Highly parallel genetic approaches in Mendelian and complex diseases

The recent advance in high-throughput sequencing and genotyping protocols allows rapid investigation of Mendelian and complex diseases on a scale not previously been possible. In my thesis research I took advantage of these modern techniques to study retinitis pigmentosa (RP), a rare inherited disease characterized by progressive loss of photoreceptors and leading to blindness; and hypertension, a common condition affecting 30% of the adult population. Firstly, I compared the performance of different next generation sequencing (NGS) platforms in the sequencing of the RP-linked gene PRPF31. The gene contained a mutation in an intronic repetitive element, which presented difficulties for both classic sequencing methods and NGS. We showed that all NGS platforms are powerful tools to identify rare and common DNA variants, also in case of more complex sequences. Moreover, we evaluated the features of different NGS platforms that are important in re-sequencing projects. The main focus of my thesis was then to investigate the involvement of pre-mRNA splicing factors in autosomal dominant RP (adRP). I screened 5 candidate genes in a large cohort of patients by using long-range PCR as enrichment step, followed by NGS. We tested two different approaches: in one, all target PCRs from all patients were pooled and sequenced as a single DNA library; in the other, PCRs from each patient were separated within the pool by DNA barcodes. The first solution was more cost-effective, while the second one allowed obtaining faster and more accurate results, but overall they both proved to be effective strategies for gene screenings in many samples. We could in fact identify novel missense mutations in the SNRNP200 gene, encoding an essential RNA helicase for splicing catalysis. Interestingly, one of these mutations showed incomplete penetrance in one family with adRP. Thus, we started to study the possible molecular causes underlying phenotypic differences between asymptomatic and affected members of this family. For the study of hypertension, I joined a European consortium to perform genome-wide association studies (GWAS). Thanks to the use of very informative genotyping arrays and of phenotipically well-characterized cohorts, we could identify a novel susceptibility locus for hypertension in the promoter region of the endothelial nitric oxide synthase gene (NOS3). Moreover, we have proven the direct causality of the associated SNP using three different methods: 1) targeted resequencing, 2) luciferase assay, and 3) population study. - Le récent progrès dans le Séquençage à haut Débit et les protocoles de génotypage a permis une plus vaste et rapide étude des maladies mendéliennes et multifactorielles à une échelle encore jamais atteinte. Durant ma thèse de recherche, j'ai utilisé ces nouvelles techniques de séquençage afin d'étudier la retinite pigmentale (RP), une maladie héréditaire rare caractérisée par une perte progressive des photorécepteurs de l'oeil qui entraine la cécité; et l'hypertension, une maladie commune touchant 30% de la population adulte. Tout d'abord, j'ai effectué une comparaison des performances de différentes plateformes de séquençage NGS (Next Generation Sequencing) lors du séquençage de PRPF31, un gène lié à RP. Ce gène contenait une mutation dans un élément répétable intronique, qui présentait des difficultés de séquençage avec la méthode classique et les NGS. Nous avons montré que les plateformes de NGS analysées sont des outils très puissants pour identifier des variations de l'ADN rares ou communes et aussi dans le cas de séquences complexes. De plus, nous avons exploré les caractéristiques des différentes plateformes NGS qui sont importantes dans les projets de re-séquençage. L'objectif principal de ma thèse a été ensuite d'examiner l'effet des facteurs d'épissage de pre-ARNm dans une forme autosomale dominante de RP (adRP). Un screening de 5 gènes candidats issus d'une large cohorte de patients a été effectué en utilisant la long-range PCR comme étape d'enrichissement, suivie par séquençage avec NGS. Nous avons testé deux approches différentes : dans la première, toutes les cibles PCRs de tous les patients ont été regroupées et séquencées comme une bibliothèque d'ADN unique; dans la seconde, les PCRs de chaque patient ont été séparées par code barres d'ADN. La première solution a été la plus économique, tandis que la seconde a permis d'obtenir des résultats plus rapides et précis. Dans l'ensemble, ces deux stratégies se sont démontrées efficaces pour le screening de gènes issus de divers échantillons. Nous avons pu identifier des nouvelles mutations faux-sens dans le gène SNRNP200, une hélicase ayant une fonction essentielle dans l'épissage. Il est intéressant de noter qu'une des ces mutations montre une pénétrance incomplète dans une famille atteinte d'adRP. Ainsi, nous avons commencé une étude sur les causes moléculaires entrainant des différences phénotypiques entre membres affectés et asymptomatiques de cette famille. Lors de l'étude de l'hypertension, j'ai rejoint un consortium européen pour réaliser une étude d'association Pangénomique ou genome-wide association study Grâce à l'utilisation de tableaux de génotypage très informatifs et de cohortes extrêmement bien caractérisées au niveau phénotypique, un nouveau locus lié à l'hypertension a été identifié dans la région promotrice du gène endothélial nitric oxide sinthase (NOS3). Par ailleurs, nous avons prouvé la cause directe du SNP associé au moyen de trois méthodes différentes: i) en reséquençant la cible avec NGS, ii) avec des essais à la luciférase et iii) une étude de population.

