Molecular screening of ADAMTSL2 gene in 33 patients reveals the genetic heterogeneity of geleophysic dysplasia.

Background Geleophysic dysplasia (GD, OMIM 231050) is an autosomal recessive disorder characterised by short stature, small hands and feet, stiff joints, and thick skin. Patients often present with a progressive cardiac valvular disease which can lead to an early death. In a previous study including six GD families, we have mapped the disease gene on chromosome 9q34.2 and identified mutations in the A Disintegrin And Metalloproteinase with Thrombospondin repeats-like 2 gene (ADAMTSL2). Methods Following this study, we have collected the samples of 30 additional GD families, including 33 patients and identified ADAMTSL2 mutations in 14/33 patients, comprising 13 novel mutations. The absence of mutation in 19 patients prompted us to compare the two groups of GD patients, namely group 1, patients with ADAMTSL2 mutations (n=20, also including the 6 patients from our previous study), and group 2, patients without ADAMTSL2 mutations (n=19). Results The main discriminating features were facial dysmorphism and tip-toe walking, which were almost constantly observed in group 1. No differences were found concerning heart involvement, skin thickness, recurrent respiratory and ear infections, bronchopulmonary insufficiency, laryngo-tracheal stenosis, deafness, and radiographic features. Conclusions It is concluded that GD is a genetically heterogeneous condition. Ongoing studies will hopefully lead to the identification of another disease gene.

Integrated Analysis of Molecular and Clinical Prognostic Factors in Stage II/III Colon Cancer.

Background The prognostic potential of individual clinical and molecular parameters in stage II/III colon cancer has been investigated, but a thorough multivariable assessment of their relative impact is missing. Methods Tumors from patients (N = 1404) in the PETACC3 adjuvant chemotherapy trial were examined for BRAF and KRAS mutations, microsatellite instability (MSI), chromosome 18q loss of heterozygosity (18qLOH), and SMAD4 expression. Their importance in predicting relapse-free survival (RFS) and overall survival (OS) was assessed by Kaplan-Meier analyses, Cox regression models, and recursive partitioning trees. All statistical tests were two-sided. Results MSI-high status and SMAD4 focal loss of expression were identified as independent prognostic factors with better RFS (hazard ratio [HR] of recurrence = 0.54, 95% CI = 0.37 to 0.81, P = .003) and OS (HR of death = 0.43, 95% CI = 0.27 to 0.70, P = .001) for MSI-high status and worse RFS (HR = 1.47, 95% CI = 1.19 to 1.81, P < .001) and OS (HR = 1.58, 95% CI = 1.23 to 2.01, P < .001) for SMAD4 loss. 18qLOH did not have any prognostic value in RFS or OS. Recursive partitioning identified refinements of TNM into new clinically interesting prognostic subgroups. Notably, T3N1 tumors with MSI-high status and retained SMAD4 expression had outcomes similar to stage II disease. Conclusions Concomitant assessment of molecular and clinical markers in multivariable analysis is essential to confirm or refute their independent prognostic value. Including molecular markers with independent prognostic value might allow more accurate prediction of prognosis than TNM staging alone.

Molecular modeling study of the binding and signaling properties of the TCRpMHC complex

