Next-generation carrier screening

Purpose: Carrier screening for recessive Mendelian disorders traditionally employs focused genotyping to interrogate limited sets of mutations most prevalent in specific ethnic groups. We sought to develop a next-generation DNA sequencing–based workflow to enable analysis of a more comprehensive set of disease-causing mutations. Methods: We utilized molecular inversion probes to capture the protein-coding regions of 15 genes from genomic DNA isolated from whole blood and sequenced those regions using the Illumina HiSeq 2000 (Illumina, San Diego, CA). To assess the quality of the resulting data, we measured both the fraction of the targeted region yielding high-quality genotype calls, and the sensitivity and specificity of those calls by comparison with conventional Sanger sequencing across hundreds of samples. Finally, to improve the overall accuracy for detecting insertions and deletions, we introduce a novel assembly-based approach that substantially increases sensitivity without reducing specificity. Results: We generated high-quality sequence for at least 99.8% of targeted base pairs in samples derived from blood and achieved high concordance with Sanger sequencing (sensitivity >99.9%, specificity >99.999%). Our novel algorithm is capable of detecting insertions and deletions inaccessible by current methods. Conclusion: Our next-generation DNA sequencing–based approach yields the accuracy and completeness necessary for a carrier screening test

Data mining of high density genomic variant data for prediction of Alzheimer's disease risk

<p>Abstract</p> <p>Background</p> <p>The discovery of genetic associations is an important factor in the understanding of human illness to derive disease pathways. Identifying multiple interacting genetic mutations associated with disease remains challenging in studying the etiology of complex diseases. And although recently new single nucleotide polymorphisms (SNPs) at genes implicated in immune response, cholesterol/lipid metabolism, and cell membrane processes have been confirmed by genome-wide association studies (GWAS) to be associated with late-onset Alzheimer's disease (LOAD), a percentage of AD heritability continues to be unexplained. We try to find other genetic variants that may influence LOAD risk utilizing data mining methods.</p> <p>Methods</p> <p>Two different approaches were devised to select SNPs associated with LOAD in a publicly available GWAS data set consisting of three cohorts. In both approaches, single-locus analysis (logistic regression) was conducted to filter the data with a less conservative p-value than the Bonferroni threshold; this resulted in a subset of SNPs used next in multi-locus analysis (random forest (RF)). In the second approach, we took into account prior biological knowledge, and performed sample stratification and linkage disequilibrium (LD) in addition to logistic regression analysis to preselect loci to input into the RF classifier construction step.</p> <p>Results</p> <p>The first approach gave 199 SNPs mostly associated with genes in calcium signaling, cell adhesion, endocytosis, immune response, and synaptic function. These SNPs together with <it>APOE and GAB2 </it>SNPs formed a predictive subset for LOAD status with an average error of 9.8% using 10-fold cross validation (CV) in RF modeling. Nineteen variants in LD with <it>ST5, TRPC1, ATG10, ANO3, NDUFA12, and NISCH </it>respectively, genes linked directly or indirectly with neurobiology, were identified with the second approach. These variants were part of a model that included <it>APOE </it>and <it>GAB2 </it>SNPs to predict LOAD risk which produced a 10-fold CV average error of 17.5% in the classification modeling.</p> <p>Conclusions</p> <p>With the two proposed approaches, we identified a large subset of SNPs in genes mostly clustered around specific pathways/functions and a smaller set of SNPs, within or in proximity to five genes not previously reported, that may be relevant for the prediction/understanding of AD.</p

LDLR Expression and Localization Are Altered in Mouse and Human Cell Culture Models of Alzheimer's Disease

Alzheimer's disease (AD) is a chronic neurodegenerative disorder and the most common form of dementia. The major molecular risk factor for late-onset AD is expression of the ε-4 allele of apolipoprotein E (apoE), the major cholesterol transporter in the brain. The low-density lipoprotein receptor (LDLR) has the highest affinity for apoE and plays an important role in brain cholesterol metabolism.Using RT-PCR and western blotting techniques we found that over-expression of APP caused increases in both LDLR mRNA and protein levels in APP transfected H4 neuroglioma cells compared to H4 controls. Furthermore, immunohistochemical experiments showed aberrant localization of LDLR in H4-APP neuroglioma cells, Aβ-treated primary neurons, and in the PSAPP transgenic mouse model of AD. Finally, immunofluorescent staining of LDLR and of γ- and α-tubulin showed a change in LDLR localization preferentially away from the plasma membrane that was paralleled by and likely the result of a disruption of the microtubule-organizing center and associated microtubule network.These data suggest that increased APP expression and Aβ exposure alters microtubule function, leading to reduced transport of LDLR to the plasma membrane. Consequent deleterious effects on apoE uptake and function will have implications for AD pathogenesis and/or progression

Intranasal “painless” Human Nerve Growth Factors Slows Amyloid Neurodegeneration and Prevents Memory Deficits in App X PS1 Mice

