Development and application of a next-generation-sequencing (NGS) approach to detect known and novel gene defects underlying retinal diseases

Background

Inherited retinal disorders are clinically and genetically heterogeneous with more than 150 gene defects accounting for the diversity of disease phenotypes. So far, mutation detection was mainly performed by APEX technology and direct Sanger sequencing of known genes. However, these methods are time consuming, expensive and unable to provide a result if the patient carries a new gene mutation. In addition, multiplicity of phenotypes associated with the same gene defect may be overlooked.

Methods

To overcome these challenges, we designed an exon sequencing array to target 254 known and candidate genes using Agilent capture. Subsequently, 20 DNA samples from 17 different families, including four patients with known mutations were sequenced using Illumina Genome Analyzer IIx next-generation-sequencing (NGS) platform. Different filtering approaches were applied to identify the genetic defect. The most likely disease causing variants were analyzed by Sanger sequencing. Co-segregation and sequencing analysis of control samples validated the pathogenicity of the observed variants.

Results

The phenotype of the patients included retinitis pigmentosa, congenital stationary night blindness, Best disease, early-onset cone dystrophy and Stargardt disease. In three of four control samples with known genotypes NGS detected the expected mutations. Three known and five novel mutations were identified in NR2E3, PRPF3, EYS, PRPF8, CRB1, TRPM1 and CACNA1F. One of the control samples with a known genotype belongs to a family with two clinical phenotypes (Best and CSNB), where a novel mutation was identified for CSNB. In six families the disease associated mutations were not found, indicating that novel gene defects remain to be identified.

Conclusions

In summary, this unbiased and time-efficient NGS approach allowed mutation detection in 75% of control cases and in 57% of test cases. Furthermore, it has the possibility of associating known gene defects with novel phenotypes and mode of inheritance.     

Evaluating methods to quantify spatial variation in the velocity of biological invasions

Invading species rarely spread homogeneously through a landscape and invasion patterns typically display irregular frontal boundaries as the invasion progresses through space. Those irregular patterns are generally produced by local environmental factors that may slow or accelerate movement of the frontal boundary. While there is an abundant literature on species distribution modelling methods that quantify local suitability for species establishment, comparatively few studies have examined methods for measuring the local velocity of invasions that can then be statistically analysed in relation to spatially variable environmental factors. Previous studies have used simulations to compare different methods for estimating the overall rate of spread of an invasion. We adopted a similar approach of simulating invasions that resemble two real case‐studies, both in terms of their spatial resolution (i.e. considering the size of one cell as one km) and their spatial extent (> 600 000 km²). Simulations were sampled to compare how different methods used to measure local spread rate, namely the neighbouring, nearest distance and Delaunay methods, perform for spatio‐temporal comparisons. We varied the assessment using three levels of complexity of the spatio‐temporal pattern of invasion, three sample sizes (500, 1000 and 2000 points), three different spatial sampling patterns (stratified, random, aggregated), three interpolation methods (generalized linear model, kriging, thin plate spline regression) and two spatio‐temporal modelling structures (trend surface analysis and boundary displacement), resulting in a total of 486 different scenarios. The thin plate spline regression interpolation method, in combination with trend surface analysis, was found to provide the most robust local spread rate quantification as it was able to reliably accommodate different sampling conditions and invasion patterns. This best approach was successfully applied to two case‐studies, the invasion of France by the horse‐chestnut leafminer Cameraria ohridella and by the bluetongue virus, generally in agreement with previously published values of spread rates. Potential avenues for further research are discussed.     

Contrasting nitrogen uptake by diatom and Phaeocystis-dominated phytoplankton assemblages in the North Sea

This paper documents ambient concentrations of nutrients in the Belgian coastal waters of the North Sea during the spring of 1996 and 1997. The paper elaborates the differences of uptake rates of oxidised nitrogen (NO₃⁻) and reduced nitrogen (NH₄ and urea) by Phaeocystis and diatoms. The nitrogen concentrations were dominated by NO₃⁻ with a maximum concentration of 30 μM (January 1997) and 40 μM (March 1996). In 1996, Phaeocystis dominated the spring biomass with a maximum of 521 μg C l⁻¹, while maximum diatom biomass was 174 μg C l⁻¹. In 1997, the maximum Phaeocystis spring biomass was 1600 μg C l⁻¹ and diatom maximum biomass was below 100 μg C l⁻¹. A maximum bacteria biomass of about 55 μg C l⁻¹ was observed in mid-May 1996. The maximum nitrogen uptake rates were recorded during spring and were dominated by NO₃⁻ (0.005 h⁻¹ in 1996 and 0.032 h⁻¹ in 1997). Maximum specific NH₄ uptake rates were between 0.005 h⁻¹ in May 1996 and 0.006 h⁻¹ in April 1997. The NO₃⁻ uptake rates displayed exponential decrease versus increasing ambient reduced nitrogen concentrations (ammonium and urea), whereas the reduced nitrogen uptake increased but never compensated the decreased nitrate uptake. The NH₄ uptake kinetics of diatoms displayed lower v_max compared to Phaeocystis. Consequently, Phaeocystis showed ability to increase their NH₄ uptake capacity when more NH₄ became available while diatoms failed to do so, after ammonium had exceeded their saturation concentration (>1 μM). Although reduced nitrogen has a negative effect on the uptake of NO₃⁻, Phaeocystis have more advantage than diatoms on the uptake of ammonium. This might be contributing to the biomass domination shown by Phaeocystis over extended periods in spring.     