The activation of the specific immune response against tumor cells is based on the recognition by the CD8+ Cytotoxic Τ Lymphocytes (CTL), of antigenic peptides (p) presented at the surface of the cell by the class I major histocompatibility complex (MHC). The ability of the so-called T-Cell Receptors (TCR) to discriminate between self and non-self peptides constitutes the most important specific control mechanism against infected cells. The TCR/pMHC interaction has been the subject of much attention in cancer therapy since the design of the adoptive transfer approach, in which Τ lymphocytes presenting an interesting response against tumor cells are extracted from the patient, expanded in vitro, and reinfused after immunodepletion, possibly leading to cancer regression. In the last decade, major progress has been achieved by the introduction of engineered lypmhocytes. In the meantime, the understanding of the molecular aspects of the TCRpMHC interaction has become essential to guide in vitro and in vivo studies. In 1996, the determination of the first structure of a TCRpMHC complex by X-ray crystallography revealed the molecular basis of the interaction. Since then, molecular modeling techniques have taken advantage of crystal structures to study the conformational space of the complex, and understand the specificity of the recognition of the pMHC by the TCR. In the meantime, experimental techniques used to determine the sequences of TCR that bind to a pMHC complex have been used intensively, leading to the collection of large repertoires of TCR sequences that are specific for a given pMHC. There is a growing need for computational approaches capable of predicting the molecular interactions that occur upon TCR/pMHC binding without relying on the time consuming resolution of a crystal structure. This work presents new approaches to analyze the molecular principles that govern the recognition of the pMHC by the TCR and the subsequent activation of the T-cell. We first introduce TCRep 3D, a new method to model and study the structural properties of TCR repertoires, based on homology and ab initio modeling. We discuss the methodology in details, and demonstrate that it outperforms state of the art modeling methods in predicting relevant TCR conformations. Two successful applications of TCRep 3D that supported experimental studies on TCR repertoires are presented. Second, we present a rigid body study of TCRpMHC complexes that gives a fair insight on the TCR approach towards pMHC. We show that the binding mode of the TCR is correctly described by long-distance interactions. Finally, the last section is dedicated to a detailed analysis of an experimental hydrogen exchange study, which suggests that some regions of the constant domain of the TCR are subject to conformational changes upon binding to the pMHC. We propose a hypothesis of the structural signaling of TCR molecules leading to the activation of the T-cell. It is based on the analysis of correlated motions in the TCRpMHC structure. - L'activation de la réponse immunitaire spécifique dirigée contre les cellules tumorales est basée sur la reconnaissance par les Lymphocytes Τ Cytotoxiques (CTL), d'un peptide antigénique (p) présenté à la suface de la cellule par le complexe majeur d'histocompatibilité de classe I (MHC). La capacité des récepteurs des lymphocytes (TCR) à distinguer les peptides endogènes des peptides étrangers constitue le mécanisme de contrôle le plus important dirigé contre les cellules infectées. L'interaction entre le TCR et le pMHC est le sujet de beaucoup d'attention dans la thérapie du cancer, depuis la conception de la méthode de transfer adoptif: les lymphocytes capables d'une réponse importante contre les cellules tumorales sont extraits du patient, amplifiés in vitro, et réintroduits après immunosuppression. Il peut en résulter une régression du cancer. Ces dix dernières années, d'importants progrès ont été réalisés grâce à l'introduction de lymphocytes modifiés par génie génétique. En parallèle, la compréhension du TCRpMHC au niveau moléculaire est donc devenue essentielle pour soutenir les études in vitro et in vivo. En 1996, l'obtention de la première structure du complexe TCRpMHC à l'aide de la cristallographie par rayons X a révélé les bases moléculaires de l'interaction. Depuis lors, les techniques de modélisation moléculaire ont exploité les structures expérimentales pour comprendre la spécificité de la reconnaissance du pMHC par le TCR. Dans le même temps, de nouvelles techniques expérimentales permettant de déterminer la séquence de TCR spécifiques envers un pMHC donné, ont été largement exploitées. Ainsi, d'importants répertoires de TCR sont devenus disponibles, et il est plus que jamais nécessaire de développer des approches informatiques capables de prédire les interactions moléculaires qui ont lieu lors de la liaison du TCR au pMHC, et ce sans dépendre systématiquement de la résolution d'une structure cristalline. Ce mémoire présente une nouvelle approche pour analyser les principes moléculaires régissant la reconnaissance du pMHC par le TCR, et l'activation du lymphocyte qui en résulte. Dans un premier temps, nous présentons TCRep 3D, une nouvelle méthode basée sur les modélisations par homologie et ab initio, pour l'étude de propriétés structurales des répertoires de TCR. Le procédé est discuté en détails et comparé à des approches standard. Nous démontrons ainsi que TCRep 3D est le plus performant pour prédire des conformations pertinentes du TCR. Deux applications à des études expérimentales des répertoires TCR sont ensuite présentées. Dans la seconde partie de ce travail nous présentons une étude de complexes TCRpMHC qui donne un aperçu intéressant du mécanisme d'approche du pMHC par le TCR. Finalement, la dernière section se concentre sur l'analyse détaillée d'une étude expérimentale basée sur les échanges deuterium/hydrogène, dont les résultats révèlent que certaines régions clés du domaine constant du TCR sont sujettes à un changement conformationnel lors de la liaison au pMHC. Nous proposons une hypothèse pour la signalisation structurelle des TCR, menant à l'activation du lymphocyte. Celle-ci est basée sur l'analyse des mouvements corrélés observés dans la structure du TCRpMHC.

