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Introduction: Despite the high need for effective treatments for major depressive disorder (MDD), the development of novel medicines is hampered by clinical, genetic and biological heterogeneity, unclear links between symptoms and neural dysfunction, and tenuous biomarkers for clinical trial contexts of use. Methods: In this study, we examined the International Study to Predict Optimized Treatment in Depression (iSPOT-D) clinical trial database for new relationships between auditory event-related potential (ERP) responses, demographic features, and clinical symptoms and behavior, to inform strategies for biomarker-driven patient stratification that could be used to optimize future clinical trial design and drug development strategy in MDD. Results: We replicate findings from previous analyses of the classic auditory oddball task in the iSPOT-D sample showing smaller than typical N1 and P300 response amplitudes and longer P300 latencies for target and standard stimuli in patients with MDD, suggesting altered bottom-up sensory and top-down attentional processes. We further demonstrate that age is an important contributor to clinical group differences, affecting both topographic distribution of the clinically informative ERP responses and the types of the stimuli sensitive to group differences. In addition, the observed brain-behavior associations indicate that levels of anxiety and stress are major contributing factors to atypical sensory and attentional processing among patients with MDD, particularly in the older subgroups. Discussion: Our novel findings support the possibility of accelerated cognitive aging in patients with MDD and identify the frontal P300 latency as an additional candidate biomarker of MDD. These results from a large, well-phenotyped sample support the view that heterogeneity of the clinical population with MDD can be systematically characterized based on age and neural biomarkers of sensory and attentional processing, informing patient stratification strategies in the design of clinical trials., Competing Interests: TT-W and DS are employed by Alkermes. AK received consulting fees from Alkermes for contributions to the current study., (Copyright © 2023 Key, Thornton-Wells and Smith.)

Two-dimensional infrared (2DIR) spectroscopy has become an established method for generating vibrational spectra in condensed phase samples composed of mixtures that yield heavily congested infrared and Raman spectra. These condensed phase 2DIR spectrometers can provide very high temporal resolution (<1 ps), but the spectral resolution is generally insufficient for resolving rotational peaks in gas phase spectra. Conventional (1D) rovibrational spectra of gas phase molecules are often plagued by severe spectral congestion, even when the sample is not a mixture. Spectral congestion can obscure the patterns in rovibrational spectra that are needed to assign peaks in the spectra. A method for generating high resolution 2DIR spectra of gas phase molecules has now been developed and tested using methane as the sample. The 2D rovibrational patterns that are recorded resemble an asterisk with a center position that provides the frequencies of both of the two coupled vibrational levels. The ability to generate easily recognizable 2D rovibrational patterns, regardless of temperature, should make the technique useful for a wide range of applications that are otherwise difficult or impossible when using conventional 1D rovibrational spectroscopy.

High resolution coherent multidimensional spectroscopy has the ability to reduce congestion and automatically sort peaks by species and quantum numbers, even for simple mixtures and molecules that are extensively perturbed. The two-dimensional version is relatively simple to carry out, and the results are easy to interpret, but its ability to deal with severe spectral congestion is limited. Three-dimensional spectroscopy is considerably more complicated and time-consuming than two-dimensional spectroscopy, but it provides the spectral resolution needed for more challenging systems. This paper describes how to design high resolution coherent 3D spectroscopy experiments so that a small number of strategically positioned 2D scans may be used instead of recording all the data required for a 3D plot. This faster and simpler approach uses new pattern recognition methods to interpret the results. Key factors that affect the resulting patterns include the scanning strategy and the four wave mixing process. Optimum four wave mixing (FWM) processes and scanning strategies have been identified, and methods for identifying the FWM process from the observed patterns have been developed. Experiments based on nonparametric FWM processes provide significant pattern recognition and efficiency advantages over those based on parametric processes. Alternative scanning strategies that use synchronous scanning and asynchronous scanning to create new kinds of patterns have also been identified. Rotating the resulting patterns in 3D space leads to an insight into similarities in the patterns produced by different FWM processes.

The pathogenesis of HIV-associated neurocognitive impairment (NCI) may involve iron dysregulation. In 243 HIV-seropositive adults without severe comorbidities, we therefore genotyped 250 variants in 20 iron-related genes and evaluated their associations with magnetic resonance imaging measures of brain structure and metabolites, including measures previously linked to NCI. Multivariable regression analyses examined associations between genetic variants and neuroimaging measures, adjusting for relevant covariates and multiple testing. Exploratory analyses stratified by NCI (Global Deficit Score ≥ 0.5 vs. <0.5), virus detectability in plasma, and comorbidity levels were also performed. Of 27 variants (in 12 iron-regulatory genes) associated with neuroimaging measures after correction for the 37 haplotype blocks represented, 3 variants survived additional correction for the 21 neuroimaging measures evaluated and demonstrated biologically plausible associations. SLC11A1 rs7576974&#95;T was significantly associated with higher frontal gray matter N-acetylaspartate (p = 3.62e -5 ). Among individuals with detectable plasma virus, TFRC rs17091382&#95;A was associated with smaller subcortical gray matter volume (p = 3.23e -5 ), and CP rs4974389&#95;A (p = 3.52e -5 ) was associated with higher basal ganglia Choline in persons with mild comorbidities. Two other strong associations were observed for variants in SLC40A1 and ACO2 but were not robust due to low minor-allele frequencies in the study sample. Variants in iron metabolism and transport genes are associated with structural and metabolite neuroimaging measures in HIV-seropositive adults, regardless of virus suppression on antiretroviral therapy. These variants may confer susceptibility to HIV-related brain injury and NCI. Further studies are needed to determine the specificity of these findings to HIV infection and explore potential underlying mechanisms.

A new method for generating high-resolution coherent 3D (HRC3D) spectra has been developed that is based on the nonparametric four-wave mixing process MENS (multiply enhanced nonparametric spectroscopy). The resulting spectra have rotational patterns that are different from those produced previously using the parametric four-wave mixing process CARS. A change in the rotational pattern facilitates a new approach to scanning where orthogonal 2D slices in 3D space are combined to make a 3D rotational pattern. This 3D rotational pattern may then be used to calculate rotational constants for levels in the excited electronic state and upper regions of the ground electronic state. Unlike previous forms of HRC3D spectroscopy, this new approach provides a stand-alone rapid and simple tool for the rotational analysis of electronic spectra without the need for obtaining peak positions or molecular constants from other (1D or 2D) forms of spectroscopy.