Antiproliferative activity of some 1,4-dimethylcarbazoles on cells that express estrogen receptors: part I

Several 9H-carbazole derivatives are used for various pharmacological applications. Many of these compounds demonstrated cytotoxic and anticancer activities. In this work, we have investigated the cytotoxic activity of some substituted carbazoles against cancer cell lines (MCF-7, and ISK). The derivative 2a showed the highest inhibitory activity against both cell lines.     

HIV-1(Lai) genomic RNA: combined used of NMR and molecular dynamics simulation for studying the structure and internal dynamics of a mutated SL1 hairpin

The genome of all retroviruses consists of two identical copies of an RNA sequence associated in a non-covalent dimer. A region upstream from the splice donor (SL1) comprising a self-complementary sequence is responsible for the initiation of the dimerization. This region is able to dimerize in two conformations: a loop-loop complex or an extended duplex. Here, we solve by 2D NMR techniques the solution structure of a 23-nucleotide sequence corresponding to HIV-1 SL1(Lai) in which the mutation G12-->A12 is included to prevent dimerization. It is shown that this monomer adopts a stem-loop conformation with a seven base pairs stem and a nine nucleotide loop containing the G10 C11 A12 C13 G14 C15 sequence. The stem is well structured in an A-form duplex, while the loop is more flexible even though elements of structure are evident. We show that the structure adopted by the stem can be appreciably different from its relaxed structure when the adenines A8, A9 and A16 in the loop are mechanically constrained. This point could be important for the efficiency of the dimerization. This experimental study is complemented with a 10 ns molecular dynamics simulation in the presence of counterions and explicit water molecules. This simulation brings about information on the flexibility of the loop, such as a hinge motion between the stem and the loop and a labile lattice of hydrogen bonds in the loop. The bases of the nucleotides G10 to C15 were found outside of the loop during a part of the trajectory, which is certainly necessary to initiate the dimerization process of the genuine SL1(Lai) sequence.     

Rainfall patterns and population dynamics of Aedes (Aedimorphus) vexans arabiensis, Patton 1905 (Diptera: Culicidae), a potential vector of Rift Valley Fever virus in Senegal.

The importance of rainfall for the development of Aedes vexans arabiensis populations, one of the potential vectors of Rift Valley Fever in West Africa, was demonstrated in a two-year follow-up study conducted in the Ferlo region of Senegal. In 2003, the rainy season began with heavy rains and, as a result, temporary ponds, the breeding places for mosquitoes, were flooded at their maximum level immediately. In such conditions, Aedes vexans arabiensis populations are abundant at the very beginning of the season, when the majority of eggs in quiescence are flooded. Females, hatching from eggs laid the year before, quickly lay eggs on the pond's wet soil, which will undergo dormancy as the water level goes down. Rainless periods longer than seven days, the time needed for embryogenesis, followed by significant rainfall, will result in the hatching of very large numbers of new eggs. Thus, several generations of adults may exist during the same rainy season. Because of potential vertical transmission of Rift Valley Fever virus in Aedes species, viral transmission and disease risk can appear as early as the beginning of the rainy season and if late rains occur, at the end of the season. This dynamic maximizes the virus' chance to persist from one year to another, thus facilitating endemisation of Rift Valley Fever in areas where Aedes vexans arabiensis exists.     

A novel strategy for site-directed chemical reactions in single stranded DNA--absorption and NMR spectroscopic studies of model compounds.

A new and simple model enabling a chemical species to be brought to a preselected site in single strand DNA is reported. Two oligonucleotides containing a propanediol linkage were hybridized to their complementary sequences with an extra-base opposite the propanediol derivative. Absorption studies results shown that the addition of a bisacridine derivative strongly increased the stabilities of both duplexes when added in a 1:1 ratio. NMR studies on one of these duplexes brought evidence of the intercalation of the bisacridine at the position involving the propanediol linkage. These results suggest that this system could be used to target a specific reaction at a preselected position using the bisacridine derivative as carrier for the reactive species.     

Solution structure of oligonucleotides covalently linked to a psoralen derivative.

Psoralen (pso) was attached via its C-5 position to the 5'-phosphate group of an oligodeoxynucleotide d(TAAGCCG) by a hexamethylene linker (m6). Complex formation between pso-m6-d(TAAGCCG) and the complementary strands d(CGGCTTA)[7-7mer] or d(CGGCTTAT)[7-8mer] was investigated by nuclear magnetic resonance in aqueous solution. Structural informations derived from DQF-COSY and NOESY maps, revealed that the mini double helix adopts a B-form conformation and that the deoxyriboses preferentially adopt a C2'-endo conformation. The nOe connectivities observed between the protons of the bases or the sugars in each duplex, and the protons of the psoralen and the hexamethylene chain, led us to propose a model involving an equilibrium between two conformations due to different locations of the psoralen. Upon UV-irradiation, the psoralen moiety cross-linked the two DNA strands at the level of 5'TpA3' sequences. NMR studies of the single major photo-cross-linked duplex pso-m6-d(TAAGCCG) and d(CGGCTTA) were performed. The stereochemistry of the diadduct is indeed cis-syn at both cyclobutane rings. In addition, the effects of this diadduct on the helical structure are analyzed in detail.