Definition of clinically distinct molecular subtypes in estrogen receptor-positive breast carcinomas through genomic grade.

PURPOSE: A number of microarray studies have reported distinct molecular profiles of breast cancers (BC), such as basal-like, ErbB2-like, and two to three luminal-like subtypes. These were associated with different clinical outcomes. However, although the basal and the ErbB2 subtypes are repeatedly recognized, identification of estrogen receptor (ER) -positive subtypes has been inconsistent. Therefore, refinement of their molecular definition is needed. MATERIALS AND METHODS: We have previously reported a gene expression grade index (GGI), which defines histologic grade based on gene expression profiles. Using this algorithm, we assigned ER-positive BC to either high-or low-genomic grade subgroups and compared these with previously reported ER-positive molecular classifications. As further validation, we classified 666 ER-positive samples into subtypes and assessed their clinical outcome. RESULTS: Two ER-positive molecular subgroups (high and low genomic grade) could be defined using the GGI. Despite tracking a single biologic pathway, these were highly comparable to the previously described luminal A and B classification and significantly correlated to the risk groups produced using the 21-gene recurrence score. The two subtypes were associated with statistically distinct clinical outcome in both systemically untreated and tamoxifen-treated populations. CONCLUSION: The use of genomic grade can identify two clinically distinct ER-positive molecular subtypes in a simple and highly reproducible manner across multiple data sets. This study emphasizes the important role of proliferation-related genes in predicting prognosis in ER-positive BC.

Cost-effectiveness of low-molecular-weight heparin for treatment of pulmonary embolism.

BACKGROUND: Low-molecular-weight heparin (LMWH) appears to be safe and effective for treating pulmonary embolism (PE), but its cost-effectiveness has not been assessed. METHODS: We built a Markov state-transition model to evaluate the medical and economic outcomes of a 6-day course with fixed-dose LMWH or adjusted-dose unfractionated heparin (UFH) in a hypothetical cohort of 60-year-old patients with acute submassive PE. Probabilities for clinical outcomes were obtained from a meta-analysis of clinical trials. Cost estimates were derived from Medicare reimbursement data and other sources. The base-case analysis used an inpatient setting, whereas secondary analyses examined early discharge and outpatient treatment with LMWH. Using a societal perspective, strategies were compared based on lifetime costs, quality-adjusted life-years (QALYs), and the incremental cost-effectiveness ratio. RESULTS: Inpatient treatment costs were higher for LMWH treatment than for UFH (dollar 13,001 vs dollar 12,780), but LMWH yielded a greater number of QALYs than did UFH (7.677 QALYs vs 7.493 QALYs). The incremental costs of dollar 221 and the corresponding incremental effectiveness of 0.184 QALYs resulted in an incremental cost-effectiveness ratio of dollar 1,209/QALY. Our results were highly robust in sensitivity analyses. LMWH became cost-saving if the daily pharmacy costs for LMWH were < dollar 51, if > or = 8% of patients were eligible for early discharge, or if > or = 5% of patients could be treated entirely as outpatients. CONCLUSION: For inpatient treatment of PE, the use of LMWH is cost-effective compared to UFH. Early discharge or outpatient treatment in suitable patients with PE would lead to substantial cost savings.

Molecular and functional characterization of a novel cardiac-specific human tropomyosin isoform.

BACKGROUND: Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1alpha (also called alpha-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. METHODS AND RESULTS: In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1kappa. By developing a TPM1kappa-specific antibody, we found that the TPM1kappa protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1kappa in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1kappa. Incorporation of increased levels of TPM1kappa protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1kappa compared with TPM1alpha. CONCLUSIONS: This functional analysis of TPM1kappa provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients.

RAX and anophthalmia in humans: Evidence of brain anomalies.

