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1.  A Frame-Shift Mutation in CAV1 Is Associated with a Severe Neonatal Progeroid and Lipodystrophy Syndrome 
PLoS ONE  2015;10(7):e0131797.
A 3-year-old female patient presenting with an unknown syndrome of a neonatal progeroid appearance, lipodystrophy, pulmonary hypertension, cutis marmorata, feeding disorder and failure to thrive was investigated by whole-genome sequencing. This revealed a de novo, heterozygous, frame-shift mutation in the Caveolin1 gene (CAV1) (p.Phe160X). Mutations in CAV1, encoding the main component of the caveolae in plasma membranes, cause Berardinelli-Seip congenital lipodystrophy type 3 (BSCL). Although BSCL is recessive, heterozygous carriers either show a reduced phenotype of partial lipodystrophy, pulmonary hypertension, or no phenotype. To investigate the pathogenic mechanisms underlying this syndrome in more depth, we performed next generation RNA sequencing of peripheral blood, which showed several dysregulated pathways in the patient that might be related to the phenotypic progeroid features (apoptosis, DNA repair/replication, mitochondrial). Secondly, we found a significant down-regulation of known Cav1 interaction partners, verifying the dysfunction of CAV1. Other known progeroid genes and lipodystrophy genes were also dysregulated. Next, western blotting of lysates of cultured fibroblasts showed that the patient shows a significantly decreased expression of wild-type CAV1 protein, demonstrating a loss-of-function mutation, though her phenotype is more severe that other heterozygotes with similar mutations. This phenotypic variety could be explained by differences in genetic background. Indications for this are supported by additional rare variants we found in AGPAT2 and LPIN1 lipodystrophy genes. CAV1, AGPAT2 and LPIN1 all play an important role in triacylglycerol (TAG) biosynthesis in adipose tissue, and the defective function in different parts of this pathway, though not all to the same extend, could contribute to a more severe lipoatrophic phenotype in this patient. In conclusion, we report, for the first time, an association of CAV1 dysfunction with a syndrome of severe premature aging and lipodystrophy. This may contribute to a better understanding of the aging process and pathogenic mechanisms that contribute to premature aging.
PMCID: PMC4503302  PMID: 26176221
2.  Genetic implication of a novel thiamine transporter in human hypertension 
We coupled two strategies – trait extremes and genome-wide pooling – to discover a novel BP locus that encodes a previously uncharacterized thiamine transporter.
Hypertension is a heritable trait that remains the most potent and widespread cardiovascular risk factor, though details of its genetic determination are poorly understood.
Representative genomic DNA pools were created from male and female subjects in the highest and lowest 5th %iles of BP in a primary care population of >50,000 individuals. The peak associated SNPs were typed in individual DNA samples, as well as twins/siblings phenotyped for cardiovascular and autonomic traits. Biochemical properties of the associated transporter were evaluated in cellular assays.
After chip hybridization and calculation of relative allele scores, the peak associations were typed in individual samples, revealing association of hypertension, SBP, and DBP to the previously uncharacterized solute carrier SLC35F3. The BP genetic association at SLC35F3 was validated by meta-analysis in an independent sample from the original source population, as well as the ICBP (across North America and Western Europe). Sequence homology to a putative yeast thiamine (vitamin B1) transporter prompted us to express human SLC35F3 in E. coli, which catalyzed [3H]-thiamine uptake. SLC35F3 risk allele (T/T) homozygotes displayed decreased erythrocyte thiamine content on microbiological assay. In twin pairs, the SLC35F3 risk allele predicted heritable cardiovascular traits previously associated with thiamine deficiency, including elevated cardiac stroke volume with decreased vascular resistance, and elevated pressor responses to environmental (cold) stress. Allelic expression imbalance (AEI) confirmed that cis-variation at the human SLC35F3 locus influenced expression of that gene, and the AEI peak coincided with the hypertension peak.
Novel strategies were coupled to position a new hypertension susceptibility locus, uncovering a previously unsuspected thiamine transporter whose genetic variants predicted several disturbances in cardiac and autonomic function. The results have implications for the pathogenesis and treatment of systemic hypertension.
PMCID: PMC3992204  PMID: 24509276
SLC35F3; thiamine; transporter; hypertension
3.  Small cell carcinoma of the ovary, hypercalcemic type, displays frequent inactivating germline and somatic mutations in SMARCA4 
Nature genetics  2014;46(5):427-429.
Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is an extremely rare, aggressive cancer affecting children and young women. We identified germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 69% (9/13) of SCCOHT cases in addition to SMARCA4 protein loss in 82% (14/17) of SCCOHT tumors but in only 0.4% (2/485) of other primary ovarian tumors. These data implicate SMARCA4 in SCCOHT oncogenesis.
PMCID: PMC4332808  PMID: 24658001
4.  A Cellular Model of Amyloid Precursor Protein Processing and Amyloid-β Peptide Production 
A hallmark pathologic feature of Alzheimer’s disease (AD) is accumulation of neuritic senile plaques in the brain parenchyma. Neurotoxic plaque cores are composed predominantly of amyloid-β (Aβ) peptides of 40 and 42 amino acids in length, formed by sequential cleavage of amyloid precursor protein (APP) by β-, and γ-secretases. There is great interest in approaches to modulate Aβ peptide production and develop therapeutic interventions to reduce Aβ levels to halt or slow the progression of neurodegeneration.
New Method
We characterized and present the BE(2)-M17 human neuroblastoma cell line as a novel in vitro model of the APP-cleavage cascade to support future 1) functional studies of molecular regulators in Aβ production, and 2) high-throughput screening assays of new pharmacotherapeutics.
In BE(2)-M17 cells, both RNA (i.e., RT-PCR, RNA Sequencing) and protein analyses (i.e., Western blots, ELISA), show endogenous expression of critical components of the amyloidogenic pathway, APP-cleavage intermediates CTF83 and CTF99, and final cleavage products Aβ40 and Aβ42. We further report effects of retinoic acid-mediated differentiation on morphology and gene expression in this cell line.
Comparison with Existing Method(s)
In contrast to primary isolates or other cell lines reported in current literature, BE(2)-M17 not only sustains baseline expression of the full contingent of APP-processing components, but also remains stably adherent during culture, facilitating experimental manipulations.
Our evidence supports the use of BE(2)-M17 as a novel, human, cell-based model of the APP processing pathway that offers a potential streamlined approach to dissect molecular functions of endogenous regulatory pathways, and perform mechanistic studies to identify modulators of Aβ production.
PMCID: PMC3931259  PMID: 24333289
Alzheimer’s; BE(2)-M17; amyloid-beta; in vitro; secretase; TNF
5.  Characterization of X Chromosome Inactivation Using Integrated Analysis of Whole-Exome and mRNA Sequencing 
PLoS ONE  2014;9(12):e113036.
In females, X chromosome inactivation (XCI) is an epigenetic, gene dosage compensatory mechanism by inactivation of one copy of X in cells. Random XCI of one of the parental chromosomes results in an approximately equal proportion of cells expressing alleles from either the maternally or paternally inherited active X, and is defined by the XCI ratio. Skewed XCI ratio is suggestive of non-random inactivation, which can play an important role in X-linked genetic conditions. Current methods rely on indirect, semi-quantitative DNA methylation-based assay to estimate XCI ratio. Here we report a direct approach to estimate XCI ratio by integrated, family-trio based whole-exome and mRNA sequencing using phase-by-transmission of alleles coupled with allele-specific expression analysis. We applied this method to in silico data and to a clinical patient with mild cognitive impairment but no clear diagnosis or understanding molecular mechanism underlying the phenotype. Simulation showed that phased and unphased heterozygous allele expression can be used to estimate XCI ratio. Segregation analysis of the patient's exome uncovered a de novo, interstitial, 1.7 Mb deletion on Xp22.31 that originated on the paternally inherited X and previously been associated with heterogeneous, neurological phenotype. Phased, allelic expression data suggested an 83∶20 moderately skewed XCI that favored the expression of the maternally inherited, cytogenetically normal X and suggested that the deleterious affect of the de novo event on the paternal copy may be offset by skewed XCI that favors expression of the wild-type X. This study shows the utility of integrated sequencing approach in XCI ratio estimation.
PMCID: PMC4264736  PMID: 25503791
6.  Identification of Novel Genetic Risk Loci in Maltese Dogs with Necrotizing Meningoencephalitis and Evidence of a Shared Genetic Risk across Toy Dog Breeds 
PLoS ONE  2014;9(11):e112755.
