Exome Sequencing of Extended Families with Alzheimer's Disease Identifies Novel Genes Implicated in Cell Immunity and Neuronal Function.

OBJECTIVE: Alzheimer's disease (AD) is a neurodegenerative disorder for which more than 20 genetic loci have been implicated to date. However, studies demonstrate not all genetic factors have been identified. Therefore, in this study we seek to identify additional rare variants and novel genes potentially contributing to AD. METHODS: Whole exome sequencing was performed on 23 multi-generational families with an average of eight affected subjects. Exome sequencing was filtered for rare, nonsynonymous and loss-of-function variants. Alterations predicted to have a functional consequence and located within either a previously reported AD gene, a linkage peak (LOD>2), or clustering in the same gene across multiple families, were prioritized. RESULTS: Rare variants were found in known AD risk genes including AKAP9, CD33, CR1, EPHA1, INPP5D, NME8, PSEN1, SORL1, TREM2 and UNC5C. Three families had five variants of interest in linkage regions with LOD>2. Genes with segregating alterations in these peaks include CD163L1 and CLECL1, two genes that have both been implicated in immunity, CTNNA1, which encodes a catenin in the cerebral cortex and MIEF1, a gene that may induce mitochondrial dysfunction and has the potential to damage neurons. Four genes were identified with alterations in more than one family include PLEKHG5, a gene that causes Charcot-Marie-Tooth disease and THBS2, which promotes synaptogenesis. CONCLUSION: Utilizing large families with a heavy burden of disease allowed for the identification of rare variants co-segregating with disease. Variants were identified in both known AD risk genes and in novel genes.

Targeted sequencing of ABCA7 identifies splicing, stop-gain and intronic risk variants for Alzheimer disease.

Several variants in the gene ABCA7 have been identified as potential causal variants for late-onset Alzheimer's disease (LOAD). In order to replicate these findings, and search for novel causal variants, we performed targeted sequencing of this gene in cohorts of non-Hispanic White (NHW) and African-American (AA) LOAD cases and controls. We sequenced the gene ABCA7 in 291 NHW LOAD cases and 103 controls. Variants were prioritized for rare, damaging variants and previously reported variants associated with LOAD, and were follow-up genotyped in 4076 NHW and 1157 AA cases and controls. We confirm three previously associated ABCA7 risk variants and extend two of these associations to other populations, an intronic variant in NHW (P=3.0×10-3) (originally reported in a Belgian population), and a splice variant originally associated in the Icelandic population, which was significantly associated in the NHW cohort (P=1.2×10-6) and nominally associated in the AA cohort (P=0.017). We also identify a 3'-UTR splice variant that segregates in four siblings of one family and is nominally associated with LOAD (P=0.040). Multiple variants in ABCA7 contribute to LOAD risk.

GWAS analysis implicates NF-κB-mediated induction of inflammatory T cells in multiple sclerosis.

To identify genes and biologically relevant pathways associated with risk to develop multiple sclerosis (MS), the Genome-Wide Association Studies noise reduction method (GWAS-NR) was applied to MS genotyping data. Regions of association were defined based on the significance of linkage disequilibrium blocks. Candidate genes were cross-referenced based on a review of current literature, with attention to molecular function and directly interacting proteins. Supplementary annotations and pathway enrichment scores were generated using The Database for Annotation, Visualization and Integrated Discovery. The candidate set of 220 MS susceptibility genes prioritized by GWAS-NR was highly enriched with genes involved in biological pathways related to positive regulation of cell, lymphocyte and leukocyte activation (P=6.1E-15, 1.2E-14 and 5.0E-14, respectively). Novel gene candidates include key regulators of NF-κB signaling and CD4+ T helper type 1 (Th1) and T helper type 17 (Th17) lineages. A large subset of MS candidate genes prioritized by GWAS-NR were found to interact in a tractable pathway regulating the NF-κB-mediated induction and infiltration of pro-inflammatory Th1/Th17 T-cell lineages, and maintenance of immune tolerance by T-regulatory cells. This mechanism provides a biological context that potentially links clinical observations in MS to the underlying genetic landscape that may confer susceptibility.

Kinetics of Evaporation of Pinned Nanofluid Volatile Droplets at Subatmospheric Pressures.

