predispose patients to clear cell renal carcinoma. We additionally link mutations from various functional groups to metabolomics data, and show that mutations from separate categories (destabilizing, binding-site affecting, and hinge-altering) alter cellular metabolic activity differently.

Using global DNA-methylation and metabolome profiles to classify variants of unknown significance (VUS) in phaeochromocytoma and paraganglioma tumours

Abstract Background Phaeochromocytomas and paragangliomas (PPGLs) are rare but strongly heritable neuroendocrine tumours. Most patients undergo genetic testing and approximately 40% will have a germline causal mutation. In 10% of patients, genetic analysis identifies uncharacterised mutations, or variants of unknown significance (VUSs). Tests to distinguish between benign and pathogenic mutations would aid clinicians' and patients' decision-making. One test could exploit the biology of a subset of PPGLs. Mutations in genes controlling the Tricarboxylic Acid cycle cause a deranged metabolome, accumulation of oncometabolites and global DNA-hypermethylation. Data show that this stable chemical modification will be greatly elevated in pathogenic over benign mutations, and be present alongside a driver metabolome. Methods Clinical characteristics were compiled for 3 patient groups: causal germline mutations, no identified mutation (sporadic) and VUS. Archival Formalin Fixed Paraffin Embedded (FFPE) material was identified locally and also provided by the international ENS@T collaboration. Tissue analysis was optimised on mouse FFPE tissue. Laser microdisection allowed collection of precise tissue areas. DNA and metabolite extraction was optimised before analysis was undertaken by Liquid Chromatography Mass Spectrometry (LC-MS). Results 93 patients were recruited locally. Clinical characteristics were collected for 25 sporadic and 56 germline cases, including 14 VUS cases. Optimisation experiments in mouse tissue show that discrete areas can be extracted by laser microdissection. LC-MS identifies cytosine, 5'-methylcytosine and 5'-hydroxymethylcytosine in 1 μg of DNA at biologically relevant ratios. TCA cycle metabolites were detected using LC-MS: succinate, fumarate and malate were found in the metabolite extract from the same amount of tissue. Conclusions This study utilises DNA hypermethylation and metabolomics for clinical application. It represents a large repository of PPGL VUS samples nationally. Global DNA-methylation levels and metabolite profiles are shown to be quantifiable from existing FFPE patient samples, using reduced amounts of patient material. Legal entity responsible for the study Guy's and St Thomas' NHS Trust, London, UK; King's College London, UK. Funding National Institute of Health Research, King's College London, Isaac Shapera Research Trust, The Bernice Bibby Research Trust. Disclosure All authors have declared no conflicts of interest.

Functional analysis of six uncharacterised mutations in LDLR gene

Background and aimsFamilial hypercholesterolemia (FH) is a primary hyperlipemia. It is an autosomal dominant genetic disorder of lipoproteins metabolism mainly caused by mutations in the low density lipoprotein receptor gene (LDLR). We aimed to investigate the functional impact on the low density lipoprotein receptor (LDLR) activity of six uncharacterised variants located in the coding region of the LDLR gene, namely c.428G > T, c.640T > C, c.1708C > T, c.1736A > T, c.1981C > G and c.2114C > G (NM_000527.4) and to attempt to define their clinical status. MethodsFunctional studies were carried out using site-directed mutagenesis techniques and expression of LDLR protein in vitro. Results were correlated with clinical data and in silico analyses in order to assess the physiopathological role of these variants. ResultsThis work provides functional information about 6 uncharacterised mutations in LDLR. ConclusionsThe six variants studied here appeared to affect the LDLR function in vitro to different degrees, ranging from receptors with normal to slightly reduced activity to receptors exhibiting less than 10% of the wild-type activity. According to these studies and The American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines, two variants could be classified as “Likely Benign” (p.(Ala705Gly) and p.(Leu570Phe)), three variants as “Pathogenic” (p.(Asp579Val), p.(Cys143Phe) and p.(Trp214Arg)) and one variant as “Likely Pathogenic” (p.(Pro661Ala)).

