Over the last few decades, computer simulation techniques have been established as an essential tool for understanding biochemical processes. This thesis deals mainly with the application of free energy calculations to ribosomal complexes and a cardiac ion channel. The linear interaction energy (LIE) method is used to explore the energetic properties of the essential process of codon–anticodon recognition on the ribosome. The calculations show the structural and energetic consequences and effects of first, second, and third position mismatches in the ribosomal decoding center. Recognition of stop codons by ribosomal termination complexes is fundamentally different from sense codon recognition. Free energy perturbation simulations are used to study the detailed energetics of stop codon recognition by the bacterial ribosomal release factors RF1 and RF2. The calculations explain the vastly different responses to third codon position A to G substitutions by RF1 and RF2. Also, previously unknown highly specific water interactions are identified. The GGQ loop of ribosomal RFs is essential for its hydrolytic activity and contains a universally methylated glutamine residue. The structural effect of this methylation is investigated. The results strongly suggest that the methylation has no effect on the intrinsic conformation of the GGQ loop, and, thus, that its sole purpose is to enhance interactions in the ribosomal termination complex. A first microscopic, atomic level, analysis of blocker binding to the pharmaceutically interesting potassium ion channel Kv1.5 is presented. A previously unknown uniform binding mode is identified, and experimental binding data is accurately reproduced. Furthermore, problems associated with pharmacophore models based on minimized gas phase ligand conformations are highlighted. Generalized Born and Poisson–Boltzmann continuum models are incorporated into the LIE method to enable implicit treatment of solvent, in an effort to improve speed and convergence. The methods are evaluated and validated using a set of plasmepsin II inhibitors.

The presence of TP53 mutations has been associated with inferior outcome in diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). In DLBCL, the impact of the TP53 codon 72 polymorphism and MDM2 SNP309 has not been clearly elucidated, whereas MDM2 SNP309 was suggested as a poor-prognostic marker in CLL. In addition, p16INK4a promoter hypermethylation has been implicated as a negative prognostic factor in DLBCL. The aim of this thesis was to further evaluate these molecular markers in well-characterised materials of DLBCL and CLL. In paper I, we investigated the prognostic role of TP53 mutation, codon 72 polymorphism and MDM2 SNP309 in DLBCL (n=102). The presence of TP53 mutations (12.7%) correlated with a poor lymphoma-specific and progression-free survival, and a particularly pronounced effect was observed in the germinal center subtype. Neither the MDM2 SNP309 nor the TP53 codon 72 polymorphism had an impact on age of onset or survival. In paper II, we applied pyrosequencing to measure the level of p16INK4a methylation in DLBCL (n=113). Thirty-seven percent of cases displayed p16INK4a methylation; however, no clear association could be observed between degree of methylation and clinical characteristics or lymphoma-specific survival. In papers III–IV, we investigated the prognostic role of MDM2 SNP309 (n=418) and TP53 mutation (n=268) in CLL. No correlation was observed between any particular MDM2 SNP309 genotype and time to treatment and overall survival. Furthermore, no association was found between the different MDM2 SNP309 genotypes and established CLL prognostic markers. TP53 mutations were detected in 3.7% of CLL patients; where the majority showed a concomitant 17p-deletion and only three carried TP53 mutations without 17p-deletion. We confirmed a significantly shorter overall survival and time to treatment in patients with both TP53 mutation and 17p-deletion. Altogether, our studies could confirm the negative prognostic impact of TP53 mutations in DLBCL, whereas MDM2 SNP309 and TP53 codon 72 polymorphisms appear to lack clinical relevance. We also question the role of p16INKa methylation as a poor-prognostic factor in DLBCL. Finally, the presence of TP53 mutation in CLL appears to be rare at disease onset and instead arise during disease progression.