PURPOSE: To report the clinical and genetic study of two families of Egyptian origin with clinical anophthalmia. To further determine the role of the retina and anterior neural fold homeobox gene (RAX) in anophthalmia and associated cerebral malformations. METHODS: Three patients with clinical anophthalmia and first-degree relatives from two consanguineous families of Egyptian origin underwent full ophthalmologic, general and neurologic examination, and blood tests. Cerebral magnetic resonance imaging (MRI) was performed in the index cases of both families. Genomic DNA was prepared from venous leukocytes, and direct sequencing of all the exons and intron-exon junctions of RAX was performed after PCR amplification. RESULTS: Clinical bilateral anophthalmia was observed in all three patients. General and neurologic examinations were normal; obesity and delay in psychomotor development were observed in the isolated case. Orbital MRI showed a hypoplastic orbit with present but rudimentary extraocular muscles and normal lacrimal glands. Cerebral MRI showed agenesis of the optic nerves, optic tracts, and optic chiasma. In the index case of family A, the absence of the frontal and sphenoidal sinuses was also noted. In the index case of family B, only the sphenoidal sinus was absent, and there was significant cortical atrophy. The three patients carried a novel homozygous c.543+3A>G mutation (IVS2+3A>G) in RAX. Parents were healthy heterozygous carriers. No mutations were detected in orthodenticle homeobox 2 (OTX2), ventral anterior homeobox 1 (VAX1), or sex determining region Y-box 2 (SOX2). CONCLUSIONS: This is the first report of a homozygous splicing RAX mutation associated with autosomal recessive bilateral anophthalmia. To our knowledge, only two isolated cases of anophthalmia, three null and one missense case affecting nuclear localization or the DNA-binding homeodomain, have been found to be caused by compound heterozygote RAX mutations. A novel missense RAX mutation was identified in three patients with bilateral anophthalmia and a distinct systemic and neurologic phenotype. The mutation potentially affects splicing of the last exon and is thought to result in a protein that has an aberrant homeodomain and no paired-tail domain. Functional consequences of this change still need to be characterized.

Clinicopathologic and molecular analysis of a choroidal pigmented schwannoma in the context of a PTEN hamartoma tumor syndrome.

PURPOSE: To report the first case of choroidal schwannoma in a patient affected by PTEN hamartoma tumor syndrome (PHTS) and investigate the molecular involvement of the phosphatase and tensin homolog (PTEN) and neurofibromin 2 (NF2) genes in this rare intraocular tumor. DESIGN: Observational case report. PARTICIPANT: A 10-year-old girl diagnosed with PHTS. METHODS: The enucleated specimen underwent histologic, immunohistochemical, and transmission electronic microscopy. The expression of PTEN and NF2 and their protein products were evaluated by reverse transcription-polymerase chain reaction and immunohistochemistry. Somatic mutations of PTEN and NF2, as well as allelic loss, were investigated by direct sequencing of DNA extracted from the tumor. PTEN epigenetic silencing was investigated by pyrosequencing. MAIN OUTCOME MEASURES: Histopathologic and molecular characterization of a choroidal pigmented schwannoma. RESULTS: Histopathologic, immunohistochemical, and electron microscopic analysis demonstrated features consistent with a pigmented cellular schwannoma of the choroid. We found no loss of heterozygosity at the genomic level for the PTEN germline mutation and no promoter hypermethylation or other somatic intragenic mutations. However, we observed an approximate 40% reduction of PTEN expression at both the mRNA and the protein level, indicating that the tumor was nonetheless functionally deficient for PTEN. Although DNA sequencing of NF2 failed to identify any pathologic variants, its expression was abolished within the tumor. CONCLUSIONS: We report the first description of a pigmented choroidal schwannoma in the context of a PHTS. This rare tumor showed a unique combination of reduction of PTEN and absence of NF2 expression.

Distal and proximal colon cancers differ in terms of molecular, pathological, and clinical features.

BACKGROUND: Differences exist between the proximal and distal colon in terms of developmental origin, exposure to patterning genes, environmental mutagens, and gut flora. Little is known on how these differences may affect mechanisms of tumorigenesis, side-specific therapy response or prognosis. We explored systematic differences in pathway activation and their clinical implications. MATERIALS AND METHODS: Detailed clinicopathological data for 3045 colon carcinoma patients enrolled in the PETACC3 adjuvant chemotherapy trial were available for analysis. A subset of 1404 samples had molecular data, including gene expression and DNA copy number profiles for 589 and 199 samples, respectively. In addition, 413 colon adenocarcinoma from TCGA collection were also analyzed. Tumor side-effect on anti-epidermal growth factor receptor (EGFR) therapy was assessed in a cohort of 325 metastatic patients. Outcome variables considered were relapse-free survival and survival after relapse (SAR). RESULTS: Proximal carcinomas were more often mucinous, microsatellite instable (MSI)-high, mutated in key tumorigenic pathways, expressed a B-Raf proto-oncogene, serine/threonine kinase (BRAF)-like and a serrated pathway signature, regardless of histological type. Distal carcinomas were more often chromosome instable and EGFR or human epidermal growth factor receptor 2 (HER2) amplified, and more frequently overexpressed epiregulin. While risk of relapse was not different per side, SAR was much poorer for proximal than for distal stage III carcinomas in a multivariable model including BRAF mutation status [N = 285; HR 1.95, 95% CI (1.6-2.4), P < 0.001]. Only patients with metastases from a distal carcinoma responded to anti-EGFR therapy, in line with the predictions of our pathway enrichment analysis. CONCLUSIONS: Colorectal carcinoma side is associated with differences in key molecular features, some immediately druggable, with important prognostic effects which are maintained in metastatic lesions. Although within side significant molecular heterogeneity remains, our findings justify stratification of patients by side for retrospective and prospective analyses of drug efficacy and prognosis.