Necrotizing meningoencephalitis (NME) affects toy and small breed dogs causing progressive, often fatal, inflammation and necrosis in the brain. Genetic risk loci for NME previously were identified in pug dogs, particularly associated with the dog leukocyte antigen (DLA) class II complex on chromosome 12, but have not been investigated in other susceptible breeds. We sought to evaluate Maltese and Chihuahua dogs, in addition to pug dogs, to identify novel or shared genetic risk factors for NME development. Genome-wide association testing of single nucleotide polymorphisms (SNPs) in Maltese dogs with NME identified 2 regions of genome-wide significance on chromosomes 4 (chr4:74522353T>A, p = 8.1×10−7) and 15 (chr15:53338796A>G, p = 1.5×10−7). Haplotype analysis and fine-mapping suggests that ILR7 and FBXW7, respectively, both important for regulation of immune system function, could be the underlying associated genes. Further evaluation of these regions and the previously identified DLA II locus across all three breeds, revealed an enrichment of nominal significant SNPs associated with chromosome 15 in pug dogs and DLA II in Maltese and Chihuahua dogs. Meta-analysis confirmed effect sizes the same direction in all three breeds for both the chromosome 15 and DLA II loci (p = 8.6×10–11 and p = 2.5×10−7, respectively). This suggests a shared genetic background exists between all breeds and confers susceptibility to NME, but effect sizes might be different among breeds. In conclusion, we identified the first genetic risk factors for NME development in the Maltese, chromosome 4 and chromosome 15, and provide evidence for a shared genetic risk between breeds associated with chromosome 15 and DLA II. Last, DLA II and IL7R both have been implicated in human inflammatory diseases of the central nervous system such as multiple sclerosis, suggesting that similar pharmacotherapeutic targets across species should be investigated.
PMCID: PMC4231098  PMID: 25393235
7.  Genome-Wide Association Meta-analysis of Neuropathologic Features of Alzheimer's Disease and Related Dementias 
PLoS Genetics  2014;10(9):e1004606.
Alzheimer's disease (AD) and related dementias are a major public health challenge and present a therapeutic imperative for which we need additional insight into molecular pathogenesis. We performed a genome-wide association study and analysis of known genetic risk loci for AD dementia using neuropathologic data from 4,914 brain autopsies. Neuropathologic data were used to define clinico-pathologic AD dementia or controls, assess core neuropathologic features of AD (neuritic plaques, NPs; neurofibrillary tangles, NFTs), and evaluate commonly co-morbid neuropathologic changes: cerebral amyloid angiopathy (CAA), Lewy body disease (LBD), hippocampal sclerosis of the elderly (HS), and vascular brain injury (VBI). Genome-wide significance was observed for clinico-pathologic AD dementia, NPs, NFTs, CAA, and LBD with a number of variants in and around the apolipoprotein E gene (APOE). GalNAc transferase 7 (GALNT7), ATP-Binding Cassette, Sub-Family G (WHITE), Member 1 (ABCG1), and an intergenic region on chromosome 9 were associated with NP score; and Potassium Large Conductance Calcium-Activated Channel, Subfamily M, Beta Member 2 (KCNMB2) was strongly associated with HS. Twelve of the 21 non-APOE genetic risk loci for clinically-defined AD dementia were confirmed in our clinico-pathologic sample: CR1, BIN1, CLU, MS4A6A, PICALM, ABCA7, CD33, PTK2B, SORL1, MEF2C, ZCWPW1, and CASS4 with 9 of these 12 loci showing larger odds ratio in the clinico-pathologic sample. Correlation of effect sizes for risk of AD dementia with effect size for NFTs or NPs showed positive correlation, while those for risk of VBI showed a moderate negative correlation. The other co-morbid neuropathologic features showed only nominal association with the known AD loci. Our results discovered new genetic associations with specific neuropathologic features and aligned known genetic risk for AD dementia with specific neuropathologic changes in the largest brain autopsy study of AD and related dementias.
Author Summary
Alzheimer's disease (AD) and related dementias are a major public health challenge and present a therapeutic imperative for which we need additional insight into molecular pathogenesis. We performed a genome-wide association study (GWAS), as well as an analysis of known genetic risk loci for AD dementia, using data from 4,914 brain autopsies. Genome-wide significance was observed for 7 genes and pathologic features of AD and related diseases. Twelve of the 22 genetic risk loci for clinically-defined AD dementia were confirmed in our pathologic sample. Correlation of effect sizes for risk of AD dementia with effect size for hallmark pathologic features of AD were strongly positive and linear. Our study discovered new genetic associations with specific pathologic features and aligned known genetic risk for AD dementia with specific pathologic changes in a large brain autopsy study of AD and related dementias.