Nos últimos anos, inúmeros estudos epidemiológicos evidenciaram a forte associação entre o carcinoma do colo uterino e a infecção por papilomavírus humano (HPV). Esta associação deriva do reconhecimento de que estes vírus codificam oncoproteínas, dentre as quais E6 e E7, que apresentam propriedades transformantes. O produto do gene E7 se liga ao produto do gene retinoblastoma que perde a sua função de regular negativamente o ciclo celular. O produto do gene E6 se liga ao produto do gene supressor de tumor p53 levando a sua degradação pela via de proteólise dependente de ubiquitina. O gene p53 é um supressor tumoral com função de regulação do ciclo celular que apresenta vários polimorfismos distintos em diversos grupos étnicos e tem sido amplamente estudado tanto em tecidos normais quanto em tecidos tumorais. O polimorfismo do códon 72 do gene i>p53 é o mais estudado e pode apresentar três alelos diferentes na população. Um alelo codifica arginina (Arg), um codifica prolina (Pro) e outro, raramente encontrado, codifica cisteína (Cys). Em 1993 foi iniciado um estudo epidemiológico da história natural da infecção por HPV e neoplasia da cérvice uterina em uma população feminina de baixa renda em São Paulo (Brasil), uma das áreas de maior risco em todo o mundo. O estudo focaliza a infecção persistente por tipos oncogênicos de HPV como evento precursor que leva à carcinogênese do colo do útero e visa entender os atributos da história natural da infecção viral e das doenças associadas ao colo uterino. Um dos objetivos deste estudo é avaliar se o polimorfismo do códon 72 do gene p53 pode, ou não, ser utilizado como marcador de predisposição ao câncer do colo do útero uma vez que um estudo inicial relatou que pacientes portando o genótipo p53Arg homozigoto seriam 7 vezes mais susceptíveis ao desenvolvimento de neoplasia da cérvice uterina que pacientes contendo o genótipo p53Pro e heterozigoto p53Pro/ Arg. Contudo, vários estudos posteriores contradizem e corroboram esses achados. O presente projeto teve como objetivos, portanto, verificar se o polimorfismo do códon 72 do gene p53 poderia estar associado a infecções persistentes por HPV e ao risco de neoplasia do colo do útero, além de comparar metodologias de detecção utilizadas por outros estudos, visando esclarecer se os motivos que levam à discordância dos resultados podem ser atribuídos a ocorrência de erros classificatórios metodológicos. Ao todo, sete metodologias de detecção foram comparadas. Apenas uma delas, PCR alelo-específica, apresentou resultado discordante das demais utilizadas. Coincidentemente, essa metodologia foi amplamente utilizada por muitos estudos que encontraram associações tanto positivas quanto negativas. Isso poderia nos dar indícios de que os erros classificatórios dependentes de metodologia poderiam influenciar os resultados de correlação entre o polimorfismo do códon 72 e o risco de neoplasia do colo do útero. As correlações observadas por este trabalho entre este polimorfismo do códon 72 e o risco de neoplasia do colo uterino não mostraram associação deste polimorfismo com o risco de infecções persistentes por HPV e as lesões precursoras do carcinoma do colo uterino. / In recent years, a number of epidemiological studies have pointed toward a strong association between cervical cancer and infection by Human Papillomavirus (HPV). This association derives from the discovery that these viruses code for oncoproteins, among them E6 and E7 that have transforming properties. The E7 gene product associates with the retinoblastoma gene product, causing the latter to lose its function as a negative regulator of the cell cycle. The E6 gene product interacts with the tumor suppressor p53 gene product, resulting in its degradation via ubiquitin dependent proteolysis. The p53 gene is a tumor suppressor that funcions in the regulation of the cell cycle. It presents a number of distinct polymorfisms in diverse ethnic groups, and has been widely studied, both in normal and tumor tissues. The polymorfism of codon 72 is the most studied, and may present three different alleles in the population. One allele codes for arginine (Arg), another codes for proline (Pro), and a third, rarely found, codes for cystein (Cys). In 1993 an epidemilogical study of the natural history of infection by HPV and its possible association with cervical neoplasia was initiated in a population of low income females in São Paulo, Brazil, one of the areas of greatest risk in the world. The study focuses on persistent infection by oncogenic types of HPV as a precursor to carcinogenesis of the cervix, and seeks to understand the attributes of the natural history of viral infection and of illnesses associated with the cervix. One of the objectives of the study is to evaluate if the polymorfisms of codon 72 of p53 can or not be used as a marker of predisposition to cervical cancer, given the finding in the initial study that patients who were homozygous for the p53Arg genotype were 7 times more susceptible to developing cervical neoplasias than those patients who were homozygous for p53Pro, or heterozygous p53 Pro/ Arg. Previous studies have been realized both supporting and disputing these findings. The current study had two main objectives: to verify if the polymorfism of p53 codon 72 could be associated with persistent infections of HPV and the risk of cervical neoplasia, as well as to compare methods of detection used by other studies, in an attempt to clarify if the discording results of past studies could be due to methodological classification errors. Seven detection methods were compared. Only one of these, allele specific PCR, presented discording results from the rest. Coincidentally, this method was widely used in a number of studies which found both positive and negative associations. This might indicate that the method-dependent classification errors could influence the results of correlation between codon 72 polymorphism and the risk of cervical neoplasia. The correlations observed by this study did not demonstrate an association between codon 72 polymorphism and the risk of persistent HPV infection and precursor lesions of cervical cancer.

