👤 Inna Levin

The comparative roles of triglyceride-rich lipoproteins (TRLs) and low-density lipoproteins (LDLs) in abdominal aortic aneurysm (AAA) pathogenesis are unclear. To evaluate the putative causal role of TRLs in AAA, quantify the relative effect on AAA risk ("aneurysmogenicity") of TRL vs LDL particles, and prioritize lipid-lowering drug targets for AAA prevention and treatment. We performed summary-level and individual-level Mendelian randomization (MR) analyses. Genetic variants were selected from 383,983 UK Biobank participants and ranked into 10 sets of variants where set 1 predominantly affected LDL cholesterol (LDL-C) and set 10 predominantly affected TRL cholesterol (TRL-C; and with mixed effects for intermediate variant sets). AAA outcome data were obtained from AAAgen (37,214 cases), FinnGen (4,439 cases), and the VA Million Veteran Program (MVP; 23,848 cases). Multivariable MR was used to assess the independent roles of LDL-C and TRL-C in AAA. For each set of variants, MR or logistic regression was used to estimate AAA odds ratios (ORs) per 10 mg/dL higher apolipoprotein B (apoB). Interaction analyses were conducted between a statin-like LDL-C-lowering variant set (set 3) and a TRL-C-lowering variant set (set 10). Drug-target MR was performed to evaluate lipid-lowering targets relevant to LDL-C- and TRL-C-lowering. Genetically predicted LDL-C and TRL-C concentrations were each associated independently with genetic liability for AAA after mutual adjustment, with 3.0 to 5.5 times stronger associations for TRL-C compared to LDL-C on a per-cholesterol basis. In AAAgen, the AAA OR per 10 mg/dL increased apoB concentrations were 1.10 (95% CI, 1.05-1.14) for variant set 1 (LDL-C-predominant) and 1.89 (95% CI, 1.69-2.11) for variant set 10 (TRL-C-predominant). Using the ratio of log(OR) per 10 mg/dL apoB for set 10 versus set 1 as a conservative estimate of relative aneurysmogenicity, TRLs were approximately 3.2 to 6.9 times more aneurysmogenic than LDLs across the three studies. No evidence of interaction was observed between LDLs and TRLs, indicating additive contribution to AAA risk. Drug-target MR supported strong protective associations for genetically proxied inhibition of TRL-pathway targets, particularly TRLs are at least threefold more aneurysmogenic than LDLs on a per-particle basis. Therapeutic strategies targeting TRL-C -especially via Show less

Lp(a) is a genetically determined lipoprotein targeted by emerging therapies. In a UK Biobank analysis (1,026 abdominal aortic aneurysm [AAA] cases, 469,989 controls), elevated Lp(a) was associated with increased risk of AAA, including at clinically relevant thresholds while controlling for traditional risk factors, including ApoB. Multivariable Mendelian randomization confirmed a causal relationship between lipoprotein(a) [Lp(a)] and AAA independent of apolipoprotein B. These findings support Lp(a) as a modifiable risk factor and potential therapeutic target for AAA, a condition with limited medical treatment options. AAA should be considered as an outcome in future clinical trials of Lp(a)-lowering therapies. Show less

Determining apolipoprotein E (APOE) ε4 allele status, a key genetic risk factor for Alzheimer's disease (AD), requires molecular genotyping infrastructure not widely accessible beyond specialized centers. A fully automated high-throughput apoE E4 proteotyping immunoassay was evaluated for clinical performance (460 participants across three cohorts) and analytical validity. Concordance with polymerase chain reaction (PCR)-based genotyping and measures of analytical validity were reported. The apoE E4 immunoassay demonstrated 99.6% (95% confidence interval [CI]: 98.4% to 99.9%) concordance with PCR-based APOE ε4 genotype results across the pooled clinical cohort; 100.0% (95% CI: 97.1% to 100.0%) in those with AD (N = 127) and 99.4% (95% CI: 97.8% to 99.8%) in those without AD (333). The assay met analytical validity criteria for E4 isoform specificity, interference, precision, and stability. The apoE E4 immunoassay demonstrated high concordance with PCR-based genotyping and robust analytical validity, offering an accessible alternative for APOE ε4 zygosity assessment. A novel high-throughput plasma-based proteotyping immunoassay for APOE ε4 zygosity classification was developed and evaluated for clinical performance and analytical validity. The apoE E4 immunoassay demonstrated high concordance (99.6%) with PCR-based APOE ε4 genotyping across a diverse international cohort, and a robust analytical profile. An apoE E4 immunoassay may offer a more cost-effective and accessible alternative to DNA genotyping approaches currently used for AD risk evaluation and anti-amyloid treatment decisions. Show less

