Prediction of phenotypes of missense mutations in human proteins from biological assemblies

Single nucleotide polymorphisms (SNPs) are the most frequent variation in the human genome. Nonsynonymous SNPs that lead to missense mutations can be neutral or deleterious, and several computational methods have been presented that predict the phenotype of human missense mutations. These methods use sequence‐based and structure‐based features in various combinations, relying on different statistical distributions of these features for deleterious and neutral mutations. One structure‐based feature that has not been studied significantly is the accessible surface area within biologically relevant oligomeric assemblies. These assemblies are different from the crystallographic asymmetric unit for more than half of X‐ray crystal structures. We find that mutations in the core of proteins or in the interfaces in biological assemblies are significantly more likely to be disease‐associated than those on the surface of the biological assemblies. For structures with more than one protein in the biological assembly (whether the same sequence or different), we find the accessible surface area from biological assemblies provides a statistically significant improvement in prediction over the accessible surface area of monomers from protein crystal structures (P = 6e‐5). When adding this information to sequence‐based features such as the difference between wildtype and mutant position‐specific profile scores, the improvement from biological assemblies is statistically significant but much smaller (P = 0.018). Combining this information with sequence‐based features in a support vector machine leads to 82% accuracy on a balanced dataset of 50% disease‐associated mutations from SwissVar and 50% neutral mutations from human/primate sequence differences in orthologous proteins. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Mutator phenotypes of common polymorphisms and missense mutations in MSH2.

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with germline mutations in the DNA mismatch repair gene hMSH2 [1], the human homologue of the Escherichia coli MutS gene. These are mostly nonsense, frameshift or deletion mutations that result in loss of intact protein and complete inactivation of DNA mismatch repair. However, cancer is also associated with hMSH2 missense mutations that are merely inferred to be deleterious because they result in non-conservative substitutions of amino acids that are highly conserved among MutS family proteins. Moreover, sequence polymorphisms exist in hMSH2 that also change conserved amino acids but whose functional consequences and relationship to cancer are uncertain. Here, we show that yeast strains harboring putative equivalents of three hMSH2 polymorphisms have elevated mutation rates. Mutator effects were also observed for yeast equivalents of hMSH2 missense mutations found in HNPCC families and in an early onset colon tumor. Several distinct phenotypes were observed, indicating that these missense mutations have differential effects on MSH2 function(s). The results suggest that cancer may be associated with even partial loss of hMSH2 function and they are consistent with the hypothesis that polymorphisms in hMSH2 might predispose humans to disease.     

LIS1 missense mutations: variable phenotypes result from unpredictable alterations in biochemical and cellular properties

Mutations in one allele of the human LIS1 gene cause a severe brain malformation, lissencephaly. Although most LIS1 mutations involve deletions, several point mutations with a single amino acid alteration were described. Patients carrying these mutations reveal variable phenotypic manifestations. We have analyzed the functional importance of these point mutations by examining protein stability, folding, intracellular localization, and protein-protein interactions. Our data suggest that the mutated proteins were affected at different levels, and no single assay could be used to predict the lissencephaly phenotype. Most interesting are those mutant proteins that retain partial folding and interactions. In the case of LIS1 mutated in F31S, the cellular phenotype may be modified by overexpression of specific interacting proteins. Overexpression of the PAF-AH alpha1 subunit dissolved aggregates induced by this mutant protein and increased its half-life. Overexpression of NudE or NudEL localized this mutant protein to spindle poles and kinetochores but had no effect on protein stability. Our results implicate that there are probably different biochemical and cellular mechanisms obstructed in each patient yielding the varied lissencephaly phenotypes.     