Adenovirus-mediated gene transfer into selected liver segments using a vascular exclusion technique.

Adenovirus-mediated gene therapy is hampered by severe virus-related toxicity, especially to the liver. The aim of the present study was to test the ability of a vascular exclusion technique to achieve transgene expression within selected liver segments, thus minimizing both viral and transgene product toxicity to the liver. An E1-E3-deleted replication-deficient adenovirus expressing a green fluorescent protein (GFP) reporter gene was injected into the portal vein of BDIX rats, with simultaneous clamping of the portal vein tributaries to liver segments II, III, IV, V, and VIII. GFP expression and inflammatory infiltrate were measured in the different segments of the liver and compared with those of the livers of animals receiving the viral vector in the portal vein without clamping. The GFP expression was significantly higher in the selectively perfused segments of the liver as compared with the non-perfused segments (p < 0.0001) and with the livers of animals that received the vector in the portal vein without clamping (p < 0.0001). Accordingly, the inflammatory infiltrate was more intense in the selectively perfused liver segments as compared with all other groups (p < 0.0001). Fluorescence was absent in lungs and kidneys and minimal in spleen. The clinical usefulness of adenovirus-mediated gene transfer to the liver largely depends on the reduction of its liver toxicity. Clamping of selected portal vein branches during injection allows for delivery of genes of interest to targeted liver segments. Transgene expression confined to selected liver segments may be useful in the treatment of focal liver diseases, including metastases.

Temporal SNR characteristics in segmented 3D-EPI at 7T.

Three-dimensional segmented echo planar imaging (3D-EPI) is a promising approach for high-resolution functional magnetic resonance imaging, as it provides an increased signal-to-noise ratio (SNR) at similar temporal resolution to traditional multislice 2D-EPI readouts. Recently, the 3D-EPI technique has become more frequently used and it is important to better understand its implications for fMRI. In this study, the temporal SNR characteristics of 3D-EPI with varying numbers of segments are studied. It is shown that, in humans, the temporal variance increases with the number of segments used to form the EPI acquisition and that for segmented acquisitions, the maximum available temporal SNR is reduced compared to single shot acquisitions. This reduction with increased segmentation is not found in phantom data and thus likely due to physiological processes. When operating in the thermal noise dominated regime, fMRI experiments with a motor task revealed that the 3D variant outperforms the 2D-EPI in terms of temporal SNR and sensitivity to detect activated brain regions. Thus, the theoretical SNR advantage of a segmented 3D-EPI sequence for fMRI only exists in a low SNR situation. However, other advantages of 3D-EPI, such as the application of parallel imaging techniques in two dimensions and the low specific absorption rate requirements, may encourage the use of the 3D-EPI sequence for fMRI in situations with higher SNR.

A new technique to measure micromotion distribution around a cementless femoral stem.