Aging and male sex are major risk factors for abdominal aortic aneurysm (AAA), a disease characterized by vascular cell phenotypic switching and aortic wall remodeling. Mitochondrial oxidative stress has been implicated in these changes. We previously demonstrated that NOX4 (NADPH oxidase 4) expression and activity increase with age in cardiovascular cells, promoting mitochondrial oxidative stress and vascular dysfunction. This study investigates whether NOX4-driven mitochondrial oxidative stress and DNA damage promote AAA development through vascular cell reprogramming. We used mitochondria-targeted NOX4-dependent mitochondrial DNA damage and activation of DNA-sensing pathways promote SMC phenotypic switching, inflammation, and aortic wall remodeling in AAA. Targeting NOX4 and enhancing mitochondrial function may offer therapeutic strategies for AAA prevention. Show less

Imaging studies showed early atrophy of the cholinergic basal forebrain (BF) already at prodromal stages of sporadic Alzheimer's disease (AD). Women and carriers of the ApoE epsilon 4 (ApoE ε4) allele are more likely to develop the disease; however, the underlying mechanisms are still unclear. Here we aimed at exploring the impact of sex and ApoE ε4 genotype in the AD spectrum on longitudinal measures of the basal forebrain and hippocampus, as a comparison region. We leveraged the German multi-centered study DELCODE and analyzed 712 individuals (median age: 71.25 years, interquartile range [IQR] = 9.22) with follow-up MRI scans (median time: 2.8 years, [IQR] = 1.75). Diagnostic groups comprised cognitively normal ( The hippocampus, but not the basal forebrain, showed significant atrophy over time (Hipp: Our findings did not show the anticipated longitudinal effects of sex and ApoE ε4 on longitudinal basal forebrain volume. Only hippocampal atrophy progressed significantly faster in ApoE ε4 homozygote carriers. This dissociation may reflect stage-dependent neurodegenerative processes, with early basal forebrain vulnerability followed by more rapid hippocampal decline, as well as methodological and sample-related constraints. If replicated, these findings suggest that hippocampal measures may be more sensitive longitudinal biomarkers in ApoE ε4 homozygotes, while sex- and ApoE ε4-related effects on the cholinergic system may be more prominent at earlier disease stages. Show less