PMCID: PMC4154667  PMID: 25188341
8.  Genetic Susceptibility for Alzheimer’s Disease Neuritic Plaque Pathology 
JAMA neurology  2013;70(9):1150-1157.
To investigate whether Alzheimer’s disease (AD) susceptibility loci from genome-wide association studies (GWAS) impact neuritic plaque pathology and to additionally identify novel risk loci for this trait.
Candidate analysis of single nucleotide polymorphisms (SNPs) and GWAS in a joint clinicopathologic cohort study, followed by targeted validation in independent neuroimaging cohorts.
Participants and Setting
725 deceased subjects from the Religious Orders and Rush Memory and Aging Project, two prospective, community-based studies of aging; the validation neuroimaging cohort consisted of 114 subjects from multiple clinical and research centers.
Main Outcome Measures
A quantitative measure of neuritic plaque pathologic burden, based on assessments of silver-stained tissue averaged from multiple brain regions. Validation based on β-amyloid load by immunocytochemistry, and replication with fibrillar β-amyloid Positron Emission Tomography (PET) imaging with Pittsburgh Compound B or florbetapir.
Besides the previously reported APOE and CR1 loci, we find that ABCA7 (rs3764650, P=0.02) and CD2AP (rs9349407, P=0.03) AD susceptibility loci are associated with neuritic plaque burden. In addition, among the top results of our GWAS, we discovered a novel variant near the amyloid precursor protein gene (APP, rs2829887) that is associated with neuritic plaques (P=3.3×10−6). This polymorphism was associated with postmortem β-amyloid load, as well as fibrillar β-amyloid in two independent cohorts of adults with normal cognition.
These findings enhance understanding of AD risk factors by relating validated susceptibility alleles to increased neuritic plaque pathology and implicate common genetic variation at the APP locus in the earliest, pre-symptomatic stages of AD.
PMCID: PMC3773291  PMID: 23836404
9.  Transcriptomic Analysis of Tail Regeneration in the Lizard Anolis carolinensis Reveals Activation of Conserved Vertebrate Developmental and Repair Mechanisms 
PLoS ONE  2014;9(8):e105004.
Lizards, which are amniote vertebrates like humans, are able to lose and regenerate a functional tail. Understanding the molecular basis of this process would advance regenerative approaches in amniotes, including humans. We have carried out the first transcriptomic analysis of tail regeneration in a lizard, the green anole Anolis carolinensis, which revealed 326 differentially expressed genes activating multiple developmental and repair mechanisms. Specifically, genes involved in wound response, hormonal regulation, musculoskeletal development, and the Wnt and MAPK/FGF pathways were differentially expressed along the regenerating tail axis. Furthermore, we identified 2 microRNA precursor families, 22 unclassified non-coding RNAs, and 3 novel protein-coding genes significantly enriched in the regenerating tail. However, high levels of progenitor/stem cell markers were not observed in any region of the regenerating tail. Furthermore, we observed multiple tissue-type specific clusters of proliferating cells along the regenerating tail, not localized to the tail tip. These findings predict a different mechanism of regeneration in the lizard than the blastema model described in the salamander and the zebrafish, which are anamniote vertebrates. Thus, lizard tail regrowth involves the activation of conserved developmental and wound response pathways, which are potential targets for regenerative medical therapies.
PMCID: PMC4139331  PMID: 25140675
10.  Variants in triggering receptor expressed on myeloid cells 2 are associated with both behavioral variant frontotemporal lobar degeneration and Alzheimer’s disease 
Neurobiology of aging  2013;34(8):10.1016/j.neurobiolaging.2013.02.016.