Functional characterisation of naturally occurring mutations in human melanopsin

Melanopsin is a blue light-sensitive opsin photopigment involved in a range of non-image forming behaviours, including circadian photoentrainment and the pupil light response. Many naturally occurring genetic variants exist within the human melanopsin gene (OPN4), yet it remains unclear how these variants affect melanopsin protein function and downstream physiological responses to light. Here, we have used bioinformatic analysis and in vitro expression systems to determine the functional phenotypes of missense human OPN4 variants. From 1242 human OPN4 variants collated in the NCBI Short Genetic Variation database (dbSNP), we identified 96 that lead to non-synonymous amino acid substitutions. These 96 missense mutations were screened using sequence alignment and comparative approaches to select 16 potentially deleterious variants for functional characterisation using calcium imaging of melanopsin-driven light responses in HEK293T cells. We identify several previously uncharacterised OPN4 mutations with altered functional properties, including attenuated or abolished light responses, as well as variants demonstrating abnormal response kinetics. These data provide valuable insight into the structure–function relationships of human melanopsin, including several key functional residues of the melanopsin protein. The identification of melanopsin variants with significantly altered function may serve to detect individuals with disrupted melanopsin-based light perception, and potentially highlight those at increased risk of sleep disturbance, circadian dysfunction, and visual abnormalities.

THU-409 - Functional analysis of previously uncharacterised disease-causing mutations of the bile salt export pump

THU-409 - Functional analysis of previously uncharacterised disease-causing mutations of the bile salt export pump

Atomistic and Reaction-Kinetic Modelling for Engineering of Fatty Acid Synthase

The multienzyme fatty acid synthase (FAS) system represents a metabolic cornerstone in all domains of cellular life and has been the subject of biochemical study for over five decades. Recently there has been an upswing of interest in fatty acid synthesis due to potential applications in the areas of biofuels and petrochemical substitution. Additionally, the FAS family is relevant to enzyme engineering efforts seeking to produce complex natural and / or pharmaceutically relevant products from the polyketide family, due to close structural and mechanistic homology between fatty acid and polyketide synthases.Although the individual reactions that drive fatty acid and polyketide synthesis are well understood on the biochemical level, rational engineering of reaction pathways has proved challenging, in no small part due to the complexity of the FAS reaction network. This complexity stems from the interplay of eight active sites processing a multitude of input substrates and intermediates of various length, shuttled by a monovalent ACP shuttle domain.With the aim of achieving an integrated, physics-based representation of the reaction network, and with the further objective of informing future rational engineering efforts, we constructed a computational model of the reaction network of S. cerevisiae FAS. Fitting against a set of known reaction conditions, and incorporating energetic quantities calculated from precisely targeted atomistic simulations, we were able to define an ensemble of parameter sets compatible with the known characteristics of FAS. Cross-validation against product spectra from previously uncharacterised mutations provided support for the predictive value of the model. Applying the model to an effort to engineer a custom synthesis of a simple polyketide of our choosing, we were able to derive substantial insights into counterintuitive phenomena that arose in our experiments.

Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study

SummaryBackgroundDiagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drug-susceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistance-determining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all first-line and second-line drugs for tuberculosis.MethodsBetween Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identified from the drug-resistance scientific literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance.FindingsWe characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7–93·7) and 98·4% specificity (98·1–98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identified among mutations under selection pressure in non-candidate genes.InterpretationA broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workflows, phasing out phenotypic drug-susceptibility testing while reporting drug resistance early.FundingWellcome Trust, National Institute of Health Research, Medical Research Council, and the European Union.