CLN3 disease, also called Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), or Batten disease, is an ultra‑rare, neurodegenerative lysosomal storage disorder generally affecting individuals during the first decade of life. There can be a delay in diagnosis or misdiagnosis due to a lack of awareness, and when the most common presenting symptom of visual loss is attributed to more common conditions affecting vision. We used a previously published Expert Mapping Tool (EMT) to identify multidisciplinary professionals with diagnostic or clinical management expertise, as well as patient advocates with experience of CLN3 disease. A systematic literature review of published evidence using the Preferred Reporting Items for Systematic Reviews and Meta‑Analyses (PRISMA) guidance was conducted independently and simultaneously to develop key clinical care statements. Each statement was based on the strength of the evidence. The statements formed the basis of an international modified-Delphi consensus process using a virtual meeting platform (Within3). Experts were asked to agree or disagree with each statement and suggest any changes. Statements that reached a consensus of 75% or over are the guiding statements within this manuscript. The processes and manuscript have been independently assessed using the Appraisal of Guidelines for Research and Evaluation (AGREE II) criteria. Thirty‑nine international experts from eight specialities were identified, including a patient advocate. Fifty‑three recommendation statements were developed covering eleven domains: General statements, Diagnostics, Clinical Recommendations and Management, Assessments, Social Considerations, Ocular Management, Epilepsy/Seizures, Nutrition, Respiratory Health, Sleep and Rest, and End-of-Life Care. Consensus was reached after one round of voting for all except three statements. The overall AGREE II score for developing these recommendations was 6.4, where 1 represents the lowest and 7 is the highest quality. Currently, there are no comprehensive clinical recommendations for CLN3 disease. These recommendations provide a comprehensive, evidence- and consensus‑based tool that can be used by all healthcare professionals involved in the management of CLN3 disease and other similar neurodegenerative conditions. The goal is to address the unmet clinical need for CLN3 disease management and complement other information available. The online version contains supplementary material available at 10.1186/s13023-026-04298-2. Show less

Circulating lipoprotein(a) [Lp(a)] levels are highly heritable and linked to atherosclerotic cardiovascular disease, yet clinical measurement rates remain low (<1%) in the United States. The high heritability of Lp(a) across populations makes genetic prediction an attractive approach for closing this testing gap, but existing polygenic scores transfer poorly across populations. Haplotype-based prediction models, which use standard genome-wide genotype data to capture common-, rare-, and structural-variation at the LPA locus, could bridge this gap, enabling opportunistic identification of individuals with elevated Lp(a) levels across diverse populations within existing large, genotyped cohorts. This study sought to develop and validate a haplotype-based prediction model using genome-wide genotype data to identify individuals with elevated Lp(a) levels across diverse populations. We developed an Among PMBB (n = 1856), MGBB (n = 1401), and BioMe (n = 1686) participants with available genotype and Lp(a) measurements, average age was 60 years, and 51% were female. Overall r A haplotype-based genetic model effectively identified individuals with elevated Lp(a) levels across diverse populations, with potential utility for opportunistic screening among cohorts where genotype data is available, but Lp(a) testing rates are low. Show less

Cirrhosis and hepatocellular carcinoma (HCC) are long-term complications of chronic liver disease (CLD). In this large multi-ancestry genome-wide association study of all-cause cirrhosis (35,481 cases, 2.36M controls) and HCC (6,680 cases, 1.76M controls), we identified 27 loci associated with cirrhosis (10 novel) and 11 with HCC (three novel). Three novel cirrhosis loci were replicated in independent cohorts (e.g. Show less

Identification of drug-repurposing targets with genetic and biological support is an economically and temporally efficient strategy for improving the treatment of diseases. We employed a cross-disciplinary approach to identify potential therapeutics for the prevention of metabolic-dysfunction-associated steatotic liver disease (MASLD) in at-risk individuals by using humans as a model organism. We identified 212 putative candidate genes associated with MASLD by using data from a large multi-ancestry genetic association study, of which 158 (74.5%) were previously unreported. From this set, we identified 57 genes that encode for druggable protein targets and for which the effects of increasing genetically predicted gene expression on MASLD risk align with the function of that drug on the protein target. We then used We then evaluated these potential targets for evidence of efficacy by using Mendelian randomization, pathway analysis, and protein structural modeling. Through these approaches, we present compelling evidence to suggest that the activation of FADS1 by icosapent ethyl, as well as S1PR2 by fingolimod, could be a promising therapeutic strategy for MASLD prevention. Show less

Identification of drug-repurposing targets with genetic and biological support is an economically and temporally efficient strategy for improving treatment of diseases. We employed a cross-disciplinary approach to identify potential treatments for metabolic dysfunction associated steatotic liver disease (MASLD) using humans as a model organism. We identified 212 putative causal genes associated with MASLD using data from a large multi-ancestry genetic association study, of which 158 (74.5%) are novel. From this set we identified 57 genes that encode for druggable protein targets, and where the effects of increasing genetically predicted gene expression on MASLD risk align with the function of that drug on the protein target. These potential targets were then evaluated for evidence of efficacy using Mendelian randomization, pathway analysis, and protein structural modeling. Using these approaches, we present compelling evidence to suggest activation of Show less