Recent evidence suggests that rare genetic variants within the TREM2 gene are associated with increased risk for Alzheimer’s disease. TREM2 mutations are the genetic basis for a condition characterized by polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL) and an early-onset dementia syndrome. TREM2 is important in the phagocytosis of apoptotic neuronal cells by microglia in the brain. Loss of function might lead to an impaired clearance and accumulation of necrotic debris and subsequent neurodegeneration. In this study, we investigated a consanguineous family segregating autosomal recessive behavioral variant FTLD from Antioquia, Colombia. Exome sequencing identified a nonsense mutation in TREM2 (p.Trp198X) segregating with disease. Next, using a cohort of clinically characterized and neuropathologically verified sporadic AD cases and controls we report replication of the AD risk association at rs75932628 within TREM2. These data suggest that mutational burden in TREM2 may serve as a risk factor for neurodegenerative disease in general and that potentially this class of TREM2 variant carriers with dementia should be considered a molecularly distinct form of neurodegenerative disease.
PMCID: PMC3830921  PMID: 23582655
11.  Initial Assessment of the Pathogenic Mechanisms of the recently identified Alzheimer Risk Loci 
Annals of human genetics  2013;77(2):85-105.
Recent genome wide association studies have identified CLU, CR1, ABCA7 BIN1, PICALM and MS4A6A/MS4A6E in addition to the long established APOE, as loci for Alzheimer’s disease. We have systematically examined each of these loci to assess whether common coding variability contributes to the risk of disease. We have also assessed the regional expression of all the genes in the brain and whether there is evidence of an eQTL explaining the risk. In agreement with other studies we find that coding variability may explain the ABCA7 association, but common coding variability does not explain any of the other loci. We were not able to show that any of the loci had eQTLs within the power of this study. Furthermore the regional expression of each of the loci did not match the pattern of brain regional distribution in Alzheimer pathology.
Although these results are mainly negative, they allow us to start defining more realistic alternative approaches to determine the role of all the genetic loci involved in Alzheimer’s disease.
PMCID: PMC3578142  PMID: 23360175
Alzheimer’s disease; genetic risk; GWAS
12.  A coding variant in CR1 interacts with APOE-ɛ4 to influence cognitive decline 
Human Molecular Genetics  2012;21(10):2377-2388.
Complement receptor 1 (CR1) is an Alzheimer's disease (AD) susceptibility locus that also influences AD-related traits such as episodic memory decline and neuritic amyloid plaque deposition. We implemented a functional fine-mapping approach, leveraging intermediate phenotypes to identify functional variant(s) within the CR1 locus. Using 1709 subjects (697 deceased) from the Religious Orders Study and the Rush Memory and Aging Project, we tested 41 single-nucleotide polymorphisms (SNPs) within the linkage disequilibrium block containing the published CR1 AD SNP (rs6656401) for associations with episodic memory decline, and then examined the functional consequences of the top result. We report that a coding variant in the LHR-D (long homologous repeat D) region of the CR1 gene, rs4844609 (Ser1610Thr, minor allele frequency = 0.02), is associated with episodic memory decline and accounts for the known effect of the index SNP rs6656401 (D′ = 1, r2= 0.084) on this trait. Further, we demonstrate that the coding variant's effect is largely dependent on an interaction with APOE-ɛ4 and mediated by an increased burden of AD-related neuropathology. Finally, in our data, this coding variant is also associated with AD susceptibility (joint odds ratio = 1.4). Taken together, our analyses identify a CR1 coding variant that influences episodic memory decline; it is a variant known to alter the conformation of CR1 and points to LHR-D as the functional domain within the CR1 protein that mediates the effect on memory decline. We thus implicate C1q and MBL, which bind to LHR-D, as likely targets of the variant's effect and suggest that CR1 may be an important intermediate in the clearance of Aβ42 particles by C1q.
PMCID: PMC3335317  PMID: 22343410
13.  A genome-wide scan for common variants affecting the rate of age-related cognitive decline 
Neurobiology of Aging  2011;33(5):1017.e1-1017.e15.