Distinct FTDP-17 missense mutations in tau produce tau aggregates and other pathological phenotypes in transfected CHO cells

Multiple tau gene mutations are pathogenic for hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), with filamentous tau aggregates as the major lesions in the CNS of these patients. Recent studies have shown that bacterially expressed recombinant tau proteins with FTDP-17 missense mutations cause functional impairments, i.e., a reduced ability of mutant tau to bind to or promote the assembly of microtubules. To investigate the biological consequences of FTDP-17 tau mutants and assess their ability to form filamentous aggregates, we engineered Chinese hamster ovary cell lines to stably express tau harboring one or several different FTDP-17 mutations and showed that different tau mutants produced distinct pathological phenotypes. For example, delta K, but not several other single tau mutants (e.g., V337 M, P301L, R406W), developed insoluble amorphous and fibrillar aggregates, whereas a triple tau mutant (VPR) containing V337M, P301L, and R406W substitutions also formed similar aggregates. Furthermore, the aggregates increased in size over time in culture. Significantly, the formation of aggregated delta K and VPR tau protein correlated with reduced affinity of these mutants to bind microtubules. Reduced phosphorylation and altered proteolysis was also observed in R406W and delta K tau mutants. Thus, distinct pathological phenotypes, including the formation of insoluble filamentous tau aggregates, result from the expression of different FTDP-17 tau mutants in transfected Chinese hamster ovary cells and implies that these missense mutations cause diverse neurodegenerative FTDP-17 syndromes by multiple mechanisms.     

Effect of PKD1 gene missense mutations on polycystin-1 membrane topogenesis

Polycystin-1 (PC1), the product of the polycystic kidney disease-1 (PKD1) gene, has a number of reported missense mutations whose pathogenicity is indeterminate. Previously, we utilized N-linked glycosylation reporter tags along with membrane insertion and topology assays to define the 11 membrane-spanning domains (I-XI) of PC1. In this report, we utilize glycosylation assays to determine whether two reported human polymorphisms/missense mutations within transmembrane (TM) domains VI and X affect the membrane topology of PC1. M3677T within TM VI had no effect on the topology of this TM domain as shown by the ability of two native N-linked glycosylation sites within the extracellular loop following TM VI to be glycosylated. In contrast, G4031D, within TM X, decreased the glycosylation of TM X reporter constructs, demonstrating that the substitution affected the C-terminal translocating activity of TM X. Furthermore, G4031D reduced the membrane association of TM X and XI together. These results suggest that G4031D affects the membrane insertion and topology of the C-terminal portion of polycystin-1 and represents a bona fide pathogenic mutation.     

Uncoupling the pleiotropic phenotypes of clk-1 with tRNA missense suppressors in Caenorhabditis elegans

clk-1 encodes a demethoxyubiquinone (DMQ) hydroxylase that is necessary for ubiquinone biosynthesis. When Caenorhabditis elegans clk-1 mutants are grown on bacteria that synthesize ubiquinone (UQ), they are viable but have a pleiotropic phenotype that includes slowed development, behaviors, and aging. However, when grown on UQ-deficient bacteria, the mutants arrest development transiently before growing up to become sterile adults. We identified nine suppressors of the missense mutation clk-1(e2519), which harbors a Glu-to-Lys substitution. All suppress the mutant phenotypes on both UQ-replete and UQ-deficient bacteria. However, each mutant suppresses a different subset of phenotypes, indicating that most phenotypes can be uncoupled from each other. In addition, all suppressors restore the ability to synthesize exceedingly small amounts of UQ, although they still accumulate the precursor DMQ, suggesting that the presence of DMQ is not responsible for the Clk-1 phenotypes. We cloned six of the suppressors, and all encode tRNA(Glu) genes whose anticodons are altered to read the substituted Lys codon of clk-1(e2519). To our knowledge, these suppressors represent the first missense suppressors identified in any metazoan. The pattern of suppression we observe suggests that the individual members of the tRNA(Glu) family are expressed in different tissues and at different levels.     