The ribosome is a macromolecular machine that produces proteins in all kingdoms of life. The proteins, in turn, control the biochemical processes within the cell. It is thus of extreme importance that the machine that makes the proteins works with high precision. By using three dimensional structures of the ribosome and homology modelling, we have applied molecular dynamics simulations and free-energy calculations to study the codon specificity of protein synthesis in initiation and termination on an atomistic level. In addition, we have examined the binding of small molecules to riboswitches, which can change the expression of an mRNA. The relative affinities on the ribosome between the eukaryotic initiator tRNA to the AUG start codon and six near-cognate codons were determined. The free-energy calculations show that the initiator tRNA has a strong preference for the start codon, but requires assistance from initiation factors 1 and 1A to uphold discrimination against near-cognate codons. When instead a stop codon (UAA, UGA or UAG) is positioned in the ribosomal A-site, a release factor binds and terminates protein synthesis by hydrolyzing the nascent peptide chain. However, vertebrate mitochondria have been thought to have four stop codons, namely AGA and AGG in addition to the standard UAA and UAG codons. Furthermore, two release factors have been identified, mtRF1 and mtRF1a. Free-energy calculations were used to determine if any of these two factors could bind to the two non-standard stop codons, and thereby terminate protein synthesis. Our calculations showed that the mtRF’s have similar stop codon specificity as bacterial RF1 and that it is highly unlikely that the mtRF’s are responsible for terminating at the AGA and AGG stop codons. The eukaryotic release factor 1, eRF1, on the other hand, can read all three stop codons singlehandedly. We show that eRF1 exerts a high discrimination against near-cognate codons, while having little preference for the different cognate stop codons. We also found an energetic mechanism for avoiding misreading of the UGG codon and could identify a conserved cluster of hydrophobic amino acids which prevents excessive solvent molecules to enter the codon binding site. The linear interaction energy method was used to examine binding of small molecules to the purine riboswitch and the FEP method was employed to explicitly calculate the LIE b-parameters. We show that the purine riboswitches have a remarkably high degree of electrostatic preorganization for their cognate ligands which is fundamental for discriminating against different purine analogs.

L’acquisition de données génomiques ne cesse de croître, ainsi que l’appétit pour les interpréter. Mais déterminer les processus qui ont façonné l’évolution des séquences codantes (et leur importance relative) est un défi scientifique passant par le développement de modèles statistiques de l’évolution prenant en compte de plus en plus d’hétérogénéités au niveau des processus mutationnels et de sélection. Identifier la sélection est une tâche qui nécessite typiquement de détecter un écart entre deux modèles : un modèle nulle ne permettant pas de régime évolutif adaptatif et un modèle alternatif qui lui en permet. Lorsqu’un test entre ces deux modèles rejette le modèle nulle, on considère avoir détecter la présence d’évolution adaptative. La tâche est d’autant plus difficile que le signal est faible et confondu avec diverses hétérogénéités négligées par les modèles. La détection de la sélection sur l’usage des codons spécifiquement est controversée, particulièrement chez les Vertébrés. Plusieurs raisons peuvent expliquer cette controverse : (1) il y a un biais sociologique à voir la sélection comme moteur principal de l’évolution, à un tel point que les hétérogénéités relatives aux processus de mutation sont historiquement négligées ; (2) selon les principes de la génétique des populations, la petite taille efficace des populations des Vertébrés limite le pouvoir de la sélection sur les mutations synonymes conférant elles-mêmes un avantage minime ; (3) par contre, la sélection sur l’usage des codons pourrait être très localisée le long des séquences codantes, à des sites précis, relevant de contraintes de sélection relatives à des motifs utilisés par la machinerie d’épissage, par exemple. Les modèles phylogénétiques de type mutation-sélection sont les outils de prédilection pour aborder ces questions, puisqu’ils modélisent explicitement les processus mutationnels ainsi que les contraintes de sélection. Toutes les hétérogénéités négligées par les modèles mutation-sélection de Yang and Nielsen [2008] peuvent engendrer de faux positifs allant de 20% (préférence site-spécifique en acides aminés) à 100% (hypermutabilité des transitions en contexte CpG) [Laurin-Lemay et al., 2018b]. En particulier, l’hypermutabilité des transitions du contexte CpG peut à elle seule expliquer la sélection détectée par Yang and Nielsen [2008] sur l’usage des codons. Mais, modéliser des phénomènes qui prennent en compte des interdépendances