We identified rare coding variants associated with Alzheimer's disease in a three-stage case-control study of 85,133 subjects. In stage 1, we genotyped 34,174 samples using a whole-exome microarray. In stage 2, we tested associated variants (P < 1 × 10 -4 ) in 35,962 independent samples using de novo genotyping and imputed genotypes. In stage 3, we used an additional 14,997 samples to test the most significant stage 2 associations (P < 5 × 10 -8 ) using imputed genotypes. We observed three new genome-wide significant nonsynonymous variants associated with Alzheimer's disease: a protective variant in PLCG2 (rs72824905: p.Pro522Arg, P = 5.38 × 10 -10 , odds ratio (OR) = 0.68, minor allele frequency (MAF) cases = 0.0059, MAF controls = 0.0093), a risk variant in ABI3 (rs616338: p.Ser209Phe, P = 4.56 × 10 -10 , OR = 1.43, MAF cases = 0.011, MAF controls = 0.008), and a new genome-wide significant variant in TREM2 (rs143332484: p.Arg62His, P = 1.55 × 10 -14 , OR = 1.67, MAF cases = 0.0143, MAF controls = 0.0089), a known susceptibility gene for Alzheimer's disease. These protein-altering changes are in genes highly expressed in microglia and highlight an immune-related protein-protein interaction network enriched for previously identified risk genes in Alzheimer's disease. These genetic findings provide additional evidence that the microglia-mediated innate immune response contributes directly to the development of Alzheimer's disease.

Introduction: African-American (AA) individuals have a higher risk for late-onset Alzheimer's disease (LOAD) than Americans of primarily European ancestry (EA). Recently, the largest genome-wide association study in AAs to date confirmed that six of the Alzheimer's disease (AD)-related genetic variants originally discovered in EA cohorts are also risk variants in AA; however, the risk attributable to many of the loci (e.g., APOE, ABCA7) differed substantially from previous studies in EA. There likely are risk variants of higher frequency in AAs that have not been discovered., Methods: We performed a comprehensive analysis of genetically determined local and global ancestry in AAs with regard to LOAD status., Results: Compared to controls, LOAD cases showed higher levels of African ancestry, both globally and at several LOAD relevant loci, which explained risk for AD beyond global differences., Discussion: Exploratory post hoc analyses highlight regions with greatest differences in ancestry as potential candidate regions for future genetic analyses., (Copyright © 2016 The Alzheimer's Association. All rights reserved.)

Late-onset Alzheimer disease (AD) has a complex genetic etiology, involving locus heterogeneity, polygenic inheritance, and gene-gene interactions; however, the investigation of interactions in recent genome-wide association studies has been limited. We used a biological knowledge-driven approach to evaluate gene-gene interactions for consistency across 13 data sets from the Alzheimer Disease Genetics Consortium. Fifteen single nucleotide polymorphism (SNP)-SNP pairs within 3 gene-gene combinations were identified: SIRT1 × ABCB1, PSAP × PEBP4, and GRIN2B × ADRA1A. In addition, we extend a previously identified interaction from an endophenotype analysis between RYR3 × CACNA1C. Finally, post hoc gene expression analyses of the implicated SNPs further implicate SIRT1 and ABCB1, and implicate CDH23 which was most recently identified as an AD risk locus in an epigenetic analysis of AD. The observed interactions in this article highlight ways in which genotypic variation related to disease may depend on the genetic context in which it occurs. Further, our results highlight the utility of evaluating genetic interactions to explain additional variance in AD risk and identify novel molecular mechanisms of AD pathogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The emergence of resting-state functional connectivity (rsFC) analysis, which examines temporal correlations of low-frequency (<0.1 Hz) blood oxygen level-dependent signal fluctuations between brain regions, has dramatically improved our understanding of the functional architecture of the typically developing (TD) human brain. This study examined rsFC in Down syndrome (DS) compared with another neurodevelopmental disorder, Williams syndrome (WS), and TD. Ten subjects with DS, 18 subjects with WS, and 40 subjects with TD each participated in a 3-Tesla MRI scan. We tested for group differences (DS vs. TD, DS vs. WS, and WS vs. TD) in between- and within-network rsFC connectivity for seven functional networks. For the DS group, we also examined associations between rsFC and other cognitive and genetic risk factors. In DS compared with TD, we observed higher levels of between-network connectivity in 6 out 21 network pairs but no differences in within-network connectivity. Participants with WS showed lower levels of within-network connectivity and no significant differences in between-network connectivity relative to DS. Finally, our comparison between WS and TD controls revealed lower within-network connectivity in multiple networks and higher between-network connectivity in one network pair relative to TD controls. While preliminary due to modest sample sizes, our findings suggest a global difference in between-network connectivity in individuals with neurodevelopmental disorders compared with controls and that such a difference is exacerbated across many brain regions in DS. However, this alteration in DS does not appear to extend to within-network connections, and therefore, the altered between-network connectivity must be interpreted within the framework of an intact intra-network pattern of activity. In contrast, WS shows markedly lower levels of within-network connectivity in the default mode network and somatomotor network relative to controls. These findings warrant further investigation using a task-based procedure that may help disentangle the relationship between brain function and cognitive performance across the spectrum of neurodevelopmental disorders.

Glyocogen synthase kinase 3 (GSK3) plays an important role in the pathophysiology of Alzheimer's disease (AD) through the phosphorylation of tau. Recent work has suggested that GSK3β also plays a role in the amyloid pathway of AD through genetic interactions with APP and APBB2 on in vivo measures of amyloid. This project extends the previously identified genotype interactions to an autopsy measure of amyloid, while also testing the same interactions leveraging gene expression data quantified in the prefrontal cortex. 797 participants (251 cognitively normal, 196 mild cognitive impairment, and 350 Alzheimer's disease) were drawn from the Religious Orders Study and Rush Memory and Aging Project. A mean score of amyloid load was calculated across eight brain regions, gene expression levels from frozen sections of the dorsolateral prefrontal cortex were quantified using RNA amplification, and expression signals were generated using Beadstudio. Three SNPs previously identified in genetic interactions were genotyped using the Illumina 1M genotyping chip. Covariates included age, sex, education, and diagnosis. We were able to evaluate 2 of the 3 previously identified interactions, of which the interaction between GSK3β (rs334543) and APBB2 (rs2585590) was found in this autopsy sample (p = 0.04). We observed a comparable interaction between GSK3β and APBB2 when comparing the highest tertile of gene expression to the lowest tertile, t(1) = -2.03, p = 0.043. These results provide additional evidence of a genetic interaction between GSK3β and APBB2 and further suggest that GSK3β is involved in the pathophysiology of both of the primary neuropathologies of Alzheimer's disease.

