The functional importance of disease-associated mutation

BACKGROUND: For many years, scientists believed that point mutations in genes are the genetic switches for somatic and inherited diseases such as cystic fibrosis, phenylketonuria and cancer. Some of these mutations likely alter a protein's function in a manner that is deleterious, and they should occur in functionally important regions of the protein products of genes. Here we show that disease-associated mutations occur in regions of genes that are conserved, and can identify likely disease-causing mutations. RESULTS: To show this, we have determined conservation patterns for 6185 non-synonymous and heritable disease-associated mutations in 231 genes. We define a parameter, the conservation ratio, as the ratio of average negative entropy of analyzable positions with reported mutations to that of every analyzable position in the gene sequence. We found that 84.0% of the 231 genes have conservation ratios less than one. 139 genes had eleven or more analyzable mutations and 88.0% of those had conservation ratios less than one. CONCLUSIONS: These results indicate that phylogenetic information is a powerful tool for the study of disease-associated mutations. Our alignments and analysis has been made available as part of the database at http://cancer.stanford.edu/mut-paper/. Within this dataset, each position is annotated with the analysis, so the most likely disease-causing mutations can be identified

An Ancient Evolutionary Origin of Genes Associated with Human Genetic Diseases

Several thousand genes in the human genome have been linked to a heritable genetic disease. The majority of these appear to be nonessential genes (i.e., are not embryonically lethal when inactivated), and one could therefore speculate that they are late additions in the evolutionary lineage toward humans. Contrary to this expectation, we find that they are in fact significantly overrepresented among the genes that have emerged during the early evolution of the metazoa. Using a phylostratigraphic approach, we have studied the evolutionary emergence of such genes at 19 phylogenetic levels. The majority of disease genes was already present in the eukaryotic ancestor, and the second largest number has arisen around the time of evolution of multicellularity. Conversely, genes specific to the mammalian lineage are highly underrepresented. Hence, genes involved in genetic diseases are not simply a random subset of all genes in the genome but are biased toward ancient genes

Sequence variation in G-protein-coupled receptors: analysis of single nucleotide polymorphisms

We assessed the disease-causing potential of single nucleotide polymorphisms (SNPs) based on a simple set of sequence-based features. We focused on SNPs from the dbSNP database in G-protein-coupled receptors (GPCRs), a large class of important transmembrane (TM) proteins. Apart from the location of the SNP in the protein, we evaluated the predictive power of three major classes of features to differentiate between disease-causing mutations and neutral changes: (i) properties derived from amino-acid scales, such as volume and hydrophobicity; (ii) position-specific phylogenetic features reflecting evolutionary conservation, such as normalized site entropy, residue frequency and SIFT score; and (iii) substitution-matrix scores, such as those derived from the BLOSUM62, GRANTHAM and PHAT matrices. We validated our approach using a control dataset consisting of known disease-causing mutations and neutral variations. Logistic regression analyses indicated that position-specific phylogenetic features that describe the conservation of an amino acid at a specific site are the best discriminators of disease mutations versus neutral variations, and integration of all our features improves discrimination power. Overall, we identify 115 SNPs in GPCRs from dbSNP that are likely to be associated with disease and thus are good candidates for genotyping in association studies

Multiple Property Tolerance Analysis for the Evaluation of Missense Mutations

Computational prediction of the impact of a mutation on protein function is still not accurate enough for clinical diagnostics without additional human expert analysis. Sequence alignment-based methods have been extensively used but their results highly depend on the quality of the input alignments and the choice of sequences. Incorporating the structural information with alignments improves prediction accuracy. Here, we present a conservation of amino acid properties method for mutation prediction, Multiple Properties Tolerance Analysis (MuTA), and a new strategy, MuTA/S, to incorporate the solvent accessible surface (SAS) property into MuTA. Instead of combining multiple features by machine learning or mathematical methods, an intuitive strategy is used to divide the residues of a protein into different groups, and in each group the properties used is adjusted