The interfacial micromotion is closely associated to the long-term success of cementless hip prostheses. Various techniques have been proposed to measure them, but only a few number of points over the stem surface can be measured simultaneously. In this paper, we propose a new technique based on micro-Computer Tomography (μCT) to measure locally the relative interfacial micromotions between the metallic stem and the surrounding femoral bone. Tantalum beads were stuck at the stem surface and spread at the endosteal surface. Relative micromotions between the stem and the endosteal bone surfaces were measured at different loading amplitudes. The estimated error was 10μm and the maximal micromotion was 60μm, in the loading direction, at 1400N. This pilot study provided a local measurement of the micromotions in the 3 direction and at 8 locations on the stem surface simultaneously. This technique could be easily extended to higher loads and a much larger number of points, covering the entire stem surface and providing a quasi-continuous distribution of the 3D interfacial micromotions around the stem. The new measurement method would be very useful to compare the induced micromotions of different stem designs and to optimize the primary stability of cementless total hip arthroplasty.

A novel technique using echocardiography to evaluate venous cannula performance perioperatively in CPB cardiac surgery.

OBJECTIVE: Transthoracic echocardiography (TTE) has been used clinically to disobstruct venous drainage cannula and to optimise placement of venous cannulae in the vena cava but it has never been used to evaluate performance capabilities. Also, little progress has been made in venous cannula design in order to optimise venous return to the heart lung machine. We designed a self-expandable Smartcanula (SC) and analysed its performance capability using echocardiography. METHODS: An epicardial echocardiography probe was placed over the SC or control cannula (CTRL) and a Doppler image was obtained. Mean (V(m)) and maximum (V(max)) velocities, flow and diameter were obtained. Also, pressure drop (DeltaP(CPB)) was obtained between the central venous pressure and inlet to venous reservoir. LDH and Free Hb were also compared in 30 patients. Comparison was made between the two groups using the student's t-test with statistical significance established when p<0.05. RESULTS: Age for the SC and CC groups were 61.6+/-17.6 years and 64.6+/-13.1 years, respectively. Weight was 70.3+/-11.6 kg and 72.8+/-14.4 kg, respectively. BSA was 1.80+/-0.2 m(2) and 1.82+/-0.2 m(2), respectively. CPB times were 114+/-53 min and 108+/-44 min, respectively. Cross-clamp time was 59+/-15 min and 76+/-29 min, respectively (p=NS). Free-Hb was 568+/-142 U/l versus 549+/-271 U/l post-CPB for the SC and CC, respectively (p=NS). LDH was 335+/-73 mg/l versus 354+/-116 mg/l for the SC and CC, respectively (p=NS). V(m) was 89+/-10 cm/s (SC) versus 63+/-3 cm/s (CC), V(max) was 139+/-23 cm/s (SC) versus 93+/-11 cm/s (CC) (both p<0.01). DeltaP(CPB) was 30+/-10 mmHg (SC) versus 43+/-13 mmHg (CC) (p<0.05). A Bland-Altman test showed good agreement between the two devices used concerning flow rate calculations between CPB and TTE (bias 300 ml+/-700 ml standard deviation). CONCLUSIONS: This novel Smartcanula design, due to its self-expanding principle, provides superior flow characteristics compared to classic two stage venous cannula used for adult CPB surgery. No detrimental effects were observed concerning blood damage. Echocardiography was effective in analysing venous cannula performance and velocity patterns.

Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor.

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI.

Sleep deprivation effects on circadian clock gene expression in the cerebral cortex parallel electroencephalographic differences among mouse strains.