Cell culture andDrosophilamodel systems define three classes of anaplastic lymphoma kinase mutations in neuroblastoma

SUMMARYNeuroblastoma is a childhood extracranial solid tumour that is associated with a number of genetic changes. Included in these genetic alterations are mutations in the kinase domain of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), which have been found in both somatic and familial neuroblastoma. In order to treat patients accordingly requires characterisation of these mutations in terms of their response to ALK tyrosine kinase inhibitors (TKIs). Here, we report the identification and characterisation of two novel neuroblastoma ALK mutations (A1099T and R1464STOP), which we have investigated together with several previously reported but uncharacterised ALK mutations (T1087I, D1091N, T1151M, M1166R, F1174I and A1234T). In order to understand the potential role of these ALK mutations in neuroblastoma progression, we have employed cell culture-based systems together with the model organism Drosophila as a readout for ligand-independent activity. Mutation of ALK at position 1174 (F1174I) generates a gain-of-function receptor capable of activating intracellular targets such as ERK (extracellular signal regulated kinase) and STAT3 (signal transducer and activator of transcription 3) in a ligand-independent manner. Analysis of these previously uncharacterised ALK mutants and comparison with ALKF1174 mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These classes are: (i) gain-of-function ligand-independent mutations such as ALKF1174l, (ii) kinase-dead ALK mutants, e.g. ALKI1250T (Schönherr et al., 2011a) and (iii) ALK mutations that are ligand-dependent in nature. Irrespective of the nature of the observed ALK mutants, in every case the activity of the mutant ALK receptors could be abrogated by the ALK inhibitor crizotinib (Xalkori/PF-02341066), albeit with differing levels of sensitivity.

Use of GFP fusions for the isolation of Escherichia coli strains for improved production of different target recombinant proteins

High level expression of a recombinant gene results in growth arrest, followed by overgrowth by non-productive derivatives. Two methods are described for the isolation of E. coli BL21* strains that are improved hosts for recombinant protein production. Both are based upon the observations (i) that fluorescence of a C-terminal GFP tag is a reliable reporter of the production and correct folding of the N-terminal target domain; and (ii) rare mutants arise spontaneously that remain productive during long periods of high level recombinant protein production. The first method relies upon identifying these mutants amongst colonies on agar plates; the other exploits fluorescence activated cell sorting. Although identical mutations in the regulatory region of the T7 polymerase gene were found in all of the improved host strains isolated, they differed in their ability to accumulate the outer membrane protein, Ccp, or a cytoplasmic protein, CheY-GFP. Cytochrome c peroxidase activity of recombinant Ccp from one of these strains was demonstrated. Changes in levels of T7 polymerase expression are therefore insufficient to ensure increased accumulation of all recombinant proteins. We demonstrate that the methods described allow strains to be isolated that carry other, currently uncharacterised mutations that are required depending on the target protein.

Identification of Genetic Mutations in Australian Malignant Hyperthermia Families Using Sequencing of Ryr1 Hotspots

Advances in analysis of the RYR1 gene (which encodes the skeletal muscle ryanodine receptor) show that genetic examination is a useful adjunct to the in vitro contracture test in the diagnosis of malignant hyperthermia, as defects in RYR1 have been shown to be responsible for malignant hyperthermia susceptibility. DNA from 34 malignant hyperthermia susceptible individuals and four malignant hyperthermia equivocal subjects was examined using direct sequencing of 'hot-spots' in the RYR1 gene to identify mutations associated with malignant hyperthermia. Seven different causative mutations (as defined by the European Malignant Hyperthermia Group) in nine malignant hyperthermia susceptible individuals were identified. In another six malignant hyperthermia susceptible individuals, five different published but as yet functionally uncharacterised mutations were identified. A further three as yet unpublished and functionally uncharacterised (novel) mutations were identified in three malignant hyperthermia susceptible samples. If the novel and previously published mutations prove to be functionally associated with calcium homeostasis, then this method of analysis achieved a mutation detection rate of 47%. Based on the number of relatives presenting to our unit in the study period, the muscle biopsy rate would have decreased by 25%. That we only identified a genetic defect in RYR1 in 47% of in vitro contracture test positive individuals suggests that there are other areas in RYR1 where pathogenic mutations may occur and that RYR1 may not be the sole gene associated with malignant hyperthermia. It may also reflect a less than 100% specificity of the in vitro contracture test.