A comparison of methods for differential expression analysis of RNA-seq data.

BACKGROUND: Finding genes that are differentially expressed between conditions is an integral part of understanding the molecular basis of phenotypic variation. In the past decades, DNA microarrays have been used extensively to quantify the abundance of mRNA corresponding to different genes, and more recently high-throughput sequencing of cDNA (RNA-seq) has emerged as a powerful competitor. As the cost of sequencing decreases, it is conceivable that the use of RNA-seq for differential expression analysis will increase rapidly. To exploit the possibilities and address the challenges posed by this relatively new type of data, a number of software packages have been developed especially for differential expression analysis of RNA-seq data. RESULTS: We conducted an extensive comparison of eleven methods for differential expression analysis of RNA-seq data. All methods are freely available within the R framework and take as input a matrix of counts, i.e. the number of reads mapping to each genomic feature of interest in each of a number of samples. We evaluate the methods based on both simulated data and real RNA-seq data. CONCLUSIONS: Very small sample sizes, which are still common in RNA-seq experiments, impose problems for all evaluated methods and any results obtained under such conditions should be interpreted with caution. For larger sample sizes, the methods combining a variance-stabilizing transformation with the 'limma' method for differential expression analysis perform well under many different conditions, as does the nonparametric SAMseq method.

Transmission of HIV-1 drug resistance in Switzerland: a 10-year molecular epidemiology survey.

OBJECTIVES: Representative prevalence data of transmitted drug-resistant HIV-1 are essential to establish accurate guidelines addressing resistance testing and first-line treatments. METHODS: Systematic resistance testing was carried out in individuals in Switzerland with documented HIV-1 seroconversion during 1996-2005 and available samples with RNA > 1000 copies/ml obtained within 1 year of estimated seroconversion. Resistance interpretation used the Stanford list of mutations for surveillance of transmitted drug resistance and the French National Agency for AIDS Research algorithm. RESULTS: Viral sequences from 822 individuals were available. Risk groups were men having sex with men (42%), heterosexual contacts (32%) and intravenous drug users (20%); 30% were infected with non-B subtype viruses. Overall, prevalence of transmitted resistance was 7.7% [95% confidence interval (CI), 5.9-9.5] for any drug, 5.5% (95% CI, 3.9-7.1) for nucleoside reverse transcriptase inhibitors, 1.9% (95% CI, 1.0-2.8) for non-nucleoside reverse transcriptase inhibitors and 2.7% (95% CI, 1.6-3.8) for protease inhibitors. Dual- or triple-class resistance was observed in 2% (95% CI, 0.8-2.5). No significant trend in prevalence of transmitted resistance was observed over years. There were no differences according to ethnicity, risk groups or gender, but prevalence of transmitted resistance was highest among individuals infected with subtype B virus. CONCLUSIONS: The transmission rate of drug-resistant HIV-1 has been stable since 1996, with very rare transmission of dual- or triple-class resistance. These data suggest that transmission of drug resistance in the setting of easy access to antiretroviral treatment can remain stable and be kept at a low level.

Clinical, molecular, and prognostic significance of WHO type inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and various other 3q abnormalities in acute myeloid leukemia.