Expansion from coherent 2D spectroscopy to coherent 3D spectroscopy can provide significant advantages when studying molecules that have heavily perturbed energy levels. This paper illustrates such advantages by demonstrating how high resolution coherent 3D (HRC3D) spectroscopy can be used to study a portion of the visible spectrum of nitrogen dioxide. High resolution coherent 2D spectra usually contain rotational and vibrational patterns that are easy to analyze, but severe congestion and complexity preclude its effective use for many parts of the NO2 spectrum. HRC3D spectroscopy appears to be much more effective; multidimensional rotational and vibrational patterns produced by this new technique are easy to identify even in the presence of strong perturbations. A method for assigning peaks, which is based upon analyzing the resulting multidimensional patterns, has been developed. The higher level of multidimensionality is useful for reducing uncertainty in peak assignments, improving spectral resolution, providing simultaneous information on multiple levels and states, and predicting, verifying, and categorizing peaks.

Importance: Recently, a rare variant in the amyloid precursor protein gene (APP) was described in a population from Iceland. This variant, in which alanine is replaced by threonine at position 673 (A673T), appears to protect against late-onset Alzheimer disease (AD). We evaluated the frequency of this variant in AD cases and cognitively normal controls to determine whether this variant will significantly contribute to risk assessment in individuals in the United States., Objective: To determine the frequency of the APP A673T variant in a large group of elderly cognitively normal controls and AD cases from the United States and in 2 case-control cohorts from Sweden., Design, Setting, and Participants: Case-control association analysis of variant APP A673T in US and Swedish white individuals comparing AD cases with cognitively intact elderly controls. Participants were ascertained at multiple university-associated medical centers and clinics across the United States and Sweden by study-specific sampling methods. They were from case-control studies, community-based prospective cohort studies, and studies that ascertained multiplex families from multiple sources., Main Outcomes and Measures: Genotypes for the APP A673T variant were determined using the Infinium HumanExome V1 Beadchip (Illumina, Inc) and by TaqMan genotyping (Life Technologies)., Results: The A673T variant genotypes were evaluated in 8943 US AD cases, 10 480 US cognitively normal controls, 862 Swedish AD cases, and 707 Swedish cognitively normal controls. We identified 3 US individuals heterozygous for A673T, including 1 AD case (age at onset, 89 years) and 2 controls (age at last examination, 82 and 77 years). The remaining US samples were homozygous for the alanine (A673) allele. In the Swedish samples, 3 controls were heterozygous for A673T and all AD cases were homozygous for the A673 allele. We also genotyped a US family previously reported to harbor the A673T variant and found a mother-daughter pair, both cognitively normal at ages 72 and 84 years, respectively, who were both heterozygous for A673T; however, all individuals with AD in the family were homozygous for A673., Conclusions and Relevance: The A673T variant is extremely rare in US cohorts and does not play a substantial role in risk for AD in this population. This variant may be primarily restricted to Icelandic and Scandinavian populations.

Congenital amusia is defined by marked deficits in pitch perception and production. Though historically examined only in otherwise typically developing (TD) populations, amusia has recently been documented in Williams syndrome (WS), a genetic, neurodevelopmental disorder with a unique auditory phenotype including auditory sensitivities and increased emotional responsiveness to music but variable musical skill. The current study used structural T1-weighted magnetic resonance imaging and diffusion tensor imaging to examine neural correlates of amusia in 17 individuals with WS (4 of whom met criteria for amusia). Consistent with findings from TD amusics, amusia in WS was associated with decreased fractional anisotropy (FA) in the right superior longitudinal fasciculus (SLF). The relationship between amusia and FA in the inferior component of the SLF was particularly robust, withstanding corrections for cognitive functioning, auditory sensitivities, or musical training. Though the number of individuals with amusia in the study is small, results add to evidence for the role of fronto-temporal disconnectivity in congenital amusia and suggest that novel populations with developmental differences can provide a window into understanding gene-brain-behavior relationships that underlie musical behaviors.

Background: A subset of individuals present at autopsy with the pathologic features of Alzheimer's disease having never manifest the clinical symptoms. We sought to identify genetic factors that modify the relationship between phosphorylated tau (PTau) and dilation of the lateral inferior ventricles., Methods: We used data from 700 subjects enrolled in the Alzheimer's Disease Neuroimaging Initiative (ADNI). A genome-wide association study approach was used to identify PTau × single nucleotide polymorphism (SNP) interactions. Variance explained by these interactions was quantified using hierarchical linear regression., Results: Five SNP × PTau interactions passed a Bonferroni correction, one of which (rs4728029, POT1, 2.6% of variance) was consistent across ADNI-1 and ADNI-2/GO subjects. This interaction also showed a trend-level association with memory performance and levels of interleukin-6 receptor., Conclusions: Our results suggest that rs4728029 modifies the relationship between PTau and both ventricular dilation and cognition, perhaps through an altered neuroinflammatory response., (Copyright © 2014 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

Importance: Because APOE locus variants contribute to risk of late-onset Alzheimer disease (LOAD) and to differences in age at onset (AAO), it is important to know whether other established LOAD risk loci also affect AAO in affected participants., Objectives: To investigate the effects of known Alzheimer disease risk loci in modifying AAO and to estimate their cumulative effect on AAO variation using data from genome-wide association studies in the Alzheimer Disease Genetics Consortium., Design, Setting, and Participants: The Alzheimer Disease Genetics Consortium comprises 14 case-control, prospective, and family-based data sets with data on 9162 participants of white race/ethnicity with Alzheimer disease occurring after age 60 years who also had complete AAO information, gathered between 1989 and 2011 at multiple sites by participating studies. Data on genotyped or imputed single-nucleotide polymorphisms most significantly associated with risk at 10 confirmed LOAD loci were examined in linear modeling of AAO, and individual data set results were combined using a random-effects, inverse variance-weighted meta-analysis approach to determine whether they contribute to variation in AAO. Aggregate effects of all risk loci on AAO were examined in a burden analysis using genotype scores weighted by risk effect sizes., Main Outcomes and Measures: Age at disease onset abstracted from medical records among participants with LOAD diagnosed per standard criteria., Results: Analysis confirmed the association of APOE with earlier AAO (P = 3.3 × 10(-96)), with associations in CR1 (rs6701713, P = 7.2 × 10(-4)), BIN1 (rs7561528, P = 4.8 × 10(-4)), and PICALM (rs561655, P = 2.2 × 10(-3)) reaching statistical significance (P < .005). Risk alleles individually reduced AAO by 3 to 6 months. Burden analyses demonstrated that APOE contributes to 3.7% of the variation in AAO (R(2) = 0.256) over baseline (R(2) = 0.221), whereas the other 9 loci together contribute to 2.2% of the variation (R(2) = 0.242)., Conclusions and Relevance: We confirmed an association of APOE (OMIM 107741) variants with AAO among affected participants with LOAD and observed novel associations of CR1 (OMIM 120620), BIN1 (OMIM 601248), and PICALM (OMIM 603025) with AAO. In contrast to earlier hypothetical modeling, we show that the combined effects of Alzheimer disease risk variants on AAO are on the scale of, but do not exceed, the APOE effect. While the aggregate effects of risk loci on AAO may be significant, additional genetic contributions to AAO are individually likely to be small.