We examine the effects of nanoparticle addition at low concentration on the evaporation kinetics of droplets in the constant radius mode. The evaporative behavior of deionized water and Al2O3 nanoparticle laden water on an aluminum substrate was observed at atmospheric and at different subatmospheric pressures. The two fluids exhibit the same evaporative behavior, independent of the droplet volume or the subatmospheric pressure. Moreover, the linear relationship between evaporation rate and droplet radius, initially proposed by Picknett and Bexon nearly four decades ago for droplets evaporating in the constant radius mode, is satisfied for both liquids. In addition, we have established a unified correlation solely function of fluid properties that extends this relationship to any subatmospheric pressure and fluid tested. We conclude that the addition of a small quantity of nanoparticles to the base fluid does not modify the kinetics of evaporation for pinned volatile droplets.

"Biodrop" Evaporation and Ring-Stain Deposits: The Significance of DNA Length.

Small sessile drops of water containing either long or short strands of DNA ("biodrops") were deposited on silicon substrates and allowed to evaporate. Initially, the triple line (TL) of both types of droplet remained pinned but later receded. The TL recession mode continued at constant speed until almost the end of drop lifetime for the biodrops with short DNA strands, whereas those containing long DNA strands entered a regime of significantly lower TL recession. We propose a tentative explanation of our observations based on free energy barriers to unpinning and increases in the viscosity of the base liquid due to the presence of DNA molecules. In addition, the structure of DNA deposits after evaporation was investigated by AFM. DNA self-assembly in a series of perpendicular and parallel orientations was observed near the contact line for the long-strand DNA, while, with the short-stranded DNA, smoother ring-stains with some nanostructuring but no striations were evident. At the interior of the deposits, dendritic and faceted crystals were formed from short and long strands, respectively, due to diffusion and nucleation limited processes, respectively. We suggest that the above results related to the biodrop drying and nanostructuring are indicative of the importance of DNA length, i.e., longer DNA chains consisting of linearly bonded shorter, rod-like DNA strands.

A novel Alzheimer disease locus located near the gene encoding tau protein.

APOE ɛ4, the most significant genetic risk factor for Alzheimer disease (AD), may mask effects of other loci. We re-analyzed genome-wide association study (GWAS) data from the International Genomics of Alzheimer's Project (IGAP) Consortium in APOE ɛ4+ (10 352 cases and 9207 controls) and APOE ɛ4- (7184 cases and 26 968 controls) subgroups as well as in the total sample testing for interaction between a single-nucleotide polymorphism (SNP) and APOE ɛ4 status. Suggestive associations (P<1 × 10(-4)) in stage 1 were evaluated in an independent sample (stage 2) containing 4203 subjects (APOE ɛ4+: 1250 cases and 536 controls; APOE ɛ4-: 718 cases and 1699 controls). Among APOE ɛ4- subjects, novel genome-wide significant (GWS) association was observed with 17 SNPs (all between KANSL1 and LRRC37A on chromosome 17 near MAPT) in a meta-analysis of the stage 1 and stage 2 data sets (best SNP, rs2732703, P=5·8 × 10(-9)). Conditional analysis revealed that rs2732703 accounted for association signals in the entire 100-kilobase region that includes MAPT. Except for previously identified AD loci showing stronger association in APOE ɛ4+ subjects (CR1 and CLU) or APOE ɛ4- subjects (MS4A6A/MS4A4A/MS4A6E), no other SNPs were significantly associated with AD in a specific APOE genotype subgroup. In addition, the finding in the stage 1 sample that AD risk is significantly influenced by the interaction of APOE with rs1595014 in TMEM106B (P=1·6 × 10(-7)) is noteworthy, because TMEM106B variants have previously been associated with risk of frontotemporal dementia. Expression quantitative trait locus analysis revealed that rs113986870, one of the GWS SNPs near rs2732703, is significantly associated with four KANSL1 probes that target transcription of the first translated exon and an untranslated exon in hippocampus (P ⩽ 1.3 × 10(-8)), frontal cortex (P ⩽ 1.3 × 10(-9)) and temporal cortex (P⩽1.2 × 10(-11)). Rs113986870 is also strongly associated with a MAPT probe that targets transcription of alternatively spliced exon 3 in frontal cortex (P=9.2 × 10(-6)) and temporal cortex (P=2.6 × 10(-6)). Our APOE-stratified GWAS is the first to show GWS association for AD with SNPs in the chromosome 17q21.31 region. Replication of this finding in independent samples is needed to verify that SNPs in this region have significantly stronger effects on AD risk in persons lacking APOE ɛ4 compared with persons carrying this allele, and if this is found to hold, further examination of this region and studies aimed at deciphering the mechanism(s) are warranted.

Effect of Poly(ethylene oxide) Molecular Weight on the Pinning and Pillar Formation of Evaporating Sessile Droplets: The Role of the Interface.