Heart failure is a complex trait, influenced by environmental and genetic factors, affecting over 30 million individuals worldwide. Here we report common-variant and rare-variant association studies of all-cause heart failure and examine how different classes of genetic variation impact its heritability. We identify 176 common-variant risk loci at genome-wide significance in 2,358,556 individuals and cluster these signals into five broad modules based on pleiotropic associations with anthropomorphic traits/obesity, blood pressure/renal function, atherosclerosis/lipids, immune activity and arrhythmias. In parallel, we uncover exome-wide significant associations for heart failure and rare predicted loss-of-function variants in TTN, MYBPC3, FLNC and BAG3 using exome sequencing of 376,334 individuals. We find that total burden heritability of rare coding variants is highly concentrated in a small set of Mendelian cardiomyopathy genes, while common-variant heritability is diffusely spread throughout the genome. Finally, we show that common-variant background modifies heart failure risk among carriers of rare pathogenic truncating variants in TTN. Together, these findings discern genetic links between dysregulated metabolism and heart failure and highlight a polygenic component to heart failure not captured by current clinical genetic testing. Show less

Heart failure (HF) is a complex trait, influenced by environmental and genetic factors, which affects over 30 million individuals worldwide. Historically, the genetics of HF have been studied in Mendelian forms of disease, where rare genetic variants have been linked to familial cardiomyopathies. More recently, genome-wide association studies (GWAS) have successfully identified common genetic variants associated with risk of HF. However, the relative importance of genetic variants across the allele-frequency spectrum remains incompletely characterized. Here, we report the results of common- and rare-variant association studies of all-cause heart failure, applying recently developed methods to quantify the heritability of HF attributable to different classes of genetic variation. We combine GWAS data across multiple populations including 207,346 individuals with HF and 2,151,210 without, identifying 176 risk loci at genome-wide significance (P-value < 5×10 Show less

Lipoprotein(a) (Lp(a)) is a circulating apolipoprotein B (ApoB) containing particle that has been observationally linked to atherosclerotic cardiovascular disease and is the target of emerging therapeutics. Recent work has highlighted the role of circulating lipoproteins in abdominal aortic aneurysm (AAA). We sought to triangulate human observational and genetic evidence to evaluate the role of Lp(a) in AAA. We tested the association between circulating levels of Lp(a) and clinically diagnosed abdominal aortic aneurysms while controlling for traditional AAA risk factors and levels of ApoB using logistic regression among 795 individuals with and 374,772 individuals without AAA in the UK Biobank (UKB). Multivariable Mendelian randomization (MVMR) was used to test for putatively causal associations between Lp(a) and AAA controlling for ApoB. Genetic instruments for Lp(a) and ApoB were created from genome-wide association studies (GWAS) of Lp(a) and ApoB comprising 335,796 and 418,505 UKB participants, respectively. The instruments were tested for association with AAA using data from a GWAS of 39,221 individuals with and 1,086,107 without AAA. Elevated Lp(a) levels were observationally associated with an increased risk of AAA (OR 1.04 per 10 nmol/L Lp(a); 95%CI 1.02-1.05; P<0.01). Clinically elevated Lp(a) levels (>150nmol/L) were likewise associated with an increased risk of AAA (OR 1.47; 95% CI 1.15-1.88; P < 0.01) when compared to individuals with Lp(a) levels <150nmol/L. MVMR confirmed a significant, ApoB-independent association between increased Lp(a) and increased risk of AAA (OR 1.13 per SD increase in Lp(a); 95%CI 1.02-1.24; P<0.02). Both observational and genetic analyses support an association between increased Lp(a) and AAA risk that is independent of ApoB. These findings suggest that Lp(a) may be a therapeutic target for AAA and drive the inclusion of AAA as an outcome in clinical trials of Lp(a) antagonists. Show less