Diversity of the basic defect of homozygous CFTR mutation genotypes in humans

Background:Knowledge of how CFTR mutations other than F508del translate into the basic defect in cystic fibrosis (CF) is scarce due to the low incidence of homozygous index cases.Methods:17 individuals who...

Characterisation of 6 Factor XI Missense Mutations.

Factor XI deficiency (MIM 264900 ) is an autosomal bleeding disorder of variable clinical severity. In contrast to haemophilia A or B the clinical symptoms do not correlate well with plasma levels of factor XI; it is therefore difficult to predict the bleeding tendency from either the factor level or the molecular defect. Causative mutations, outside of the Ashkenazi Jewish population, are highly heterogeneous, with 79 different mutations currently recorded on the Factor XI Deficiency Mutation Database (http://www.factorxi.com/ ), and this number being added to at a rapid rate. The identification of novel missense mutations leaves the dilemma as to whether these are causative or not. The in vitro expression of novel missense mutations is currently the preferred method for assigning causality.We have utilised site-directed mutagenesis and lipofectamine transfection to express novel factor XI missense mutations in a Baby Hamster Kidney cell line (BHK 570). Three novel /previously uncharacterised missense mutations, Met-18Ile, Met102Thr and Thr575Met, and a change previously reported as a polymorphism, Arg378Cys, were expressed in vitro. Two further missense mutations, on which expression data had been reported, were also expressed as expression negative (Tyr133Ser) and expression positive (Pro520Leu) controls, along with the wild-type FXI cDNA vector. A sandwich ELISA assay failed to detect factor XI antigen in the conditioned media of cell lines expressing the Met-18Ile, Met102Thr or the Tyr133Ser mutant FXI cDNAs but detected good FXI expression from the wild-type FXI cDNA. FXI antigen was detected in the cell lysates of all three cell lines, at similar levels to the wild-type cell line, demonstrating that factor XI was being synthesised in but not being secreted by these cell lines. Preliminary data for the Thr575Met, Arg378Cys and Pro520Leu cell lines shows that a significant amount of FXI antigen is being secreted into the conditioned media, with levels broadly similar for all three cell lines. However, levels in the wild-type cell line are approximately 6-fold higher. Further work is on-going to see whether this is a true reflection of secretion efficiency or the effect of differences in cell growth, plasmid incorporation etc. Levels of FXI antigen in cell lysates from these cell lines were broadly similar. Functional studies on these proteins is on-going. The Arg378Cys ‘mutation', although previously reported as a polymorphism, was detected in a factor XI deficient father and daughter, with no other genetic variation detectable. Secretion of the Arg378Cys protein was surprising as the creation of a free Cysteine residue was expected to cause structural disruption resulting in a CRM- phenotype, as seen in the Arg308Cys and Trp501Cys mutants. We await the results of the function studies with interest.The Met-18Ile mutation was of particular interest as the mutation results in the loss of the initiator Methionine. Western blot analysis failed to detect any size difference between factor XI antigen recovered from cell lysates from the Met-18Ile and wild-type cell lines. We intend to purify the M-18I FXI protein and subject it to tandem MS analysis to try and determine the amnio acid sequence of this mutant protein.

Gaucher disease in Romanian patients: incidence of the most common mutations and phenotypic manifestations.