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease (AD) treatment but the clinical application is hindered by its potent pro-nociceptive activity. Thus, to reduce systemic exposure that would induce pain, in recent clinical studies NGF was administered through an invasive intracerebral gene-therapy approach. Our group demonstrated the feasibility of a non-invasive intranasal delivery of NGF in a mouse model of neurodegeneration. NGF therapeutic window could be further increased if its nociceptive effects could be avoided altogether. In this study we exploit forms of NGF, mutated at residue R100, inspired by the human genetic disease HSAN V (Hereditary Sensory Autonomic Neuropathy Type V), which would allow increasing the dose of NGF without triggering pain. We show that “painless” hNGF displays full neurotrophic and anti-amyloidogenic activities in neuronal cultures, and a reduced nociceptive activity in vivo. When administered intranasally to APPxPS1 mice ( n = 8), hNGFP61S/R100E prevents the progress of neurodegeneration and of behavioral deficits. These results demonstrate the in vivo neuroprotective and anti-amyloidogenic properties of hNGFR100 mutants and provide a rational basis for the development of “painless” hNGF variants as a new generation of therapeutics for neurodegenerative diseases

Effect of changing temperature on the ionic permeation through the cyclic GMP-gated channel from vertebrate photoreceptors.

Native cGMP-gated channels were studied in rod outer segments of the larval tiger salamander Ambystoma tigrinum. The alpha subunit of the cGMP-gated channel from bovine rods, here referred to as the wild type (w.t.), and mutant channels were heterologously expressed in Xenopus laevis oocytes. These channels were studied in excised membrane patches in the inside-out configuration and were activated by the addition of 100 or 500 microM cGMP. The effect of temperature on the ionic permeation was studied. The macroscopic current flowing through the native channel at +100 mV had an activation energy of 35.8, 30, 31.8, 34.5, 41.3, and 22.4 kJ mol-1 in the presence of Li+, Na+, K+, Rb+, Cs+, and NH4+, respectively. The macroscopic current flowing through the w.t. channel at +100 mV had an activation energy of 45.2, 38.2, 37.5, 47.3, 49.4, and 38.9 kJ mol-1 in the presence of Li+, Na+, K+, Rb+, Cs+, and NH4+, respectively. The activation energy of the macroscopic current flowing through the native and w.t. channels did not vary significantly when the ionic concentration of the permeant ion was changed between 2.5 and 110 mM. The activation energy of the single-channel current of the w.t. channel at +100 mV was 40.4 and 33 kJ mol-1 for Na+ and NH4+, respectively. The reversal potential of biionic solutions changed significantly with temperature. These results can be used to obtain an estimate of the enthalpic and entropic contributions to the barrier of the Gibbs free energy experienced by an ion during its permeation through the open channel. These estimates indicate that the ionic permeation and selectivity of the cGMP-gated channel are controlled both by enthalpic and entropic factors and that the selectivity of the native channel for Li+ over Na+ is primarily caused by entropic effects

Single-channel properties of ionic channel gated by cyclic nucleotides

This paper presents an extensive analysis of single-channel properties of cyclic nucleotide gated (CNG) channels, obtained by injecting into Xenopus laevis oocytes the mRNA encoding for the alpha and beta subunits from bovine rods. When the alpha and beta subunits of the CNG channel are coexpressed, at least three types of channels with different properties are observed. One type of channel has well-resolved, multiple conductive levels at negative voltages, but not at positive voltages. The other two types of channel are characterized by flickering openings, but are distinguished because they have a low and a high conductance. The alpha subunit of CNG channels has a well-defined conductance of about 28 pS, but multiple conductive levels are observed in mutant channels E363D and T364M. The conductance of these open states is modulated by protons and the membrane voltage, and has an activation energy around 44 kJ/mol. The relative probability of occupying any of these open states is independent of the cGMP concentration, but depends on extracellular protons. The open probability in the presence of saturating cGMP was 0.78, 0.47, 0.5, and 0.007 in the w.t. and mutants E363D, T364M, and E363G, and its dependence on temperature indicates that the thermodynamics of the transition between the closed and open state is also affected by mutations in the pore region. These results suggest that CNG channels have different conductive levels, leading to the existence of multiple open states in homomeric channels and to the flickering behavior in heteromeric channels, and that the pore is an essential part of the gating of CNG channels

Functional properties of sodium channels do not depend on the cytoskeleton integrity.

Several observations suggest an interaction of the sodium channel alpha-subunit with the cytoskeletal structures. However, there is a wide variability in the results of experiments of heterologous expression in Xenopus oocytes and studies on mammalian cells are sometimes contradictory. In general, there has been no direct demonstration that ad hoc large perturbations of the cytoskeleton modify the intrinsic properties of the sodium channels expressed endogenously or heterologously in plasma membranes. We have studied in CHO cells transfected with the rat muscle sodium channel alpha-subunit the effects of two substances expected to produce drastic perturbations of the cytoskeletal structure: Cytochalasin-D, which depolymerizes microfilaments, and Colchicine, which inhibits the microtubules polymerization. We observed no significant differences in the voltage dependence, kinetic parameters and surface density of the expressed sodium channels after treatment of the cells with these substances. We conclude that the two known main components of the cytoskeleton do not interfere directly with the sodium channel function or with the heterologous expression of channels in the cell membrane