Plants have evolved photosynthetic regulatory mechanisms to maintain homeostasis in response to light changes during diurnal transitions and those caused by passing clouds or by wind. One such adaptation directs photosynthetic electron flow to a cyclic pathway to alleviate excess energy surges. Here, we assign a function to regulatory cysteines of PGR5-like protein 1A (PGRL1A), a constituent of the PROTON GRADIENT REGULATION5 (PGR5)-dependent cyclic electron flow (CEF) pathway. During step increases from darkness to low light intensity in Arabidopsis (Arabidopsis thaliana), the intermolecular disulfide of the PGRL1A 59-kDa complex was reduced transiently within seconds to the 28-kDa form. In contrast, step increases from darkness to high light stimulated a stable, partially reduced redox state in PGRL1A. Mutations of 2 cysteines in PGRL1A, Cys82 and Cys183, resulted in a constitutively pseudo-reduced state. The mutant displayed higher proton motive force (PMF) and nonphotochemical quenching (NPQ) than the wild type (WT) and showed altered donor and acceptor dynamic flow around PSI. These changes were found to correspond with the redox state of PGRL1A. Continuous light regimes did not affect mutant growth compared to the WT. However, under fluctuating regimes of high light, the mutant showed better growth than the WT. In contrast, in fluctuating regimes of low light, the mutant displayed a growth penalty that can be attributed to constant stimulation of CEF under low light. Treatment with photosynthetic inhibitors indicated that PGRL1A redox state control depends on the penultimate Fd redox state. Our results showed that redox state changes in PGRL1A are crucial to optimize photosynthesis. Show less

Plasma triglycerides (TGs) are causally associated with coronary artery disease and acute pancreatitis. Apolipoprotein A-V (apoA-V, gene We used hydrogen-deuterium exchange mass spectrometry to determine the secondary structure of human apoA-V in lipid-free and lipid-associated conditions and identified a C-terminal hydrophobic face. Then, we used genomic data in the Penn Medicine Biobank to identify a rare variant, Q252X, predicted to specifically eliminate this region. We interrogated the function of apoA-V Q252X using recombinant protein Human apoA-V Q252X carriers exhibited elevated plasma TG levels consistent with loss of function. Deletion of apoA-V's C-terminus leads to reduced apoA-V bioavailability Show less

The study aimed to discover novel genetic loci for atrial fibrillation (AF), explore the shared genetic etiologies between AF and other cardiovascular and cardiometabolic traits, and uncover AF pathogenesis using Mendelian randomization analysis. We conducted a genome-wide association study meta-analysis including 109,787 AF cases and 1,165,920 controls of European ancestry and identified 215 loci, among which 91 were novel. We performed Genomic Structural Equation Modeling analysis between AF and four cardiovascular comorbidities (coronary artery disease, ischemic stroke, heart failure, and vneous thromboembolism) and found 189 loci shared across these diseases as well as a universal genetic locus shared by atherosclerotic outcomes (i.e., rs1537373 near This genome-wide association study and trans-omic Mendelian randomization analysis provides insights into disease risk prediction, pathophysiology and downstream sequelae. Show less