High-resolution coherent multidimensional spectroscopy provides an alternative to conventional methods for generating rotationally resolved electronic spectra of gas phase molecules. In addition to revealing information such as the relationships among peaks, it can provide clearly recognizable patterns for spectra that otherwise appear patternless due to rotational congestion. Despite this improvement, high-resolution coherent 2D spectroscopy can still exhibit congestion problems; expansion to the second dimension is often not sufficient to prevent overlapping of peaks from different patterns. A new 3D version of the technique that provides improved resolution and selectivity to help address cases with severe congestion was recently demonstrated. The experimental design and interpretation of data for the 3D technique are significantly more complicated than that for the 2D version. The purpose of this paper is to provide important information needed to plan, run, and interpret results from high-resolution coherent 3D spectroscopy experiments.

Background: While a great deal of work has gone into understanding the relationship between Cerebrospinal fluid (CSF) biomarkers, brain atrophy, and disease progression, less work has attempted to investigate how genetic variation modifies these relationships. The goal of this study was two-fold. First, we sought to identify high-risk vs. low-risk individuals based on their CSF tau and Aβ load and characterize these individuals with regard to brain atrophy in an AD-relevant region of interest. Next, we sought to identify genetic variants that modified the relationship between biomarker classification and neurodegeneration., Methods: Participants were categorized based on established cut-points for biomarker positivity. Mixed model regression was used to quantify longitudinal change in the left inferior lateral ventricle. Interaction analyses between single nucleotide polymorphisms (SNPs) and biomarker group status were performed using a genome wide association study (GWAS) approach. Correction for multiple comparisons was performed using the Bonferroni procedure., Results: One intergenic SNP (rs4866650) and one SNP within the SPTLC1 gene (rs7849530) modified the association between amyloid positivity and neurodegeneration. A transcript variant of WDR11-AS1 gene (rs12261764) modified the association between tau positivity and neurodegeneration. These effects were consistent across the two sub-datasets and explained approximately 3% of variance in ventricular dilation. One additional SNP (rs6887649) modified the association between amyloid positivity and baseline ventricular volume, but was not observed consistently across the sub-datasets., Conclusions: Genetic variation modifies the association between AD biomarkers and neurodegeneration. Genes that regulate the molecular response in the brain to oxidative stress may be particularly relevant to neural vulnerability to the damaging effects of amyloid-β.

Imaging genetics is an emerging methodological field that combines genetic information with medical imaging-derived metrics to understand how genetic factors impact observable phenotypes. In order for a trait to be a reasonable phenotype in an imaging genetics study, it must be heritable: at least some proportion of its variance must be due to genetic influences. The Sequential Oligogenic Linkage Analysis Routines (SOLAR) imaging genetics software can estimate the heritability of a trait in complex pedigrees. We investigate the ability of SOLAR to accurately estimate heritability and common environmental effects on simulated imaging phenotypes in various family structures. We found that heritability is reliably estimated with small family-based studies of 40 to 80 individuals, though subtle differences remain between the family structures. In an imaging application analysis, we found that with 80 subjects in any of the family structures, estimated heritability of white matter fractional anisotropy was biased by <10% for every region of interest. Results from these studies can be used when investigators are evaluating power in planning genetic analyzes.

Background: Alzheimer's disease is a common debilitating dementia with known heritability, for which 20 late onset susceptibility loci have been identified, but more remain to be discovered. This study sought to identify new susceptibility genes, using an alternative gene-wide analytical approach which tests for patterns of association within genes, in the powerful genome-wide association dataset of the International Genomics of Alzheimer's Project Consortium, comprising over 7 m genotypes from 25,580 Alzheimer's cases and 48,466 controls., Principal Findings: In addition to earlier reported genes, we detected genome-wide significant loci on chromosomes 8 (TP53INP1, p = 1.4×10-6) and 14 (IGHV1-67 p = 7.9×10-8) which indexed novel susceptibility loci., Significance: The additional genes identified in this study, have an array of functions previously implicated in Alzheimer's disease, including aspects of energy metabolism, protein degradation and the immune system and add further weight to these pathways as potential therapeutic targets in Alzheimer's disease.

The driving theoretical framework of Alzheimer's disease (AD) has been built around the amyloid-β (Aβ) cascade in which amyloid pathology precedes and drives tau pathology. Other evidence has suggested that tau and amyloid pathology may arise independently. Both lines of research suggest that there may be epistatic relationships between genes involved in amyloid and tau pathophysiology. In the current study, we hypothesized that genes coding glycogen synthase kinase 3 (GSK-3) and comparable tau kinases would modify genetic risk for amyloid plaque pathology. Quantitative amyloid positron emission tomography data from the Alzheimer's Disease Neuroimaging Initiative served as the quantitative outcome in regression analyses, covarying for age, gender, and diagnosis. Three interactions reached statistical significance, all involving the GSK3β single nucleotide polymorphism rs334543-2 with APBB2 (rs2585590, rs3098914) and 1 with APP (rs457581). These interactions explained 1.2%, 1.5%, and 1.5% of the variance in amyloid deposition respectively. Our results add to a growing literature on the role of GSK-3 activity in amyloid processing and suggest that combined variation in GSK3β and APP-related genes may result in increased amyloid burden., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The genetic etiology of late-onset Alzheimer's disease (LOAD) has proven complex, involving clinical and genetic heterogeneity and gene-gene interactions. Recent genome wide association studies in LOAD have led to the discovery of novel genetic risk factors; however, the investigation of gene-gene interactions has been limited. Conventional genetic studies often use binary disease status as the primary phenotype, but for complex brain-based diseases, neuroimaging data can serve as quantitative endophenotypes that correlate with disease status and closely reflect pathological changes. In the Alzheimer's Disease Neuroimaging Initiative cohort, we tested for association of genetic interactions with longitudinal MRI measurements of the inferior lateral ventricles (ILVs), which have repeatedly shown a relationship to LOAD status and progression. We performed linear regression to evaluate the ability of pathway-derived SNP-SNP pairs to predict the slope of change in volume of the ILVs. After Bonferroni correction, we identified four significant interactions in the right ILV (RILV) corresponding to gene-gene pairs SYNJ2-PI4KA, PARD3-MYH2, PDE3A-ABHD12B, and OR2L13-PRKG1 and one significant interaction in the left ILV (LILV) corresponding to SYNJ2-PI4KA. The SNP-SNP interaction corresponding to SYNJ2-PI4KA was identical in the RILV and LILV and was the most significant interaction in each (RILV: p = 9.13 × 10(-12); LILV: p = 8.17 × 10(-13)). Both genes belong to the inositol phosphate signaling pathway which has been previously associated with neurodegeneration in AD and we discuss the possibility that perturbation of this pathway results in a down-regulation of the Akt cell survival pathway and, thereby, decreased neuronal survival, as reflected by increased volume of the ventricles.