Sleep deprivation (SD) results in increased electroencephalographic (EEG) delta power during subsequent non-rapid eye movement sleep (NREMS) and is associated with changes in the expression of circadian clock-related genes in the cerebral cortex. The increase of NREMS delta power as a function of previous wake duration varies among inbred mouse strains. We sought to determine whether SD-dependent changes in circadian clock gene expression parallel this strain difference described previously at the EEG level. The effects of enforced wakefulness of incremental durations of up to 6 h on the expression of circadian clock genes (bmal1, clock, cry1, cry2, csnk1epsilon, npas2, per1, and per2) were assessed in AKR/J, C57BL/6J, and DBA/2J mice, three strains that exhibit distinct EEG responses to SD. Cortical expression of clock genes subsequent to SD was proportional to the increase in delta power that occurs in inbred strains: the strain that exhibits the most robust EEG response to SD (AKR/J) exhibited dramatic increases in expression of bmal1, clock, cry2, csnkIepsilon, and npas2, whereas the strain with the least robust response to SD (DBA/2) exhibited either no change or a decrease in expression of these genes and cry1. The effect of SD on circadian clock gene expression was maintained in mice in which both of the cryptochrome genes were genetically inactivated. cry1 and cry2 appear to be redundant in sleep regulation as elimination of either of these genes did not result in a significant deficit in sleep homeostasis. These data demonstrate transcriptional regulatory correlates to previously described strain differences at the EEG level and raise the possibility that genetic differences underlying circadian clock gene expression may drive the EEG differences among these strains.

Optimum power allocation for parallel Gaussian channels with arbitrary input distributions

The mutual information of independent parallel Gaussian-noise channels is maximized, under an average power constraint, by independent Gaussian inputs whose power is allocated according to the waterfilling policy. In practice, discrete signalling constellations with limited peak-to-average ratios (m-PSK, m-QAM, etc) are used in lieu of the ideal Gaussian signals. This paper gives the power allocation policy that maximizes the mutual information over parallel channels with arbitrary input distributions. Such policy admits a graphical interpretation, referred to as mercury/waterfilling, which generalizes the waterfilling solution and allows retaining some of its intuition. The relationship between mutual information of Gaussian channels and nonlinear minimum mean-square error proves key to solving the power allocation problem.

Parallel Research, Multiple Intellectual Property Right Protection Instruments, and the Correlation among R&D Projects, September 2005

The choice of a research path in attacking scientific and technological problems is a significant component of firms’ R&D strategy. One of the findings of the patent races literature is that, in a competitive market setting, firms’ noncooperative choices of research projects display an excessive degree of correlation, as compared to the socially optimal level. The paper revisits this question in a context in which firms have access to trade secrets, in addition to patents, to assert intellectual property rights (IPR) over their discoveries. We find that the availability of multiple IPR protection instruments can move the paths chosen by firms engaged in an R&D race toward the social optimum.

The effect of femoral component rotation on the five-year outcome of cemented mobile bearing total knee arthroplasty.

PURPOSE: Performing total knee replacement, accurate alignment and neutral rotation of the femoral component are widely believed to be crucial for the ultimate success. Contrary to absolute bone referenced alignment, using a ligament balancing technique does not automatically rotate the femoral component parallel to the transepicondylar axis. In this context we established the hypothesis that rotational alignment of the femoral component parallel to the transepicondylar axis (0° ± 3°) results in better outcome than alignment outside of this range. METHODS: We analysed 204 primary cemented mobile bearing total knee replacements five years postoperatively. Femoral component rotation was measured on axial radiographs using the condylar twist angle (CTA). Knee society score, range of motion as well as subjective rating documented outcome. RESULTS: In 96 knees the femoral component rotation was within the range 0 ± 3° (neutral rotation group), and in 108 knees the five-year postoperative rotational alignment of the femoral component was outside of this range (outlier group). Postoperative CTA showed a mean of 2.8° (±3.4°) internal rotation (IR) with a range between 6° external rotation (ER) and 15° IR (CI 95). No difference with regard to subjective and objective outcome could be detected. CONCLUSION: The present work shows that there is a large given natural variability in optimal rotational orientation, in this study between 6° ER and 15° IR, with numerous co-factors determining correct positioning of the femoral component. Further studies substantiating pre- and postoperative determinants are required to complete the understanding of resulting biomechanics in primary TKA.