Insights on variant analysis in silico tools for pathogenicity prediction

Molecular biology is currently a fast-advancing science. Sequencing techniques are getting cheaper, but the interpretation of genetic variants requires expertise and computational power, therefore is still a challenge. Next-generation sequencing releases thousands of variants and to classify them, researchers propose protocols with several parameters. Here we present a review of several in silico pathogenicity prediction tools involved in the variant prioritization/classification process used by some international protocols for variant analysis and studies evaluating their efficiency

Tutkimus LRP5-geenin merkityksestä luustoon sekä sokeri- ja rasva-aineenvaihduntaan

Bone mass accrual and maintenance are regulated by a complex interplay between genetic and environmental factors. Recent studies have revealed an important role for the low-density lipoprotein receptor-related protein 5 (LRP5) in this process. The aim of this thesis study was to identify novel variants in the LRP5 gene and to further elucidate the association of LRP5 and its variants with various bone health related clinical characteristics. The results of our studies show that loss-of-function mutations in LRP5 cause severe osteoporosis not only in homozygous subjects but also in the carriers of these mutations, who have significantly reduced bone mineral density (BMD) and increased susceptibility to fractures. In addition, we demonstrated for the first time that a common polymorphic LRP5 variant (p.A1330V) was associated with reduced peak bone mass, an important determinant of BMD and osteoporosis in later life. The results from these two studies are concordant with results seen in other studies on LRP5 mutations and in association studies linking genetic variation in LRP5 with BMD and osteoporosis. Several rare LRP5 variants were identified in children with recurrent fractures. Sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses revealed no disease-causing mutations or whole-exon deletions. Our findings from clinical assessments and family-based genotype-phenotype studies suggested that the rare LRP5 variants identified are not the definite cause of fractures in these children. Clinical assessments of our study subjects with LPR5 mutations revealed an unexpectedly high prevalence of impaired glucose tolerance and dyslipidaemia. Moreover, in subsequent studies we discovered that common polymorphic LRP5 variants are associated with unfavorable metabolic characteristics. Changes in lipid profile were already apparent in pre-pubertal children. These results, together with the findings from other studies, suggest an important role for LRP5 also in glucose and lipid metabolism. Our results underscore the important role of LRP5 not only in bone mass accrual and maintenance of skeletal health but also in glucose and lipid metabolism. The role of LRP5 in bone metabolism has long been studied, but further studies with larger study cohorts are still needed to evaluate the specific role of LRP5 variants as metabolic risk factors.Osteoporoosi eli luukato on luuston sairaus, jolle ominaista on luun massan vähenemisestä ja luun rakenteellisista muutoksista aiheutuva murtuma-alttius. Ympäristötekijät, kuten ravinto ja liikunta, vaikuttavat merkittävästi luun massan kertymiseen ja sen ylläpitoon, mutta myös perintötekijöillä on suuri merkitys. Tämän väitöskirjan tutkimusten pääkohteena oli LRP5-geeni, jonka merkitys luun tiheyden säätelijänä on osoitettu useissa kansainvälisissä tutkimuksissa. Tässä väitöskirjatyössä tutkittiin LRP5-geenin mutaatioiden ja polymorfismien vaikutusta luumassan kertymiseen, lapsuusiän murtumiin, sekä sokeritasapainoon ja rasva-aineenvaihduntaan. Tutkimuksemme osoittivat, että LRP5-geenissä sijaitseva A1330V -polymorfismi assosioituu matalaan luun mineraalitiheyteen. Tutkimuksemme oli ensimmäinen, jossa osoitettiin LRP5-geenin merkitys nuoruusiässä saavutettavan luun huippumassan kehittymiseen. Tulokset ovat merkittäviä, sillä eräs aikuisiän osteoporoosin keskeisimmistä ennustetekijöistä on nuoruudessa saavutettu luun huippumassa. Osoitimme myös, että homotsygoottiset mutaatiot LRP5-geenissä aiheuttavat vaikean osteoporoosin osteoporosis pseudoglioma (OPPG) potilaille ja että myös näiden mutaatioiden heterotsygoottisilla kantajilla on selvästi alentunut luuntiheys ja siten suurempi alttius luunmurtumille. Koska matala luumassa altistaa murtumille, tutkimme myös LRP5-geenin merkitystä lasten murtumien synnyssä. Tutkimuksemme oli osa ainutlaatuista 1390 lapsen murtuma-aineistoa, josta LRP5-tutkimukseen valittiin 66 toistuvia murtumia saanutta lasta. Tutkimuksemme osoittivat, että murtumien syntymekanismin taustalla ei ole LRP5-geenin mutaatioita. Geenitutkimuksissa löytyi kuitenkin useita yleisiä ja harvinaisia LRP5-geenin polymorfismeja, jotka saattavat myötävaikuttaa murtuma-alttiuteen. Kliiniset tutkimuksemme osoittivat, että OPPG-potilailla ja LRP5-geenin mutaatioiden kantajilla on tavanomaista useammin alentunut sokerinsieto sekä kohonneet kolesterolipitoisuudet. Jatkotutkimukset terveillä suomalaisilla lapsilla osoittivat, että myös eräät LRP5-geenin polymorfismit assosioituvat ei-toivottujen metabolisten riskitekijöiden, kuten korkean kolesterolitason ja verenpainnen kanssa. Tulokset ovat merkittäviä ja osoittavat, että LRP5-geenillä on vaikutusta metabolisten riskitekijöiden syntyyn jo lapsuusiässä. Tämän väitöskirjan tutkimustulokset toivat uutta tietoa LRP5-geenin merkityksestä luustoon sekä sokeri- ja rasva-aineenvaihduntaan. Lisätutkimuksia kuitenkin tarvitaan, jotta ymmärretään riittävästi näiden tekijöiden toiminnasta molekyylitasolla. Geenivirheiden tunnistaminen ja analysoiminen solutasolla mahdollistavat osteoporoosipotilaiden varhaisen diagnosoinnin ja osteoporoottisten murtumien tehokkaan ennaltaehkäisyn riskiryhmissä