Late-onset Alzheimer's disease (LOAD) is known to have a complex, oligogenic etiology, with considerable genetic heterogeneity. We investigated the influence of genetic interactions between genes in the Alzheimer's disease (AD) pathway on amyloid-beta (Aβ) deposition as measured by PiB or AV-45 ligand positron emission tomography (PET) to aid in understanding LOAD's genetic etiology. Subsets of the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohorts were used for discovery and for two independent validation analyses. A significant interaction between RYR3 and CACNA1C was confirmed in all three of the independent ADNI datasets. Both genes encode calcium channels expressed in the brain. The results shown here support previous animal studies implicating interactions between these calcium channels in amyloidogenesis and suggest that the pathological cascade of this disease may be modified by interactions in the amyloid-calcium axis. Future work focusing on the mechanisms of such relationships may inform targets for clinical intervention.

Background: Individuals with Down Syndrome (DS) are reported to experience early onset of brain aging. However, it is not well understood how pre-existing neurodevelopmental effects versus neurodegenerative processes might be contributing to the observed pattern of brain atrophy in younger adults with DS. The aims of the current study were to: (1) to confirm previous findings of age-related changes in DS compared to adults with typical development (TD), (2) to test for an effect of these age-related changes in a second neurodevelopmental disorder, Williams syndrome (WS), and (3) to identify a pattern of regional age-related effects that are unique to DS., Methods: High-resolution T1-weighted MRI of the brains of subjects with DS, WS, and TD controls were segmented, and estimates of regional brain volume were derived using FreeSurfer. A general linear model was employed to test for age-related effects on volume between groups. Secondary analyses in the DS group explored the relationship between brain volume and neuropsychological tests and APOE., Results: Consistent with previous findings, the DS group showed significantly greater age-related effects relative to TD controls in total gray matter and in regions of the orbitofrontal cortex and the parietal cortex. Individuals with DS also showed significantly greater age-related effects on volume of the left and right inferior lateral ventricles (LILV and RILV, respectively). There were no significant differences in age-related effects on volume when comparing the WS and TD groups. In the DS group, cognitive tests scores measuring signs of dementia and APOE ϵ4 carrier status were associated with LILV and RILV volume., Conclusions: Individuals with DS demonstrated a unique pattern of age-related effects on gray matter and ventricular volume, the latter of which was associated with dementia rating scores in the DS group. Results may indicate that early onset of brain aging in DS is primarily due to DS-specific neurodegenerative processes, as opposed to general atypical neurodevelopment.

G-protein coupled inwardly rectifying potassium (GIRK) channels are effectors determining degree of analgesia experienced upon opioid receptor activation by endogenous and exogenous opioids. The impact of GIRK-related genetic variation on human pain responses has received little research attention. We used a tag single nucleotide polymorphism (SNP) approach to comprehensively examine pain-related effects of KCNJ3 (GIRK1) and KCNJ6 (GIRK2) gene variation. Forty-one KCNJ3 and 69 KCNJ6 tag SNPs were selected, capturing the known variability in each gene. The primary sample included 311 white patients undergoing total knee arthroplasty in whom postsurgical oral opioid analgesic medication order data were available. Primary sample findings were then replicated in an independent white sample of 63 healthy pain-free individuals and 75 individuals with chronic low back pain (CLBP) who provided data regarding laboratory acute pain responsiveness (ischemic task) and chronic pain intensity and unpleasantness (CLBP only). Univariate quantitative trait analyses in the primary sample revealed that 8 KCNJ6 SNPs were significantly associated with the medication order phenotype (P < .05); overall effects of the KCNJ6 gene (gene set-based analysis) just failed to reach significance (P = .054). No significant KCNJ3 effects were observed. A continuous GIRK Related Risk Score (GRRS) was derived in the primary sample to summarize each individual's number of KCNJ6 "pain risk" alleles. This GRRS was applied to the replication sample, which revealed significant associations (P < .05) between higher GRRS values and lower acute pain tolerance and higher CLBP intensity and unpleasantness. Results suggest further exploration of the impact of KCNJ6 genetic variation on pain outcomes is warranted., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Eleven susceptibility loci for late-onset Alzheimer's disease (LOAD) were identified by previous studies; however, a large portion of the genetic risk for this disease remains unexplained. We conducted a large, two-stage meta-analysis of genome-wide association studies (GWAS) in individuals of European ancestry. In stage 1, we used genotyped and imputed data (7,055,881 SNPs) to perform meta-analysis on 4 previously published GWAS data sets consisting of 17,008 Alzheimer's disease cases and 37,154 controls. In stage 2, 11,632 SNPs were genotyped and tested for association in an independent set of 8,572 Alzheimer's disease cases and 11,312 controls. In addition to the APOE locus (encoding apolipoprotein E), 19 loci reached genome-wide significance (P < 5 × 10(-8)) in the combined stage 1 and stage 2 analysis, of which 11 are newly associated with Alzheimer's disease.

Mitochondrial DNA (mtDNA) variation can affect phenotypic variation; therefore, knowing its distribution within and among individuals is of importance to understanding many human diseases. Intra-individual mtDNA variation (heteroplasmy) has been generally assumed to be random. We used massively parallel sequencing to assess heteroplasmy across ten tissues and demonstrate that in unrelated individuals there are tissue-specific, recurrent mutations. Certain tissues, notably kidney, liver and skeletal muscle, displayed the identical recurrent mutations that were undetectable in other tissues in the same individuals. Using RFLP analyses we validated one of the tissue-specific mutations in the two sequenced individuals and replicated the patterns in two additional individuals. These recurrent mutations all occur within or in very close proximity to sites that regulate mtDNA replication, strongly implying that these variations alter the replication dynamics of the mutated mtDNA genome. These recurrent variants are all independent of each other and do not occur in the mtDNA coding regions. The most parsimonious explanation of the data is that these frequently repeated mutations experience tissue-specific positive selection, probably through replication advantage., Competing Interests: The authors have declared that no competing interests exist.