We report on the drying process of sessile droplets of aqueous poly(ethylene oxide) (PEO) solutions studied by contact angle analysis. Liquid samples were prepared with the same initial concentration of four different molecular weights, Mw, of PEO. Droplets with initial volumes of between 1 and 5 µL were left to evaporate while temperature, pressure, and relative humidity were kept constant. Residues were formed with either a disklike puddle or a distinctive tall conical pillar shape. The latter occurred following a four-stage deposition process: pinned drying, during which the contact line is stationary; pseudodewetting, where the receding contact line is induced by precipitation; bootstrap building, during which the liquid droplet is lifted on freshly precipitated solid; and late drying. Contact angle analysis allowed us to monitor all stages during drying and consider transitions between stages for different molecular weights. We illustrate the mechanisms taking place during the crucial stages of pinning and depinning, revealing the effect of adhesion and contact line friction for high molecular weights and its influence on the final morphology of the dried PEO solute. To this end, we performed PEO solution droplet evaporation on PEO and PTFE films demonstrating the importance of interfacial interaction phenomena. We show that the formation of disklike puddles for high molecular weights on glass is associated with continuous droplet contact line pinning. This results from the strong adhesion due to the interdigitation of the loops and tails of a polymer layer (adsorbed on glass during evaporation) with the polymer gel network inside the droplet that forms as water evaporates.

Neuropathic ocular pain: an important yet underevaluated feature of dry eye.

Dry eye has gained recognition as a public health problem given its prevalence, morbidity, and cost implications. Dry eye can have a variety of symptoms including blurred vision, irritation, and ocular pain. Within dry eye-associated ocular pain, some patients report transient pain whereas others complain of chronic pain. In this review, we will summarize the evidence that chronicity is more likely to occur in patients with dysfunction in their ocular sensory apparatus (ie, neuropathic ocular pain). Clinical evidence of dysfunction includes the presence of spontaneous dysesthesias, allodynia, hyperalgesia, and corneal nerve morphologic and functional abnormalities. Both peripheral and central sensitizations likely play a role in generating the noted clinical characteristics. We will further discuss how evaluating for neuropathic ocular pain may affect the treatment of dry eye-associated chronic pain.

Genome-wide scan demonstrates significant linkage for male sexual orientation.

BACKGROUND: Findings from family and twin studies support a genetic contribution to the development of sexual orientation in men. However, previous studies have yielded conflicting evidence for linkage to chromosome Xq28. METHOD: We conducted a genome-wide linkage scan on 409 independent pairs of homosexual brothers (908 analyzed individuals in 384 families), by far the largest study of its kind to date. RESULTS: We identified two regions of linkage: the pericentromeric region on chromosome 8 (maximum two-point LOD = 4.08, maximum multipoint LOD = 2.59), which overlaps with the second strongest region from a previous separate linkage scan of 155 brother pairs; and Xq28 (maximum two-point LOD = 2.99, maximum multipoint LOD = 2.76), which was also implicated in prior research. CONCLUSIONS: Results, especially in the context of past studies, support the existence of genes on pericentromeric chromosome 8 and chromosome Xq28 influencing development of male sexual orientation.

Effect of particle geometry on triple line motion of nano-fluid drops and deposit nano-structuring.

We illustrate the importance of particle geometry on droplet contact line pinning, 'coffee-stain' formation and nano-structuring within the resulting rings. We present the fundamentals of pure liquid droplet evaporation and then discuss the effect of particles on the evaporation process. The resulting coffee-stain patterns and particle structuring within them are presented and discussed. In the second part, we turn our attention to the effect of particle geometry on the evaporation process. A wide range of particle shapes, categorised according to aspect ratio, from the simple shape of a sphere to the highly irregular shapes of platelets and tubes is discussed. Particle geometry effect on evaporation behaviour was quantified in terms of change in contact angle and contact radius for the stick-slip cases. Consequently the hysteretic energy barrier pinning the droplets was estimated, showing an increasing trend with particle aspect ratio. The three-phase contact line (TL) motion kinetics are complemented with analysis of the nano-structuring behaviour of each shape, leading to the identification of the two main parameters affecting nanoparticle self-assembly behaviour at the wedge. Flow velocity and wedge constraints were found to have antagonist effects on particle deposition, although these varied with particle shape. This description should help in understanding the drying behaviour of more complex fluids. Furthermore, knowing the fundamentals of this simple and inexpensive surface patterning technique should permit its tailoring to the needs of many potential applications.

Recalcitrant bubbles.