dans les données (par exemple l’hypermutabilité du contexte CpG) augmente de beaucoup la complexité des fonctions de vraisemblance. D’autre part, aujourd’hui le niveau de sophistication des modèles fait en sorte que des vecteurs de paramètres de haute dimensionnalité sont nécessaires pour modéliser l’hétérogénéité des processus étudiés, dans notre cas de contraintes de sélection sur la protéine. Le calcul bayésien approché (Approximate Bayesian Computation ou ABC) permet de contourner le calcul de la vraisemblance. Cette approche diffère de l’échantillonnage par Monte Carlo par chaîne de Markov (MCMC) communément utilisé pour faire l’approximation de la distribution a posteriori. Nous avons exploré l’idée de combiner ces approches pour une problématique spécifique impliquant des paramètres de haute dimensionnalité et de nouveaux paramètres prenant en compte des dépendances entre sites. Dans certaines conditions, lorsque les paramètres de haute dimensionnalité sont faiblement corrélés aux nouveaux paramètres d’intérêt, il est possible d’inférer ces mêmes paramètres de haute dimensionnalité avec la méthode MCMC, et puis les paramètres d’intérêt au moyen de l’ABC. Cette nouvelle approche se nomme CABC [Laurin-Lemay et al., 2018a], pour calcul bayésien approché conditionnel (Conditional Approximate Bayesian Computation : CABC). Nous avons pu vérifier l’efficacité de la méthode CABC en étudiant un cas d’école, soit celui de l’hypermutabilité des transitions en contexte CpG chez les Eutheria [Laurin-Lemay et al., 2018a]. Nous trouvons que 100% des 137 gènes testés possèdent une hypermutabilité des transitions significative. Nous avons aussi montré que les modèles incorporant l’hypermutabilité des transitions en contexte CpG prédisent un usage des codons plus proche de celui des gènes étudiés. Ceci suggère qu’une partie importante de l’usage des codons peut être expliquée à elle seule par les processus mutationnels et non pas par la sélection. Finalement nous explorons plusieurs pistes de recherche suivant nos développements méthodologiques : l’application de la détection de l’hypermutabilité des transitions en contexte CpG à l’échelle des Vertébrés ; l’expansion du modèle pour reconnaître des contextes autres que seul le CpG (e.g., hypermutabilité des transitions et transversions en contexte CpG et TpA) ; ainsi que des perspectives méthodologiques d’amélioration de la performance du CABC. / The acquisition of genomic data continues to grow, as does the appetite to interpret them. But determining the processes that shaped the evolution of coding sequences (and their relative importance) is a scientific challenge that requires the development of statistical models of evolution that increasingly take into account heterogeneities in mutation and selection processes. Identifying selection is a task that typically requires comparing two models: a null model that does not allow for an adaptive evolutionary regime and an alternative model that allows it. When a test between these two models rejects the null, we consider to have detected the presence of adaptive evolution. The task is all the more difficult as the signal is weak and confounded with various heterogeneities neglected by the models. The detection of selection on codon usage is controversial, particularly in Vertebrates. There are several reasons for this controversy: (1) there is a sociological bias in seeing selection as the main driver of evolution, to such an extent that heterogeneities relating to mutation processes are historically neglected; (2) according to the principles of population genetics, the small effective size of vertebrate populations limits the power of selection over synonymous mutations conferring a minimal advantage; (3) On the other hand, selection on the use of codons could be very localized along the coding sequences, at specific sites, subject to selective constraints related to DNA patterns used by the splicing machinery, for example. Phylogenetic mutation-selection models are the preferred tools to address these issues, as they explicitly model mutation processes and selective constraints. All the heterogeneities neglected by the mutation-selection models of Yang and Nielsen [2008] can generate false positives, ranging from 20% (site-specific amino acid preference) to 100% (hypermutability of transitions in CpG context)[Laurin-Lemay et al., 2018b]. In particular, the hypermutability of transitions in the CpG context alone can explain the selection on codon usage detected by Yang and Nielsen [2008]. However, modelling phenomena that take into account data interdependencies (e.g., hypermutability of the CpG context) greatly increases the complexity of the likelihood function. On the other hand, today’s sophisticated models require high-dimensional parameter vectors to model the heterogeneity of the processes studied, in our case selective constraints on the protein. Approximate Bayesian Computation (ABC) is used to bypass the calculation of the likelihood function. This approach differs from the Markov Chain Monte