In the past, high-resolution spectroscopy has been limited to small, simple molecules that yield relatively uncongested spectra. Larger and more complex molecules have a higher density of peaks and are susceptible to complications (e.g., effects from conical intersections) that can obscure the patterns needed to resolve and assign peaks. Recently, high-resolution coherent two-dimensional (2D) spectroscopy has been used to resolve and sort peaks into easily identifiable patterns for molecules where pattern-recognition has been difficult. For very highly congested spectra, however, the ability to resolve peaks using coherent 2D spectroscopy is limited by the bandwidth of instrumentation. In this article, we introduce and investigate high-resolution coherent three-dimensional spectroscopy (HRC3D) as a method for dealing with heavily congested systems. The resulting patterns are unlike those in high-resolution coherent 2D spectra. Analysis of HRC3D spectra could provide a means for exploring the spectroscopy of large and complex molecules that have previously been considered too difficult to study.

Aims: To establish success and complication rates of excimer laser coronary atherectomy (ELCA) in a contemporary series of patients with balloon failure during percutaneous coronary intervention (PCI) of both chronic total occlusions (CTO) and lesions with distal TIMI 3 flow., Methods and Results: We identified 58 cases of balloon failure treated with ELCA±rotational atherectomy (RA) over four years, representing 0.84% of all PCI performed in our centre during this period. Balloon failures were classified according to: (i) mechanism of balloon failure; and (ii) whether this occurred in the context of treating a CTO. ELCA was performed following balloon failure using the CVX-300 Excimer Laser System and a 0.9 mm catheter with saline flush. For the entire cohort, procedure success was achieved in 91% (with ELCA successful: alone in 76.1%, after RA failure in 6.8% and in combination with RA for 8.6%). Only in one case did RA succeed where ELCA had failed. There were four procedure-related complications, including transient no-reflow, side branch occlusion and two coronary perforations, of which one was directly attributable to ELCA and led to subsequent mortality., Conclusions: ELCA provides safe and effective adjunctive therapy in contemporary PCI to treat lesions associated with balloon failure due to an inability either to cross the lesion or to expand a balloon sufficiently to permit stenting. ELCA was successful in the majority of these selected cases when used independently with further effectiveness achieved when combined with RA or after RA failure.

Missing heritability in late onset Alzheimer disease can be attributed, at least in part, to heterogeneity in disease status and to the lack of statistical analyses exploring genetic interactions. In the current study, we use quantitative intermediate phenotypes derived from magnetic resonance imaging data available from the Alzheimer's Disease Neuroimaging Initiative, and we test for association with gene-gene interactions within biological pathways. Regional brain volumes from the hippocampus (HIP) and entorhinal cortex (EC) were estimated from baseline and 12-month magnetic resonance imaging scans. Approximately 560,000 single nucleotide polymorphisms (SNPs) were available genome-wide. We tested all pairwise SNP-SNP interactions (approximately 151 million) within 212 Kyoto Encyclopedia of Genes and Genomes pathways for association with 12-month regional atrophy rates using linear regression, with sex, APOE ε4 carrier status, age, education, and clinical status as covariates. A total of 109 SNP-SNP interactions were associated with right HIP atrophy, and 125 were associated with right EC atrophy. Enrichment analysis indicated significant SNP-SNP interactions were overrepresented in the calcium signaling and axon guidance pathways for both HIP and EC atrophy and in the ErbB signaling pathway for HIP atrophy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Williams syndrome (WS) is a rare genetic disorder caused by the deletion of approximately 25 genes at 7q11.23 that involves mild to moderate intellectual disability (ID). When using functional magnetic resonance imaging (fMRI) to compare individuals with ID to typically developing individuals, there is a possibility that differences in IQ contribute to between-group differences in BOLD signal. If IQ is correlated with BOLD signal, then group-level analyses should adjust for IQ, or else IQ should be matched between groups. If, however, IQ is not correlated with BOLD signal, no such adjustment or criteria for matching (and exclusion) based on IQ is necessary., Methods: In this study, we aimed to test this hypothesis systematically using four extant fMRI datasets in WS. Participants included 29 adult subjects with WS (17 men) demonstrating a wide range of standardized IQ scores (composite IQ mean = 67, SD = 17.2). We extracted average BOLD activation for both cognitive and task-specific anatomically defined regions of interest (ROIs) in each individual and correlated BOLD with composite IQ scores, verbal IQ scores and non-verbal IQ scores in Spearman rank correlation tests., Results: Of the 312 correlations performed, only six correlations (2%) in four ROIs reached statistical significance at a P value < 0.01, but none survived correction for multiple testing. All six correlations were positive. Therefore, none supports the hypothesis that IQ is negatively correlated with BOLD response., Conclusions: These data suggest that the inclusion of subjects with below normal IQ does not introduce a confounding factor, at least for some types of fMRI studies with low cognitive load. By including subjects who are representative of IQ range for the targeted disorder, findings are more likely to generalize to that population.