Age-related cognitive decline is likely promoted by accumulated brain injury due to chronic conditions of aging, including neurodegenerative and vascular disease. Since common neuronal mechanisms may mediate the adaptation to diverse cerebral insults, we hypothesized that susceptibility for age-related cognitive decline may be due in part to a shared genetic network. We have therefore performed a genome-wide association study using a quantitative measure of global cognitive decline slope, based on repeated measures of 17 cognitive tests in 749 subjects from the Religious Orders Study. Top results were evaluated in three independent replication cohorts, consisting of 2,279 additional subjects with repeated cognitive testing. As expected, we find that the Alzheimer’s disease (AD) susceptibility locus, APOE, is strongly associated with rate of cognitive decline (PDISC=5.6×10−9; PJOINT=3.7×10−27). We additionally discover a variant, rs10808746, which shows consistent effects in the replication cohorts and modestly improved evidence of association in the joint analysis (PDISC=6.7×10−5; PREP=9.4×10−3; PJOINT=2.3×10−5). This variant influences the expression of two adjacent genes, PDE7A and MTFR1, which are potential regulators of inflammation and oxidative injury, respectively. Using aggregate measures of genetic risk, we find that known susceptibility loci for cardiovascular disease, type II diabetes, and inflammatory diseases are not significantly associated with cognitive decline in our cohort. Our results suggest that intermediate phenotypes, when coupled with larger sample sizes, may be a useful tool to dissect susceptibility loci for age-related cognitive decline and uncover shared molecular pathways with a role in neuronal injury.
PMCID: PMC3307898  PMID: 22054870
14.  Plasma cytokine profiling in sibling pairs discordant for autism spectrum disorder 
Converging lines of evidence point to the existence of immune dysfunction in autism spectrum disorder (ASD), which could directly affect several key neurodevelopmental processes. Previous studies have shown higher cytokine levels in patients with autism compared with matched controls or subjects with other developmental disorders. In the current study, we used plasma-cytokine profiling for 25 discordant sibling pairs to evaluate whether these alterations occur within families with ASD.
Plasma-cytokine profiling was conducted using an array-based multiplex sandwich ELISA for simultaneous quantitative measurement of 40 unique targets. We also analyzed the correlations between cytokine levels and clinically relevant quantitative traits (Vineland Adaptive Behavior Scale in Autism (VABS) composite score, Social Responsiveness Scale (SRS) total T score, head circumference, and full intelligence quotient (IQ)). In addition, because of the high phenotypic heterogeneity of ASD, we defined four subgroups of subjects (those who were non-verbal, those with gastrointestinal issues, those with regressive autism, and those with a history of allergies), which encompass common and/or recurrent endophenotypes in ASD, and tested the cytokine levels in each group.
None of the measured parameters showed significant differences between children with ASD and their related typically developing siblings. However, specific target levels did correlate with quantitative clinical traits, and these were significantly different when the ASD subgroups were analyzed. It is notable that these differences seem to be attributable to a predisposing immunogenetic background, as no other significant differences were noticed between discordant sibling pairs. Interleukin-1β appears to be the cytokine most involved in quantitative traits and clinical subgroups of ASD.
In the present study, we found a lack of significant differences in plasma-cytokine levels between children with ASD and in their related non-autistic siblings. Thus, our results support the evidence that the immune profiles of children with autism do not differ from their typically developing siblings. However, the significant association of cytokine levels with the quantitative traits and the clinical subgroups analyzed suggests that altered immune responses may affect core feature of ASD.
PMCID: PMC3616926  PMID: 23497090
15.  Genome reannotation of the lizard Anolis carolinensis based on 14 adult and embryonic deep transcriptomes 
BMC Genomics  2013;14:49.
The green anole lizard, Anolis carolinensis, is a key species for both laboratory and field-based studies of evolutionary genetics, development, neurobiology, physiology, behavior, and ecology. As the first non-avian reptilian genome sequenced, A. carolinesis is also a prime reptilian model for comparison with other vertebrate genomes. The public databases of Ensembl and NCBI have provided a first generation gene annotation of the anole genome that relies primarily on sequence conservation with related species. A second generation annotation based on tissue-specific transcriptomes would provide a valuable resource for molecular studies.
Here we provide an annotation of the A. carolinensis genome based on de novo assembly of deep transcriptomes of 14 adult and embryonic tissues. This revised annotation describes 59,373 transcripts, compared to 16,533 and 18,939 currently for Ensembl and NCBI, and 22,962 predicted protein-coding genes. A key improvement in this revised annotation is coverage of untranslated region (UTR) sequences, with 79% and 59% of transcripts containing 5’ and 3’ UTRs, respectively. Gaps in genome sequence from the current A. carolinensis build (Anocar2.0) are highlighted by our identification of 16,542 unmapped transcripts, representing 6,695 orthologues, with less than 70% genomic coverage.