PURPOSE: Acute myeloid leukemia (AML) with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) [inv(3)/t(3;3)] is recognized as a distinctive entity in the WHO classification. Risk assignment and clinical and genetic characterization of AML with chromosome 3q abnormalities other than inv(3)/t(3;3) remain largely unresolved. PATIENTS AND METHODS: Cytogenetics, molecular genetics, therapy response, and outcome analysis were performed in 6,515 newly diagnosed adult AML patients. Patients were treated on Dutch-Belgian Hemato-Oncology Cooperative Group/Swiss Group for Clinical Cancer Research (HOVON/SAKK; n = 3,501) and German-Austrian Acute Myeloid Leukemia Study Group (AMLSG; n = 3,014) protocols. EVI1 and MDS1/EVI1 expression was determined by real-time quantitative polymerase chain reaction. RESULTS: 3q abnormalities were detected in 4.4% of AML patients (288 of 6,515). Four distinct groups were defined: A: inv(3)/t(3;3), 32%; B: balanced t(3q26), 18%; C: balanced t(3q21), 7%; and D: other 3q abnormalities, 43%. Monosomy 7 was the most common additional aberration in groups (A), 66%; (B), 31%; and (D), 37%. N-RAS mutations and dissociate EVI1 versus MDS1/EVI1 overexpression were associated with inv(3)/t(3;3). Patients with inv(3)/t(3;3) and balanced t(3q21) at diagnosis presented with higher WBC and platelet counts. In multivariable analysis, only inv(3)/t(3;3), but not t(3q26) and t(3q21), predicted reduced relapse-free survival (hazard ratio [HR], 1.99; P < .001) and overall survival (HR, 1.4; P = .006). This adverse prognostic impact of inv(3)/t(3;3) was enhanced by additional monosomy 7. Group D 3q aberrant AML also had a poor outcome related to the coexistence of complex and/or monosomal karyotypes and cryptic inv(3)/t(3;3). CONCLUSION: Various categories of 3q abnormalities in AML can be distinguished according to their clinical, hematologic, and genetic features. AML with inv(3)/t(3;3) represents a distinctive subgroup with unfavorable prognosis.

Broadening the horizon--level 2.5 of the HUPO-PSI format for molecular interactions.

BACKGROUND: Molecular interaction Information is a key resource in modern biomedical research. Publicly available data have previously been provided in a broad array of diverse formats, making access to this very difficult. The publication and wide implementation of the Human Proteome Organisation Proteomics Standards Initiative Molecular Interactions (HUPO PSI-MI) format in 2004 was a major step towards the establishment of a single, unified format by which molecular interactions should be presented, but focused purely on protein-protein interactions. RESULTS: The HUPO-PSI has further developed the PSI-MI XML schema to enable the description of interactions between a wider range of molecular types, for example nucleic acids, chemical entities, and molecular complexes. Extensive details about each supported molecular interaction can now be captured, including the biological role of each molecule within that interaction, detailed description of interacting domains, and the kinetic parameters of the interaction. The format is supported by data management and analysis tools and has been adopted by major interaction data providers. Additionally, a simpler, tab-delimited format MITAB2.5 has been developed for the benefit of users who require only minimal information in an easy to access configuration. CONCLUSION: The PSI-MI XML2.5 and MITAB2.5 formats have been jointly developed by interaction data producers and providers from both the academic and commercial sector, and are already widely implemented and well supported by an active development community. PSI-MI XML2.5 enables the description of highly detailed molecular interaction data and facilitates data exchange between databases and users without loss of information. MITAB2.5 is a simpler format appropriate for fast Perl parsing or loading into Microsoft Excel.

Molecular modeling of peptide-MHC class I complexes : applications to peptide based immunotherapy