We demonstrate that thermocapillary forces may drive bubbles against liquid flow in 'anomalous' mixtures. Unlike 'ordinary' liquids, in which bubbles migrate towards higher temperatures, we have observed vapour bubbles migrating towards lower temperatures, therefore against the flow. This unusual behaviour may be explained by the temperature dependence of surface tension of these binary mixtures. Bubbles migrating towards their equilibrium position follow an exponential trend. They finally settle in a stationary position just 'downstream' of the minimum in surface tension. The exponential trend for bubbles in 'anomalous' mixtures and the linear trend in pure liquids can be explained by a simple model. For larger bubbles, oscillations were observed. These oscillations can be reasonably explained by including an inertial term in the equation of motion (neglected for smaller bubbles).

Evaporation of nanofluid droplets with applied DC potential.

A considerable growth of interest in electrowetting (EW) has stemmed from the potential exploitation of this technique in numerous industrial and biological applications, such as microfluidics, lab-on-a-chip, electronic paper, and bioanalytical techniques. The application of EW to droplets of liquids containing nanoparticles (nanofluids) is a new area of interest. Understanding the effects of electrowetting at the fundamental level and being able to manipulate deposits from nanofluid droplets represents huge potential. In this work, we study the complete evaporation of nanofluid droplets under DC conditions. Different evolutions of contact angle and contact radius, as well as deposit patterns, are revealed. When a DC potential is applied, continuous and smoother receding of the contact line during the drying out of TiO2 nanofluids and more uniform patterning of the deposit are observed, in contrast to the typical "stick-slip" behavior and rings stains. Furthermore, the mechanisms for nanoparticle interactions with the applied DC potential differ from those proposed for the EW of droplets under AC conditions. The more uniform patterns of particle deposits resulting from DC potential are a consequence of a shorter timescale for electrophoretic mobility than advection transport driven by evaporation.

Structural transitions in a ring stain created at the contact line of evaporating nanosuspension sessile drops.

Monodisperse nanosuspension droplets, placed on a flat surface, evaporated following the stick-slip motion of the three-phase contact line. Unexpectedly, a disordered region formed at the exterior edge of a closely packed nanocolloidal crystalline structure during the "stick" period. In order to assess the role of particle velocity on particle structuring, we did experiments in a reduced pressure environment which allowed the enhancement of particle velocity. These experiments revealed the promotion of hexagonal packing at the very edge of the crystallite with increasing velocity. Quantification of particle velocity and comparison with measured deposit shape for each case allowed us to provide a tentative description of the underlying mechanisms that govern particle deposition of nanoparticles at the triple line of an evaporating droplet. Behavior is governed by an interplay between the fluid, and hence particle, flow velocity (main ordering parameter) and wedge constraints, and consequently disjoining pressure (main disordering parameter). Furthermore, the formation of a second disordered particle region at the interior edge of the deposit (towards bulk fluid) was found and attributed to the rapid motion of the triple line during the "slip" regime. Additionally, the magnitude of the pinning forces acting on the triple line of the same drops was calculated. These findings provide further insight into the mechanisms of the phenomenon and could facilitate its exploitation in various nanotechnological applications.

Inertial to viscoelastic transition in early drop spreading on soft surfaces.

It has been known for many years that a spreading liquid droplet can be appreciably slowed on a soft, viscoelastic substrate by the appearance of a "wetting ridge" or protuberance of the solid near the triple phase contact line because of capillary forces. Viscoelastic dissipation in the solid surface can outweigh that of liquid viscosity and, therefore, dominate wetting dynamics. In this paper, we show that a short, rapid spreading stage exists after initial contact. The requisite balance determining the speed of motion is between capillary forces and inertial effects. As spreading proceeds, however, inertia lessens and the lower spreading speed allow for viscoelastic effects in the solid to increase. The transition between early inertial and viscoelastic regimes is studied with high-speed photography and explained by a simple theory.

On the behavior of dew drops.

It may be observed that, when dew drops form, although they may be positioned randomly on flat leaves, they tend to accumulate at the pointed ends of thin, slightly conical growths. We discuss here the basic physics leading to this phenomenon.

Stick-slip of evaporating droplets: substrate hydrophobicity and nanoparticle concentration.