Nanodiscs are emerging to serve as transfer vectors for the insertion of recombinant membrane proteins into membranes of living cells. In combination with cell-free expression technologies, this novel process opens new perspectives to analyze the effects of even problematic targets such as toxic, hard-to-express, or artificially modified membrane proteins in complex cellular environments of different cell lines. Furthermore, transferred cells must not be genetically engineered and primary cell lines or cancer cells could be implemented as well. We have systematically analyzed the basic parameters of the nanotransfer approach and compared the transfer efficiencies from nanodiscs with that from Salipro particles. The transfer of five membrane proteins was analyzed: the prokaryotic proton pump proteorhodopsin, the human class A family G-protein coupled receptors for endothelin type B, prostacyclin, free fatty acids type 2, and the orphan GPRC5B receptor as a class C family member. The membrane proteins were cell-free synthesized with a detergent-free strategy by their cotranslational insertion into preformed nanoparticles containing defined lipid environments. The purified membrane protein/nanoparticles were then incubated with mammalian cells. We demonstrate that nanodiscs disassemble and only lipids and membrane proteins, not the scaffold protein, are transferred into cell membranes. The process is detectable within minutes, independent of the nanoparticle lipid composition, and the transfer efficiency directly correlates with the membrane protein concentration in the transfer mixture and with the incubation time. Transferred membrane proteins insert in both orientations, N-terminus in and N-terminus out, in the cell membrane, and the ratio can be modulated by engineering. The viability of cells is not notably affected by the transfer procedure, and transferred membrane proteins stay detectable in the cell membrane for up to 3 days. Transferred G-protein coupled receptors retained their functionality in the cell environment as shown by ligand binding, induction of internalization, and specific protein interactions. In comparison to transfection, the cellular membrane protein concentration is better controllable and more uniformly distributed within the analyzed cell population. A further notable difference to transfection is the accumulation of transferred membrane proteins in clusters, presumably determined by microdomain structures in the cell membranes. Show less

'Genome-first' approaches to analyzing rare variants can reveal new insights into human biology and disease. Because pathogenic variants are often rare, new discovery requires aggregating rare coding variants into 'gene burdens' for sufficient power. However, a major challenge is deciding which variants to include in gene burden tests. Pathogenic variants in MYBPC3 and MYH7 are well-known causes of hypertrophic cardiomyopathy (HCM), and focusing on these 'positive control' genes in a genome-first approach could help inform variant selection methods and gene burdening strategies for other genes and diseases. Integrating exome sequences with electronic health records among 41 759 participants in the Penn Medicine BioBank, we evaluated the performance of aggregating predicted loss-of-function (pLOF) and/or predicted deleterious missense (pDM) variants in MYBPC3 and MYH7 for gene burden phenome-wide association studies (PheWAS). The approach to grouping rare variants for these two genes produced very different results: pLOFs but not pDM variants in MYBPC3 were strongly associated with HCM, whereas the opposite was true for MYH7. Detailed review of clinical charts revealed that only 38.5% of patients with HCM diagnoses carrying an HCM-associated variant in MYBPC3 or MYH7 had a clinical genetic test result. Additionally, 26.7% of MYBPC3 pLOF carriers without HCM diagnoses had clear evidence of left atrial enlargement and/or septal/LV hypertrophy on echocardiography. Our study shows the importance of evaluating both pLOF and pDM variants for gene burden testing in future studies to uncover novel gene-disease relationships and identify new pathogenic loss-of-function variants across the human genome through genome-first analyses of healthcare-based populations. Show less

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7's enzyme activity with high affinity (IC Show less

Protein complexes often represent an ensemble of different assemblies with distinct functions and regulation. This increased complexity is enabled by the variety of protein diversification mechanisms that exist at every step of the protein biosynthesis pathway, such as alternative splicing and post transcriptional and translational modifications. The resulting variation in subunits can generate compositionally distinct protein assemblies. These different forms of a single protein complex may comprise functional variances that enable response and adaptation to varying cellular conditions. Despite the biological importance of this layer of complexity, relatively little is known about the compositional heterogeneity of protein complexes, mostly due to technical barriers of studying such closely related species. Here, we show that native mass spectrometry (MS) offers a way to unravel this inherent heterogeneity of protein assemblies. Our approach relies on the advanced Orbitrap mass spectrometer capable of multistage MS analysis across all levels of protein organization. Specifically, we have implemented a two-step fragmentation process in the inject flatapole device, which was converted to a linear ion trap, and can now probe the intact protein complex assembly, through its constituent subunits, to the primary sequence of each protein. We demonstrate our approach on the yeast homotetrameric FBP1 complex, the rate-limiting enzyme in gluconeogenesis. We show that the complex responds differently to changes in growth conditions by tuning phosphorylation dynamics. Our methodology deciphers, on a single instrument and in a single measurement, the stoichiometry, kinetics, and exact position of modifications, contributing to the exposure of the multilevel diversity of protein complexes. Show less