Gaucher disease (GD) is an inherited glycolipid storage disorder resulting from the deficiency of glucocerebrosidase. It is the most frequent lysosomal storage disease in Romania, accounting for 70% of all lysosomal disorders diagnosed since 1997 in this country. The prevalence of six common mutations (N370S, L444P, R463C, 84GG, recNciI and recTL) and their phenotypic impact were studied in 20 type 1 GD patients of non-Jewish origin. Mutation analysis identified 77.8% of the GD alleles. The N370S mutation had the highest prevalence (50%), followed by the L444P (22.2%) and the recNciI (5.6%) alleles. Mutations R463C, 84GG and recTL have not been found in our patients. Rare or novel mutations likely accounted for 22.2% of the disease-producing uncharacterised alleles. Our study indicates a high prevalence of type 1 among Romanian GD patients. Clinical phenotype and disease severity were evaluated according to the standardised severity score index. Genotype-phenotype correlations were similar to those reported for other Caucasian non-Jewish populations. The absence of neuronopathic disease in patients presenting at least one copy of the N370S allele was confirmed, but the relative mildness of N370S homozygotes was not a constant feature among our patients. The presence of the L444P or of uncharacterised sporadic mutations was always associated with severe clinical manifestations, even in compound heterozygotes with the N370S allele. A large degree of phenotypic variability was observed in patients displaying the same genotype. The particularities of genotype-phenotype correlations may suggest the impact of other genetic or non-genetic factors on the clinical picture.

HLA Genotype of Patients with Severe Haemophilia A due to Intron 22 Inversion with and without Inhibitors of Factor VIII

Molecular genetic studies have shown that development of antibodies to factor VIII (inhibitors) occurs most frequently in patients with severe haemophilia due to major gene lesions including inversions, stop codons and large deletions. Previous studies of HLA type were performed on inhibitor and non-inhibitor patients with diverse uncharacterized mutations which may have confounded detection of significant associations. We therefore selected a group of patients with a single mutation type, the prevalent intron 22 inversion, with or without inhibitors, to determine HLA genotype. Seventy-one such patients, 42 without and 29 with inhibitors (13 high, 9 low and 7 transient responders) were genotyped for MHC Class I HLA-A, -B, -C and Class II HLA-DQA, -DQB and -DRB loci. No strong correlation of any HLA-allele to inhibitor or non-inhibitor status was found. However, alleles of the haplotype HLA-A3, HLA-B7, HLA-C7, HLA-DQA0102, HLA-DQB0602, HLA-DR15 occurred more often in inhibitor patients. Since the alleles of this extended haplotype are common in the North European population only a very strong association would achieve statistical significance. Further studies of groups of patients similar to those studied here will be needed to confirm or exclude this association.

Genotype analysis of adult cystic fibrosis patients.

To assess the relationship between the genotype and phenotype of adult CF patients we have selected from a group of 512 CF patients attending centres in France, all these of greater than 35 years. We have analysed the entire coding sequence of their CFTR genes. The complete genotype was determined in 7 of the 8 patients and clinical data regarding pancreatic, respiratory and reproductive function were carefully evaluated. All these patients are compound heterozygote, seven carrying the delta F508 and one the G542X on one allele. The other allele carried is: (i) a missense mutation located in exons coding for transmembrane region in five patients [R334W (1); I336K (2); R117H (1); H1054D (1)]; (ii) a splice mutation in two patients [2789 + 5G-->A], (iii) an uncharacterised mutations in one patient. These results strongly suggest less severe CF phenotype to be associated with these mutations and strengthen the hypothesis that less severe phenotype are genetically determined.

The cystic fibrosis ΔF508 mutation in the French population

French families (n = 129) with at least one cystic fibrosis (CF) affected child and 44 unrelated subjects from the general population were tested for the presence of the delta F508 mutation by the polymerase chain reaction. The delta F508/CF mutation ratio (CF: uncharacterised CF mutations) was tested in the CF families with and without meconium ileus. The association between delta F508 and CF mutations and restriction fragment length polymorphism haplotypes (XV2c and KM19) has been estimated; these data suggest that the CF chromosomes include a panel of independent and probably different mutations.