Testing computational prediction of missense mutation phenotypes: Functional characterization of 204 mutations of human cystathionine beta synthase

Predicting the phenotypes of missense mutations uncovered by large‐scale sequencing projects is an important goal in computational biology. High‐confidence predictions can be an aid in focusing experimental and association studies on those mutations most likely to be associated with causative relationships between mutation and disease. As an aid in developing these methods further, we have derived a set of random mutations of the enzymatic domains of human cystathionine beta synthase. This enzyme is a dimeric protein that catalyzes the condensation of serine and homocysteine to produce cystathionine. Yeast missing this enzyme cannot grow on medium lacking a source of cysteine, while transfection of functional human CBS into yeast strains missing endogenous enzyme can successfully complement for the missing gene. We used PCR mutagenesis with error‐prone Taq polymerase to produce 948 colonies and compared cell growth in the presence or absence of a cysteine source as a measure of CBS function. We were able to infer the phenotypes of 204 single‐site mutants, 79 of them deleterious and 125 neutral. This set was used to test the accuracy of six publicly available prediction methods for phenotype prediction of missense mutations: SIFT, PolyPhen, PMut, SNPs3D, PhD‐SNP, and nsSNPAnalyzer. The top methods are PolyPhen, SIFT, and nsSNPAnalyzer, which have similar performance. Using kernel discriminant functions, we found that the difference in position‐specific scoring matrix values is more predictive than the wild‐type PSSM score alone, and that the relative surface area in the biologically relevant complex is more predictive than that of the monomeric proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Structural impact of three Parkinsonism-associated missense mutations on human DJ-1

A number of missense mutations in the oxidative stress response protein DJ-1 are implicated in rare forms of familial Parkinsonism. The best-characterized Parkinsonian DJ-1 missense mutation, L166P, disrupts homodimerization and results in a poorly folded protein. The molecular basis by which the other Parkinsonism-associated mutations disrupt the function of DJ-1, however, is incompletely understood. In this study we show that three different Parkinsonism-associated DJ-1 missense mutations (A104T, E163K, and M26I) reduce the thermal stability of DJ-1 in solution by subtly perturbing the structure of DJ-1 without causing major folding defects or loss of dimerization. Atomic resolution X-ray crystallography shows that the A104T substitution introduces water and a discretely disordered residue into the core of the protein, E163K disrupts a key salt bridge with R145, and M26I causes packing defects in the core of the dimer. The deleterious effect of each Parkinsonism-associated mutation on DJ-1 is dissected by analysis of engineered substitutions (M26L, A104V, and E163K/R145E) that partially alleviate each of the defects introduced by the A104T, E163K and M26I mutations. In total, our results suggest that the protective function of DJ-1 can be compromised by diverse perturbations in its structural integrity, particularly near the junctions of secondary structural elements.     

Functional consequences of PRODH missense mutations

PRODH maps to 22q11 in the region deleted in the velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS) and encodes proline oxidase (POX), a mitochondrial inner-membrane enzyme that catalyzes the first step in the proline degradation pathway. At least 16 PRODH missense mutations have been identified in studies of type I hyperprolinemia (HPI) and schizophrenia, 10 of which are present at polymorphic frequencies. The functional consequences of these missense mutations have been inferred by evolutionary conservation, but none have been tested directly. Here, we report the effects of these mutations on POX activity. We find that four alleles (R185Q, L289M, A455S, and A472T) result in mild (<30%), six (Q19P, A167V, R185W, D426N, V427M, and R431H) in moderate (30%-70%), and five (P406L, L441P, R453C, T466M, and Q521E) in severe (>70%) reduction in POX activity, whereas one (Q521R) increases POX activity. The POX encoded by one severe allele (T466M) shows in vitro responsiveness to high cofactor (flavin adenine dinucleotide) concentrations. Although there is limited information on plasma proline levels in individuals of known PRODH genotype, extant data suggest that severe hyperprolinemia (>800 microM) occurs in individuals with large deletions and/or PRODH missense mutations with the most-severe effect on function (L441P and R453C), whereas modest hyperprolinemia (300-500 microM) is associated with PRODH alleles with a moderate reduction in activity. Interestingly, three of the four alleles associated with or found in schizophrenia (V427M, L441P, and R453C) resulted in severe reduction of POX activity and hyperprolinemia. These observations plus the high degree of polymorphism at the PRODH locus are consistent with the hypothesis that reduction in POX function is a risk factor for schizophrenia.     