Deformation-based morphometry (DBM) is a widely used method for characterizing anatomical differences across groups. DBM is based on the analysis of the deformation fields generated by nonrigid registration algorithms, which warp the individual volumes to a DBM atlas. Although several studies have compared nonrigid registration algorithms for segmentation tasks, few studies have compared the effect of the registration algorithms on group differences that may be uncovered through DBM. In this study, we compared group atlas creation and DBM results obtained with five well-established nonrigid registration algorithms using 13 subjects with Williams syndrome and 13 normal control subjects. The five nonrigid registration algorithms include the following: (1) the adaptive bases algorithm, (2) the image registration toolkit, (3) The FSL nonlinear image registration tool, (4) the automatic registration tool, and (5) the normalization algorithm available in Statistical Parametric Mapping (SPM8). Results indicate that the choice of algorithm has little effect on the creation of group atlases. However, regions of differences between groups detected with DBM vary from algorithm to algorithm both qualitatively and quantitatively. Some regions are detected by several algorithms, but their extent varies. Others are detected only by a subset of the algorithms. Based on these results, we recommend using more than one algorithm when performing DBM studies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Williams syndrome is a neurodevelopmental disorder associated with significant non-social fears. Consistent with this elevated non-social fear, individuals with Williams syndrome have an abnormally elevated amygdala response when viewing threatening non-social stimuli. In typically-developing individuals, amygdala activity is inhibited through dense, reciprocal white matter connections with the prefrontal cortex. Neuroimaging studies suggest a functional uncoupling of normal prefrontal-amygdala inhibition in individuals with Williams syndrome, which might underlie both the extreme amygdala activity and non-social fears. This functional uncoupling might be caused by structural deficits in underlying white matter pathways; however, prefrontal-amygdala white matter deficits have yet to be explored in Williams syndrome. We used diffusion tensor imaging to investigate prefrontal-amygdala white matter integrity differences in individuals with Williams syndrome and typically-developing controls with high levels of non-social fear. White matter pathways between the amygdala and several prefrontal regions were isolated using probabilistic tractography. Within each pathway, we tested for between-group differences in three measures of white matter integrity: fractional anisotropy (FA), radial diffusivity (RD), and parallel diffusivity (λ(1)). Individuals with Williams syndrome had lower FA, compared to controls, in several of the prefrontal-amygdala pathways investigated, indicating a reduction in white matter integrity. Lower FA in Williams syndrome was explained by significantly higher RD, with no differences in λ(1), suggestive of lower fiber density or axon myelination in prefrontal-amygdala pathways. These results suggest that deficits in the structural integrity of prefrontal-amygdala white matter pathways might underlie the increased amygdala activity and extreme non-social fears observed in Williams syndrome., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Williams syndrome (WS) is a rare genetic neurodevelopmental disorder characterized by increased non-social anxiety, sensitivity to sounds and hypersociability. Previous studies have reported contradictory findings with regard to regional brain variation in WS, relying on only one type of morphological measure (usually volume) in each study. The present study aims to contribute to this body of literature and perhaps elucidate some of these discrepancies by examining concurrent measures of cortical thickness, surface area and subcortical volume between WS subjects and typically-developing (TD) controls. High resolution MRI scans were obtained on 31 WS subjects and 50 typically developing control subjects. We derived quantitative regional estimates of cortical thickness, cortical surface area, and subcortical volume using FreeSurfer software. We evaluated between-group ROI differences while controlling for total intracranial volume. In post-hoc exploratory analyses within the WS group, we tested for correlations between regional brain variation and Beck Anxiety Inventory scores. Consistent with our hypothesis, we detected complex patterns of between-group cortical variation, which included lower surface area in combination with greater thickness in the following cortical regions: post central gyrus, cuneus, lateral orbitofrontal cortex and lingual gyrus. Additional cortical regions showed between-group differences in one (but not both) morphological measures. Subcortical volume was lower in the basal ganglia and the hippocampus in WS versus TD controls. Exploratory correlations revealed that anxiety scores were negatively correlated with gray matter surface area in insula, OFC, rostral middle frontal, superior temporal and lingual gyrus. Our results were consistent with previous reports showing structural alterations in regions supporting the socio-affective and visuospatial impairments in WS. However, we also were able to effectively capture novel and complex patterns of cortical differences using both surface area and thickness. In addition, correlation results implicate specific brain regions in levels of anxiety in WS, consistent with previous reports investigating general anxiety disorders in the general population.

Diffusion tensor imaging (DTI) was used to investigate the involvement of brain white matter in Williams syndrome (WS), a genetic neurodevelopmental disorder. Whole-brain DTIs were obtained from 16 young adults with WS and 16 normal controls. A voxel-based analysis was performed to compare fractional anisotropy (FA) values between the two groups. A tract-based analysis was also performed to compare FA values between the two groups along two major white matter tracts that pass through the external capsule: the uncinate and inferior fronto-occipital fasciculi. Several regions of both increased and decreased FA were found within major white matter tracts that connect functional regions that have previously been implicated in the cognitive and neurological symptoms of the syndrome. The tract-based analysis provided additional insight into the involvement of specific white matter tracts implicated in the voxel-based analysis within the external capsule. The results from this study support previously reported changes in white matter diffusion properties in WS and demonstrate the potential usefulness for tract-based analysis in future studies of the disorder., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Williams syndrome is a neurodevelopmental disorder with an intriguing behavioral phenotype-hypersociability combined with significant non-social fears. Previous studies have demonstrated abnormalities in amygdala function in individuals with Williams syndrome compared to typically-developing controls. However, it remains unclear whether the findings are related to the atypical neurodevelopment of Williams syndrome, or are also associated with behavioral traits at the extreme end of a normal continuum. We used functional magnetic resonance imaging (fMRI) to compare amygdala blood-oxygenation-level-dependent (BOLD) responses to non-social and social images in individuals with Williams syndrome compared to either individuals with inhibited temperament (high non-social fear) or individuals with uninhibited temperament (high sociability). Individuals with Williams syndrome had larger amygdala BOLD responses when viewing the non-social fear images than the inhibited temperament control group. In contrast, when viewing both fear and neutral social images, individuals with Williams syndrome did not show smaller amygdala BOLD responses relative to the uninhibited temperament control group, but instead had amygdala responses proportionate to their sociability. These results suggest heightened amygdala response to non-social fear images is characteristic of WS, whereas, variability in amygdala response to social fear images is proportionate to, and might be explained by, levels of trait sociability.

Williams syndrome is a genetic neurodevelopmental disorder with a distinctive phenotype, including cognitive-linguistic features, nonsocial anxiety, and a strong attraction to music. we preformed functional MRI studies examining brain responses to musical and other types of stimuli in young adults with Williams syndrome and typically developing controls. In Study 1, the Williams syndrome group exhibited unforeseen activations of the visual cortex to musical stimuli, and it was this novel finding that became the focus of two subsequent studies. Using retinotopy, color localizers, and additional sound conditions, we identified specific visual areas in subjects with Williams syndrome that were activated by both musical and nonmusical auditory stimuli. The results, similar to synthetic-like experiences, have implications for cross-modal sensory processing in typical and atypical neurodevelopment.

Common diseases with a genetic basis are likely to have a very complex etiology, in which the mapping between genotype and phenotype is far from straightforward. A new comprehensive statistical and computational strategy for identifying the missing link between genotype and phenotype has been proposed, which emphasizes the need to address heterogeneity in the first stage of any analysis and gene-gene interactions in the second stage. We applied this two-stage analysis strategy to late-onset Alzheimer disease (LOAD) data, which included functional and positional candidate genes and markers in a region of interest on chromosome 10. Bayesian classification found statistically significant clusterings for independent family-based and case-control datasets, which used the same five markers in leucine-rich repeat transmembrane neuronal 3 (LRRTM3) as the most influential in determining cluster assignment. In subsequent analyses to detect main effects and gene-gene interactions, markers in three genes--urokinase-type plasminogen activator (PLAU), angiotensin 1 converting enzyme (ACE) and cell division cycle 2 (CDC2)--were found to be associated with LOAD in particular subsets of the data based on their LRRTM3 multilocus genotype. All of these genes are viable candidates for LOAD based on their known biological function, even though PLAU, CDC2 and LRRTM3 were initially identified as positional candidates. Further studies are needed to replicate these statistical findings and to elucidate possible biological interaction mechanisms between LRRTM3 and these genes.