Understanding Structure and Dynamics of PTEN and its Possible Genotype-Phenotype Correlations in Endometriosis and Cancer

The phosphatase and tensin homolog deleted on chromosome 10, (PTEN) gene encodes a tumor suppressor phosphatase frequently mutated in various human cancers. Somatic missense mutations of PTEN have recently been found in patients with endometriosis, endometrial cancer, and ovarian cancer. Here we present the first computational analysis of 13 somatic missense PTEN mutations to assess a possible genotype-phenotype correlation in endometriosis and cancer. We posit PTEN’s active site defines a possible mutation-driven allosteric region wherein a subset of mutations correlate with endometriosis, endometrial cancer, and ovarian cancer. Our data suggest that mutations within the active site disrupt the structural stability, electrostatic interaction, global dynamics and the structural communication pathway, likely contributing to the aforementioned phenotypes. Multiple in silico prediction methods were utilized to calculate protein structural stability changes produced by each mutation; decreases in protein structure stability were seen in each mutation with an increase in dynamics across the phosphatase-C2 domain interface of R130G/L/Q and R173C/H mutations. To assess the impact on intrinsic and global dynamics, elastic network models (ENMs) were employed demonstrating changes from wild-type “hinge-bending” to “zipper-like” global motions induced by each mutation. All-atom molecular dynamics (MD) simulations revealed large conformational changes that affect the global dynamics of the active site loops and the CBR3 loop in the C2 domain. Interestingly, mutations G36E/R, C124S, G129R, R130L/Q, R173C/H, and V191A dramatically affected the principal motions of the active site loops and inter-domain interface. Overall, the global dynamics induced by each mutation effects reveal unique long-range perturbations that may impair PTEN’s function. We further investigated structural communication within each mutant system using protein structure network (PSN) analysis and found that R130 and R173 play critical roles in controlling salient communication pathways suggesting a compelling interplay between the two positions involving a potential mutation-driven allosteric interface. The results of this research provide a greater understanding of the mechanistic role of mutated PTEN associated with endometriosis and cancer. It is our hope that these results will aid in a better clinical-molecular classification of the resulting phenotypes allowing for translation into improved diagnostic and therapeutic approaches.Biology and Biochemistry, Department o