The dynamics of the three-phase contact line for water and ethanol is experimentally investigated using substrates of various hydrophobicities. Different evolutions of the droplet profile (contact line, R, and contact angle, θ) are found to be dependent on the hydrophobicity of the substrate. A simple theoretical approach based on the unbalanced Young force is used to explain the depinning of the contact line on hydrophilic surfaces or the monotonic slip on hydrophobic substrates. The second part of the article involves the addition of different quantities of titanium oxide nanoparticles to water, and a comparison of the evaporative behavior of these novel fluids with the base liquid (water) on substrates varying in hydrophobicity (i.e., silicon, Cytop, and PTFE) is presented. The observed stick-slip behavior is found to be dependent on the nanoparticle concentration. The evaporation rate is closely related to the dynamics of the contact line. These findings may have an important impact when considering the evaporation of droplets on different substrates and/or those containing nanoparticles.

Capillary rise of superspreaders.

Trisiloxane surfactants, known as 'superspreaders', are commonly employed in numerous applications where enhanced wetting is of the utmost importance. The underlying mechanisms of superspreader wetting have been a focus of scientific interest for ca. 2 decades, and a number of mechanisms have been proposed to explain the unique trisiloxane dynamics. We have studied trisiloxane behaviour in thin capillaries to get further insight into their interfacial activity. Additionally, our knowledge of the capillary rise of superspreaders is surprisingly limited, and the effect of this extraordinary group of surfactants on capillary phenomena has been largely overlooked. Diffusion was confirmed to be the limiting factor of trisiloxane behaviour. A tentative theoretical explanation for the phenomenon studied and an appropriate mathematical model are presented. It is concluded that the enhancement of wetting due to surfactant addition is also a function of geometry: the effect is clear for a sessile drop, but more complex and less beneficial in a capillary.

SeqEM: an adaptive genotype-calling approach for next-generation sequencing studies.

MOTIVATION: Next-generation sequencing presents several statistical challenges, with one of the most fundamental being determining an individual's genotype from multiple aligned short read sequences at a position. Some simple approaches for genotype calling apply fixed filters, such as calling a heterozygote if more than a specified percentage of the reads have variant nucleotide calls. Other genotype-calling methods, such as MAQ and SOAPsnp, are implementations of Bayes classifiers in that they classify genotypes using posterior genotype probabilities. RESULTS: Here, we propose a novel genotype-calling algorithm that, in contrast to the other methods, estimates parameters underlying the posterior probabilities in an adaptive way rather than arbitrarily specifying them a priori. The algorithm, which we call SeqEM, applies the well-known Expectation-Maximization algorithm to an appropriate likelihood for a sample of unrelated individuals with next-generation sequence data, leveraging information from the sample to estimate genotype probabilities and the nucleotide-read error rate. We demonstrate using analytic calculations and simulations that SeqEM results in genotype-call error rates as small as or smaller than filtering approaches and MAQ. We also apply SeqEM to exome sequence data in eight related individuals and compare the results to genotypes from an Illumina SNP array, showing that SeqEM behaves well in real data that deviates from idealized assumptions. CONCLUSION: SeqEM offers an improved, robust and flexible genotype-calling approach that can be widely applied in the next-generation sequencing studies. AVAILABILITY AND IMPLEMENTATION: Software for SeqEM is freely available from our website: www.hihg.org under Software Download.

Association and gene-gene interaction of SLC6A4 and ITGB3 in autism.

Autism is a heritable neurodevelopmental disorder with substantial genetic heterogeneity. Studies point to possible links between autism and two serotonin related genes: SLC6A4 and ITGB3 with a sex-specific genetic effect and interaction between the genes. Despite positive findings, inconsistent results have complicated interpretation. This study seeks to validate and clarify previous findings in an independent dataset taking into account sex, family-history (FH) and gene-gene effects. Family-based association analysis was performed within each gene. Gene-gene interactions were tested using extended multifactor dimensionality reduction (EMDR) and MDR-phenomics (MDR-P) using sex of affecteds and FH as covariates. No significant associations with individual SNPs were found in the datasets stratified by sex, but associations did emerge when we stratified by family history. While not significant in the overall dataset, nominally significant association was identified at RS2066713 (P = 0.006) within SLC6A4 in family-history negative (FH-) families, at RS2066713 (P = 0.038) in family-history positive (FH+) families but with the opposite risk allele as in the FH- families. For ITGB3, nominally significant association was identified at RS3809865 overall (P = 0.040) and within FH+ families (P = 0.031). However, none of the associations survived the multiple testing correction. MDR-P confirmed gene-gene effects using sex of affecteds (P = 0.023) and family history (P = 0.014, survived the multiple testing corrections) as covariates. Our results indicate the extensive heterogeneity within these two genes among families. The potential interaction between SLC6A4 and ITGB3 may be clarified using family history as an indicator of genetic architecture, illustrating the importance of covariates as markers of heterogeneity in genetic analyses.