Krit1 missense mutations lead to splicing errors in cerebral cavernous malformation

At least 40% of families affected with cerebral cavernous malformation have a mutation in Krit1. We previously identified two point mutations in Krit1 leading to changes in amino acids (D137G and Q210E) in two different families. Further RNA analysis reveals that both point mutations actually activate cryptic splice-donor sites, causing aberrant splicing and leading to a frameshift and protein truncation. To date, no simple missense mutations have been detected in Krit1.     

Phenylketonuria missense mutations in the Mediterranean.

Two missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of an Italian phenylketonuria (PKU) patient. Both mutations occurred in exon 7 of the PAH gene, resulting in the substitution of Trp for Arg at amino acid 252 (R252W) and of Leu for Pro (P281L) at amino acid 281 of the protein. Expression vectors containing either the normal human PAH cDNA or mutant cDNAs were constructed and transfected into cultured mammalian cells. Extracts from cells transfected with either mutant construct showed negligible enzyme activity and undetectable levels of immunoreactive PAH protein as compared to the normal construct. These results are compatible with the severe classical PKU phenotype observed in this patient. Population genetic studies in the Italian population revealed that both the R252W and the P281L mutations are in linkage disequilibrium with mutant restriction fragment length polymorphism (RFLP) haplotype 1, which is the most prevalent RFLP haplotype in this population. The R252W mutation is present in 10% and the P281L mutation is present in 20% of haplotype 1 mutant chromosomes. These mutations are both very rare among other European populations, suggesting a Mediterranean origin for these mutant chromosomes.     

Haploinsufficiency of cyfip1 produces fragile x-like phenotypes in mice

Background

Copy number variation (CNV) at the 15q11.2 region, which includes a gene that codes for CYFIP1 (cytoplasmic FMR1 interacting protein 1), has been implicated in autism, intellectual disability and additional neuropsychiatric phenotypes. In the current study we studied the function of Cyfip1 in synaptic physiology and behavior, using mice with a disruption of the Cyfip1 gene.

Methodology/Principal Findings

We observed that in Cyfip1 heterozygous mice metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) induced by paired-pulse low frequency stimulation (PP-LFS) was significantly increased in comparison to wildtype mice. In addition, mGluR-LTD was not affected in the presence of protein synthesis inhibitor in the Cyfip1 heterozygous mice, while the same treatment inhibited LTD in wildtype littermate controls. mGluR-agonist (RS)-3,5-dihydroxyphenylglycine (DHPG)-induced LTD was also significantly increased in hippocampal slices from Cyfip1 heterozygous mice and again showed independence from protein synthesis only in the heterozygous animals. Furthermore, we observed that the mammalian Target of Rapamycin (mTOR) inhibitor rapamycin was only effective at reducing mGluR-LTD in wildtype animals. Behaviorally, Cyfip1 heterozygous mice showed enhanced extinction of inhibitory avoidance. Application of both mGluR5 and mGluR1 antagonist to slices from Cyfip1 heterozygous mice reversed the increase in DHPG-induced LTD in these mice.

Conclusions/Significance

These results demonstrate that haploinsufficiency of Cyfip1 mimics key aspects of the phenotype of Fmr1 knockout mice and are consistent with the hypothesis that these effects are mediated by interaction of Cyfip1 and Fmrp in regulating activity-dependent translation. The data provide support for a model where CYFIP1 haploinsufficiency in patients results in intermediate phenotypes increasing risk for neuropsychiatric disorders.     