Dramatic advances in genotyping technology have established a need for fast, flexible analysis methods for genetic association studies. Common complex diseases, such as Parkinson's disease or multiple sclerosis, are thought to involve an interplay of multiple genes working either independently or together to influence disease risk. Also, multiple underlying traits, each its own genetic basis may be defined together as a single disease. These effects - trait heterogeneity, locus heterogeneity, and gene-gene interactions (epistasis) - contribute to the complex architecture of common genetic diseases. Association Rule Discovery (ARD) searches for frequent itemsets to identify rule-based patterns in large scale data. In this study, we apply Apriori (an ARD algorithm) to simulated genetic data with varying degrees of complexity. Apriori using information difference to prior as a rule measure shows good power to detect functional effects in simulated cases of simple trait heterogeneity, trait heterogeneity and epistasis, and moderate power in cases of trait heterogeneity and locus heterogeneity. Also, we illustrate that bootstrapping the rule induction process does not considerably improve the power to detect these effects. These results show that ARD is a framework with sufficient flexibility to characterize complex genetic effects.

Background: Trait heterogeneity, which exists when a trait has been defined with insufficient specificity such that it is actually two or more distinct traits, has been implicated as a confounding factor in traditional statistical genetics of complex human disease. In the absence of detailed phenotypic data collected consistently in combination with genetic data, unsupervised computational methodologies offer the potential for discovering underlying trait heterogeneity. The performance of three such methods--Bayesian Classification, Hypergraph-Based Clustering, and Fuzzy k-Modes Clustering--appropriate for categorical data were compared. Also tested was the ability of these methods to detect trait heterogeneity in the presence of locus heterogeneity and/or gene-gene interaction, which are two other complicating factors in discovering genetic models of complex human disease. To determine the efficacy of applying the Bayesian Classification method to real data, the reliability of its internal clustering metrics at finding good clusterings was evaluated using permutation testing., Results: Bayesian Classification outperformed the other two methods, with the exception that the Fuzzy k-Modes Clustering performed best on the most complex genetic model. Bayesian Classification achieved excellent recovery for 75% of the datasets simulated under the simplest genetic model, while it achieved moderate recovery for 56% of datasets with a sample size of 500 or more (across all simulated models) and for 86% of datasets with 10 or fewer nonfunctional loci (across all simulated models). Neither Hypergraph Clustering nor Fuzzy k-Modes Clustering achieved good or excellent cluster recovery for a majority of datasets even under a restricted set of conditions. When using the average log of class strength as the internal clustering metric, the false positive rate was controlled very well, at three percent or less for all three significance levels (0.01, 0.05, 0.10), and the false negative rate was acceptably low (18 percent) for the least stringent significance level of 0.10., Conclusion: Bayesian Classification shows promise as an unsupervised computational method for dissecting trait heterogeneity in genotypic data. Its control of false positive and false negative rates lends confidence to the validity of its results. Further investigation of how different parameter settings may improve the performance of Bayesian Classification, especially under more complex genetic models, is ongoing.

Acute pericarditis is usually a benign self-limiting condition, often of unexplained or viral aetiology, involving inflammation of the pericardial layers. It is often part of the differential diagnosis in patients admitted with acute chest pain and can be confused with acute myocardial infarction, acute pulmonary embolism and pleurisy. Occasionally it can result in cardiac tamponade and, if associated with myocarditis, in heart failure. This article sets out how to diagnose acute pericarditis, the common underlying causes, the possible treatment options and outcomes.

Molecular biologists and geneticists alike now acknowledge that most common human diseases with a genetic component are likely to have complex etiologies. Yet despite this belief, many statistical geneticists continue applying, in slightly new and different ways, methodologies that were developed to dissect much simpler etiologies. In this article, we characterize, with examples, the various factors that can complicate genetic analysis and demonstrate their shared features and how they affect genetic analysis. We describe a variety of approaches that are currently available, revealing methodological gaps and suggesting new directions for method development. Finally, we propose a comprehensive two-step approach to analysis that systemically addresses the different genetic factors that are likely to underlie complex diseases.

In the primary quinone (Q(A)) binding site of Rb. sphaeroides reaction centers (RCs), isoleucine M265 is in extensive van der Waals contact with the ubiquinone headgroup. Substitution of threonine or serine for this residue (mutants M265IT and M265IS), but not valine (mutant M265IV), lowers the redox midpoint potential of Q(A) by about 100 mV (Takahashi et al. (2001) Biochemistry 40, 1020-1028). The unexpectedly large effect of the polar substitutions is not due to reorientation of the methoxy groups as similar redox potential changes are seen for these mutants with either ubiquinone or anthraquinone as Q(A). Using FTIR spectroscopy to compare Q(A)(-)/Q(A) IR difference spectra for wild type and the M265 mutant RCs, we found changes in the polar mutants (M265IT and M265IS) in the quinone C[double bond]O and C[double bond]C stretching region (1600-1660 cm(-1)) and in the semiquinone anion band (1440-1490 cm(-1)), as well as in protein modes. Modeling the mutations into the X-ray structure of the wild-type RC indicates that the hydroxyl group of the mutant polar residues, Thr and Ser, is hydrogen bonded to the peptide C[double bond]O of Thr(M261). It is suggested that the mutational effect is exerted through the extended backbone region that includes Ala(M260), the hydrogen bonding partner to the C1 carbonyl of the quinone headgroup. The resulting structural perturbations are likely to include lengthening of the hydrogen bond between the quinone C1[double bond]O and the peptide NH of Ala(M260). Possible origins of the IR spectroscopic and redox potential effects are discussed.

Attenuated total reflection (ATR) spectroscopy brings an added dimension to studies of structural changes of cytochrome c oxidase (CcO) because it enables the recording of reaction-induced infrared difference spectra under a wide variety of controlled conditions (e.g. pH and chemical composition), without relying on light or potentiometric changes to trigger the reaction. We have used the ATR method to record vibrational difference spectra of CcO with reduction induced by flow-exchange of the aqueous buffer. Films of CcO prepared from Rhodobacter sphaeroides and beef heart mitochondria by reconstitution with lipid were adhered to the internal reflection element of the ATR device and retained their full functionality as evidenced by visible spectroscopy and time-resolved vibrational spectroscopy. These results demonstrate that the technique of perfusion-induced Fourier-transform infrared difference spectroscopy can be successfully applied to a large, complex enzyme, such as CcO, with sufficient signal/noise to probe vibrational changes in individual residues of the enzyme under various conditions.