Carlo (MCMC) sampling commonly used to approximate the posterior distribution. We explored the idea of combining these approaches for a specific problem involving high-dimensional parameters and new parameters taking into account dependencies between sites. Under certain conditions, when the high dimensionality parameters are weakly correlated to the new parameters of interest, it is possible to infer the high dimensionality parameters with the MCMC method, and then the parameters of interest using the ABC. This new approach is called Conditional Approximate Bayesian Computation (CABC) [Laurin-Lemay et al., 2018a]. We were able to verify the effectiveness of the CABC method in a case study, namely the hypermutability of transitions in the CpG context within Eutheria [Laurin-Lemay et al.,2018a]. We find that 100% of the 137 genes tested have significant hypermutability of transitions. We have also shown that models incorporating hypermutability of transitions in CpG contexts predict a codon usage closer to that of the genes studied. This suggests that a significant part of codon usage can be explained by mutational processes alone. Finally, we explore several avenues of research emanating from our methodological developments: the application of hypermutability detection of transitions in CpG contexts to the Vertebrate scale; the expansion of the model to recognize contexts other than only CpG (e.g., hypermutability of transitions and transversions in CpG and TpA context); and methodological perspectives to improve the performance of the CABC approach.

No description available.

The development of shape in multicellular organisms has intrigued human minds for millenia. Empowered by modern genetic techniques, molecular biologists are now striving to not only dissect developmental processes, but to exploit their modularity for the design of custom living systems used in bioproduction, remediation, and regenerative medicine. Currently, our capacity to harness this potential is fundamentally limited by a lack of spatiotemporal control over gene expression in multicellular systems. While several synthetic genetic circuits for control of multicellular patterning have been reported, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its fundamental role in biological self-organization. In this thesis, I introduce the first synthetic genetic system implementing population-based AND logic for programmed hierarchical patterning of bacterial populations of Escherichia coli, and address fundamental prerequisites for implementation of an analogous genetic circuit into the emergent multicellular plant model Marchantia polymorpha. In both model systems, I explore the use of bacteriophage T7 RNA polymerase as a gene expression engine to control synthetic patterning across populations of cells. In E. coli, I developed a ratiometric assay of bacteriophage T7 RNA polymerase activity, which I used to systematically characterize different intact and split enzyme variants. I utilized the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. I validated the AND gate-like behavior of this system both in cell suspension and in surface culture. Then, I used the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations. To prepare the adaption of bacteriophage T7 RNA polymerase-driven synthetic patterning from the prokaryote E. coli to the eukaryote M. polymorpha, I developed a toolbox of genetic elements for spatial gene expression control in the liverwort: I analyzed codon usage across the transcriptome of M. polymorpha, and used insights gained to design codon-optimized fluorescent reporters successfully expressed from its nuclear and chloroplast genomes. For targeting of bacteriophage T7 RNA polymerase to these cellular compartments, I functionally validated nuclear localization signals and chloroplast transit peptides. For spatiotemporal control of bacteriophage T7 RNA polymerase in M. polymorpha, I characterized spatially restricted and inducible promoters. For facilitated posttranscriptional processing of target transcripts, I functionally validated viral enhancer sequences in M. polymorpha. Taking advantage of this genetic toolbox, I introduced inducible nuclear-targeted bacteriophage T7 RNA polymerase into M. polymorpha. I showed implementation of the bacteriophage T7 RNA polymerase/PT7 expression system accompanied by hypermethylation of its target nuclear transgene. My observations suggest operation of efficient epigenetic gene silencing in M. polymorpha, and guide future efforts in chassis engineering of this multicellular plant model. Furthermore, my results encourage utilization of spatiotemporally controlled bacteriophage T7 RNA polymerase as a targeted silencing system for functional genomic studies and morphogenetic engineering in the liverwort. Taken together, the work presented enhances our capacity for spatiotemporal gene expression control in bacterial populations and plants, facilitating future efforts in synthetic morphogenesis for applications in synthetic biology and metabolic engineering.