Bioinformatic analysis of pathogenic missense mutations of activin receptor like kinase 1 ectodomain

Activin A receptor, type II-like kinase 1 (also called ALK1), is a serine-threonine kinase predominantly expressed on endothelial cells surface. Mutations in its ACVRL1 encoding gene (12q11-14) cause type 2 Hereditary Haemorrhagic Telangiectasia (HHT2), an autosomal dominant multisystem vascular dysplasia. The study of the structural effects of mutations is crucial to understand their pathogenic mechanism. However, while an X-ray structure of ALK1 intracellular domain has recently become available (PDB ID: 3MY0), structure determination of ALK1 ectodomain (ALK1(EC)) has been elusive so far. We here describe the building of a homology model for ALK1(EC), followed by an extensive bioinformatic analysis, based on a set of 38 methods, of the effect of missense mutations at the sequence and structural level. ALK1(EC) potential interaction mode with its ligand BMP9 was then predicted combining modelling and docking data. The calculated model of the ALK1(EC) allowed mapping and a preliminary characterization of HHT2 associated mutations. Major structural changes and loss of stability of the protein were predicted for several mutations, while others were found to interfere mainly with binding to BMP9 or other interactors, like Endoglin (CD105), whose encoding ENG gene (9q34) mutations are known to cause type 1 HHT. This study gives a preliminary insight into the potential structure of ALK1(EC) and into the structural effects of HHT2 associated mutations, which can be useful to predict the potential effect of each single mutation, to devise new biological experiments and to interpret the biological significance of new mutations, private mutations, or non-synonymous polymorphisms.     

Functional analysis of HNPCC-related missense mutations in MSH2

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in the human DNA mismatch repair (MMR) genes, most frequently MSH2 and MLH1. The majority of HNPCC mutations cause truncations and thus loss of function of the affected polypeptide. However, a significant proportion of MMR mutations found in HNPCC patients are single amino acid substitutions and the functional consequences of many of these mutations in DNA repair are unclear. We have examined the consequences of seven MSH2 missense mutations found in HNPCC families by testing the MSH2 mutant proteins in functional assays as well as by generating equivalent missense mutations in Escherichia coli MutS and analyzing the phenotypes of these mutants. Here we show that two mutant proteins, MSH2-P622L and MSH2-C697F confer multiple biochemical defects, namely in mismatch binding, in vivo interaction with MSH6 and EXO1, and in nuclear localization in the cell. Mutation G674R, located in the ATP-binding region of MSH2, appears to confer resistance to ATP-dependent mismatch release. Mutations D167H and H639R show reduced mismatch binding. Results of in vivo experiments in E. coli with MutS mutants show that one additional mutant, equivalent of MSH2-A834T that do not show any defects in MSH2 assays, is repair deficient. In conclusion, all mutant proteins (except for MSH2-A305T) have defects; either in mismatch binding, ATP-release, mismatch repair activity, subcellular localization or protein-protein interactions.     

Variable expression of Dkc1 mutations in mice

In humans mutations in DKC1, cause the rare bone marrow failure syndrome dyskeratosis congenita. We have used gene targeting to produce mouse ES cells with Dkc1 mutations that cause DC when in humans. The mutation A353V, the most common human mutation, causes typical DC to very severe DC in humans. Male chimeric mice carrying this mutation do not pass the mutated allele to their offspring. The mutation G402E accounts for a single typical case of DC in a human family. The allele carrying this mutation was transmitted to the offspring with high efficiency. Expression of RNA and protein was reduced compared to wild type animals, but no abnormalities of growth and development or in blood values were found in mutant mice. Thus Dkc1 mutations have variable expression inmice, as in humans. genesis 47:366–373, 2009. © 2009 Wiley‐Liss, Inc.     

Hidden phenotypes of PINK1/Parkin knockout mice

PINK1, a serine/threonine ubiquitin kinase, and Parkin, an E3 ubiquitin ligase, work in coordination to target damaged mitochondria to the lysosome in a process called mitophagy. This review will cover what we have learned from PINK1 and Parkin knockout (KO) mice. Systemic PINK1 and Parkin KO mouse models haven’t faithfully recapitulated early onset forms of Parkinson’s disease found in humans with recessive mutations in these genes. However, the utilization of these mouse models has given us insight into how PINK1 and Parkin contribute to mitochondrial quality control and function in different tissues beyond the brain such as in heart and adipose tissue. Although PINK1 and Parkin KO mice have been generated over a decade ago, these models are still being used today to creatively elucidate cell-type specific functions. Recently, these mouse models have uncovered that these proteins contribute to innate immunity and cancer phenotypes.