Summary The specific CD8+ T cell immune response against tumors relies on the recognition by the T cell receptor (TCR) on cytotoxic T lymphocytes (CTL) of antigenic peptides bound to the class I major histocompatibility complex (MHC) molecule. Such tumor associated antigenic peptides are the focus of tumor immunotherapy with peptide vaccines. The strategy for obtaining an improved immune response often involves the design of modified tumor associated antigenic peptides. Such modifications aim at creating higher affinity and/or degradation resistant peptides and require precise structures of the peptide-MHC class I complex. In addition, the modified peptide must be cross-recognized by CTLs specific for the parental peptide, i.e. preserve the structure of the epitope. Detailed structural information on the modified peptide in complex with MHC is necessary for such predictions. In this thesis, the main focus is the development of theoretical in silico methods for prediction of both structure and cross-reactivity of peptide-MHC class I complexes. Applications of these methods in the context of immunotherapy are also presented. First, a theoretical method for structure prediction of peptide-MHC class I complexes is developed and validated. The approach is based on a molecular dynamics protocol to sample the conformational space of the peptide in its MHC environment. The sampled conformers are evaluated using conformational free energy calculations. The method, which is evaluated for its ability to reproduce 41 X-ray crystallographic structures of different peptide-MHC class I complexes, shows an overall prediction success of 83%. Importantly, in the clinically highly relevant subset of peptide-HLAA*0201 complexes, the prediction success is 100%. Based on these structure predictions, a theoretical approach for prediction of cross-reactivity is developed and validated. This method involves the generation of quantitative structure-activity relationships using three-dimensional molecular descriptors and a genetic neural network. The generated relationships are highly predictive as proved by high cross-validated correlation coefficients (0.78-0.79). Together, the here developed theoretical methods open the door for efficient rational design of improved peptides to be used in immunotherapy. Résumé La réponse immunitaire spécifique contre des tumeurs dépend de la reconnaissance par les récepteurs des cellules T CD8+ de peptides antigéniques présentés par les complexes majeurs d'histocompatibilité (CMH) de classe I. Ces peptides sont utilisés comme cible dans l'immunothérapie par vaccins peptidiques. Afin d'augmenter la réponse immunitaire, les peptides sont modifiés de façon à améliorer l'affinité et/ou la résistance à la dégradation. Ceci nécessite de connaître la structure tridimensionnelle des complexes peptide-CMH. De plus, les peptides modifiés doivent être reconnus par des cellules T spécifiques du peptide natif. La structure de l'épitope doit donc être préservée et des structures détaillées des complexes peptide-CMH sont nécessaires. Dans cette thèse, le thème central est le développement des méthodes computationnelles de prédiction des structures des complexes peptide-CMH classe I et de la reconnaissance croisée. Des applications de ces méthodes de prédiction à l'immunothérapie sont également présentées. Premièrement, une méthode théorique de prédiction des structures des complexes peptide-CMH classe I est développée et validée. Cette méthode est basée sur un échantillonnage de l'espace conformationnel du peptide dans le contexte du récepteur CMH classe I par dynamique moléculaire. Les conformations sont évaluées par leurs énergies libres conformationnelles. La méthode est validée par sa capacité à reproduire 41 structures des complexes peptide-CMH classe I obtenues par cristallographie aux rayons X. Le succès prédictif général est de 83%. Pour le sous-groupe HLA-A*0201 de complexes de grande importance pour l'immunothérapie, ce succès est de 100%. Deuxièmement, à partir de ces structures prédites in silico, une méthode théorique de prédiction de la reconnaissance croisée est développée et validée. Celle-ci consiste à générer des relations structure-activité quantitatives en utilisant des descripteurs moléculaires tridimensionnels et un réseau de neurones couplé à un algorithme génétique. Les relations générées montrent une capacité de prédiction remarquable avec des valeurs de coefficients de corrélation de validation croisée élevées (0.78-0.79). Les méthodes théoriques développées dans le cadre de cette thèse ouvrent la voie du design de vaccins peptidiques améliorés.

Phenotypic and molecular assessment of seven patients with 6p25 deletion syndrome: relevance to ocular dysgenesis and hearing impairment.

BACKGROUND: Thirty-nine patients have been described with deletions involving chromosome 6p25. However, relatively few of these deletions have had molecular characterization. Common phenotypes of 6p25 deletion syndrome patients include hydrocephalus, hearing loss, and ocular, craniofacial, skeletal, cardiac, and renal malformations. Molecular characterization of deletions can identify genes that are responsible for these phenotypes. METHODS: We report the clinical phenotype of seven patients with terminal deletions of chromosome 6p25 and compare them to previously reported patients. Molecular characterization of the deletions was performed using polymorphic marker analysis to determine the extents of the deletions in these seven 6p25 deletion syndrome patients. RESULTS: Our results, and previous data, show that ocular dysgenesis and hearing impairment are the two most highly penetrant phenotypes of the 6p25 deletion syndrome. While deletion of the forkhead box C1 gene (FOXC1) probably underlies the ocular dysgenesis, no gene in this region is known to be involved in hearing impairment. CONCLUSIONS: Ocular dysgenesis and hearing impairment are the two most common phenotypes of 6p25 deletion syndrome. We conclude that a locus for dominant hearing loss is present at 6p25 and that this locus is restricted to a region distal to D6S1617. Molecular characterization of more 6p25 deletion patients will aid in refinement of this locus and the identification of a gene involved in dominant hearing loss.