No description available.

Deux approches principales existent pour évaluer la robustesse des codes génétiques et des séquences de codage. L'approche statistique est basée sur des estimations empiriques de probabilité calculées à partir d'échantillons aléatoires de permutations représentant les affectations d'acides aminés aux codons, alors que l'approche basée sur l'optimisation repose sur le pourcentage d’optimisation, généralement calculé en utilisant des métaheuristiques. Nous proposons une méthode basée sur les deux premiers moments de la distribution des valeurs de robustesse pour tous les codes génétiques possibles. En se basant sur une instance polynomiale du Problème d'Affectation Quadratique, nous proposons un algorithme vorace exact pour trouver la valeur minimale de la robustesse génomique. Pour réduire le nombre d'opérations de calcul des scores et de la borne supérieure de Cantelli, nous avons développé des méthodes basées sur la structure de voisinage du code génétique et sur la comparaison par paires des codes génétiques, entre autres. Pour calculer la robustesse des codes génétiques naturels et des génomes procaryotes, nous avons choisi 23 codes génétiques naturels, 235 propriétés d'acides aminés, ainsi que 324 procaryotes thermophiles et 418 procaryotes non thermophiles. Parmi nos résultats, nous avons constaté que bien que le code génétique standard soit plus robuste que la plupart des codes génétiques, certains codes génétiques mitochondriaux et nucléaires sont plus robustes que le code standard aux troisièmes et premières positions des codons, respectivement. Nous avons observé que l'utilisation des codons synonymes tend à être fortement optimisée pour amortir l'impact des changements d'une seule base, principalement chez les procaryotes thermophiles. / There are two main approaches to assess the robustness of genetic codes and coding sequences. The statistical approach is based on empirical estimates of probabilities computed from random samples of permutations representing assignments of amino acids to codons, whereas, the optimization-based approach relies on the optimization percentage frequently computed by using metaheuristics. We propose a method based on the first two moments of the distribution of robustness values for all possible genetic codes. Based on a polynomially solvable instance of the Quadratic Assignment Problem, we propose also an exact greedy algorithm to find the minimum value of the genome robustness. To reduce the number of operations for computing the scores and Cantelli’s upper bound, we developed methods based on the genetic code neighborhood structure and pairwise comparisons between genetic codes, among others. For assessing the robustness of natural genetic codes and genomes, we have chosen 23 natural genetic codes, 235 amino acid properties, as well as 324 thermophilic and 418 non-thermophilic prokaryotes. Among our results, we found that although the standard genetic code is more robust than most genetic codes, some mitochondrial and nuclear genetic codes are more robust than the standard code at the third and first codon positions, respectively. We also observed that the synonymous codon usage tends to be highly optimized to buffer the impact of single-base changes, mainly, in thermophilic prokaryotes.

No / Heterozygous germline defects in a gene encoding a type II receptor for bone morphogenetic proteins (BMPR-II) underlie the majority of inherited cases of the vascular disorder known as pulmonary arterial hypertension (PAH). However, the precise molecular consequences of PAH causing mutations on the function of the receptor complex remain unclear. We employed novel enzymatic and fluorescence activity based techniques to assess the impact of PAH mutations on pre-mRNA splicing, nonsense-mediated decay (NMD) and receptor complex interactions. We demonstrate that nonsense and frameshift mutations trigger NMD, providing further evidence that haplo-insufficiency is a major molecular consequence of disease-related BMPR2 mutations. We identified heterogeneous functional defects in BMPR-II activity, including impaired type I receptor phosphorylation, receptor interactions and altered receptor complex stoichiometry leading to perturbation of downstream signalling pathways. Importantly, these studies demonstrate that the intracellular domain of BMPR-II is both necessary and sufficient for receptor complex interaction. Finally and to address the potential for resolution of stoichiometric balance, we investigated an agent that promotes translational readthrough of a BMPR2 nonsense reporter construct without interfering with the NMD pathway. We propose that stoichiometric imbalance, due to either haplo-insufficiency or loss of optimal receptor-receptor interactions impairs BMPR-II mediated signalling in PAH. Taken together, these studies have identified an important target for early therapeutic intervention in familial PAH.