Reciprocal copy-number variation (CNV) of a 593 kb region of 16p11.2 is a common genetic cause of autism spectrum disorder (ASD), yet it is not completely penetrant and can manifest in a wide array of phenotypes. To explore its molecular consequences, we performed RNA sequencing of cerebral cortex from mouse models with CNV of the syntenic 7qF3 region and lymphoblast lines from 34 members of 7 multiplex ASD-affected families harboring the 16p11.2 CNV. Expression of all genes in the CNV region correlated well with their DNA copy number, with no evidence of dosage compensation. We observed effects on gene expression outside the CNV region, including apparent positional effects in cis and in trans at genomic segments with evidence of physical interaction in Hi-C chromosome conformation data. One of the most significant positional effects was telomeric to the 16p11.2 CNV and includes the previously described "distal" 16p11.2 microdeletion. Overall, 16p11.2 CNV was associated with altered expression of genes and networks that converge on multiple hypotheses of ASD pathogenesis, including synaptic function (e.g., NRXN1, NRXN3), chromatin modification (e.g., CHD8, EHMT1, MECP2), transcriptional regulation (e.g., TCF4, SATB2), and intellectual disability (e.g., FMR1, CEP290). However, there were differences between tissues and species, with the strongest effects being consistently within the CNV region itself. Our analyses suggest that through a combination of indirect regulatory effects and direct effects on nuclear architecture, alteration of 16p11.2 genes disrupts expression networks that involve other genes and pathways known to contribute to ASD, suggesting an overlap in mechanisms of pathogenesis. Show less

How do intricate multi-residue features such as protein-protein interfaces evolve? To address this question, we evolved a new colicin-immunity binding interaction. We started with Im9, which inhibits its cognate DNase ColE9 at 10(-14) M affinity, and evolved it toward ColE7, which it inhibits promiscuously (Kd > 10(-8) M). Iterative rounds of random mutagenesis and selection toward higher affinity for ColE7, and selectivity (against ColE9 inhibition), led to an approximately 10(5)-fold increase in affinity and a 10(8)-fold increase in selectivity. Analysis of intermediates along the evolved variants revealed that changes in the binding configuration of the Im protein uncovered a latent set of interactions, thus providing the key to the rapid divergence of new Im7 variants. Overall, protein-protein interfaces seem to share the evolvability features of enzymes, that is, the exploitation of promiscuous interactions and alternative binding configurations via 'generalist' intermediates, and the key role of compensatory stabilizing mutations in facilitating the divergence of new functions. Show less

Analysis of the three-dimensional structures of three closely related mesophilic, thermophilic, and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Clostridium beijerinckii (CbADH), Entamoeba histolytica (EhADH1), and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro100) might be crucial for maintaining the thermal stability of EhADH1. To determine whether proline substitution at this position in TbADH and CbADH would affect thermal stability, we used site-directed mutagenesis to replace the complementary residues in both enzymes with proline. The results showed that replacing Gln100 with proline significantly enhanced the thermal stability of the mesophilic ADH: DeltaT(1/2) (60 min) = + 8 degrees C (temperature of 50% inactivation after incubation for 60 min), DeltaT(1/2) (CD) = +11.5 degrees C (temperature at which 50% of the original CD signal at 218 nm is lost upon heating between 30 degrees and 98 degrees C). A His100 --> Pro substitution in the thermophilic TbADH had no effect on its thermostability. An analysis of the three-dimensional structure of the crystallized thermostable mutant Q100P-CbADH suggested that the proline residue at position 100 stabilized the enzyme by reinforcing hydrophobic interactions and by reducing the flexibility of a loop at this strategic region. Show less