Incorporation of tissue-specific transcriptome sequence into the A. carolinensis genome annotation has markedly improved its utility for comparative and functional studies. Increased UTR coverage allows for more accurate predicted protein sequence and regulatory analysis. This revised annotation also provides an atlas of gene expression specific to adult and embryonic tissues.
PMCID: PMC3561122  PMID: 23343042
Annotation; Lizard; Anolis carolinensis; Transcriptome; Genome; RNA-Seq; Gene; Vertebrate; Embryo; Tissue-specific
16.  Analysis of Copy Number Variation in Alzheimer’s Disease in a Cohort of Clinically Characterized and Neuropathologically Verified Individuals 
PLoS ONE  2012;7(12):e50640.
Copy number variations (CNVs) are genomic regions that have added (duplications) or deleted (deletions) genetic material. They may overlap genes affecting their function and have been shown to be associated with disease. We previously investigated the role of CNVs in late-onset Alzheimer's disease (AD) and mild cognitive impairment using Alzheimer’s Disease Neuroimaging Initiative (ADNI) and National Institute of Aging-Late Onset AD/National Cell Repository for AD (NIA-LOAD/NCRAD) Family Study participants, and identified a number of genes overlapped by CNV calls. To confirm the findings and identify other potential candidate regions, we analyzed array data from a unique cohort of 1617 Caucasian participants (1022 AD cases and 595 controls) who were clinically characterized and whose diagnosis was neuropathologically verified. All DNA samples were extracted from brain tissue. CNV calls were generated and subjected to quality control (QC). 728 cases and 438 controls who passed all QC measures were included in case/control association analyses including candidate gene and genome-wide approaches. Rates of deletions and duplications did not significantly differ between cases and controls. Case-control association identified a number of previously reported regions (CHRFAM7A, RELN and DOPEY2) as well as a new gene (HLA-DRA). Meta-analysis of CHRFAM7A indicated a significant association of the gene with AD and/or MCI risk (P = 0.006, odds ratio = 3.986 (95% confidence interval 1.490–10.667)). A novel APP gene duplication was observed in one case sample. Further investigation of the identified genes in independent and larger samples is warranted.
PMCID: PMC3515604  PMID: 23227193
17.  Genome-wide association between DNA methylation and alternative splicing in an invertebrate 
BMC Genomics  2012;13:480.
Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee) and Nasonia vitripennis (jewel wasp) analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data.
We generated RNA deep sequencing data (RNA-seq) to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher’s exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation.
This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice variants by positively influencing exon inclusion during transcription. The results from our cross-species homology analysis suggest that DNA methylation and alternative splicing are genetic mechanisms whose utilization could contribute to a longer gene length and a slower rate of gene evolution.
PMCID: PMC3526459  PMID: 22978521
18.  Tonic Premarin dose-dependently enhances memory, affects neurotrophin protein levels and alters gene expression in middle-aged rats 
Neurobiology of aging  2009;32(4):680-697.
Premarin™ is the most commonly prescribed estrogenic component of hormone therapy, given since 1942. The current study is the first examining cognitive effects of tonic Premarin treatment in an animal model. Middle-aged ovariectomized (Ovx) rats received vehicle or one of three doses of Premarin (12, 24 or 36 μg daily). Rats were tested on a spatial working and reference memory maze battery. Both Medium- and High- dose Premarin enhanced memory retention, while Low-dose Premarin impaired learning and memory retention. Correlations with serum hormone levels showed that as the ratio of estrone:17β-estradiol increased, animals tended to show better working memory performance. Taken together with the dissociation of dose-specific estrogenic profiles, results suggest that higher levels of estrone, in the presence of 17β-estradiol concentrations higher than that of Ovx levels, may be beneficial for memory. Moreover, Premarin exerted dose and brain-region specific effects on BDNF and NGF protein levels, with most marked changes in cingulate and perirhinal cortices. Hippocampal gene expression profiling demonstrated significant Premarin-induced transcriptional changes in genes linked to plasticity and cognition. These findings indicate that Premarin can impact memory and the brain, and that dosing should be recognized as a clinically relevant factor possibly affecting the direction and efficacy of cognitive outcome.
PMCID: PMC3016463  PMID: 19883953
Premarin; estrogen; hormone replacement; working memory; spatial memory; neurotrophins; gene expression
19.  CR1 is associated with amyloid plaque burden and age-related cognitive decline 
Annals of neurology  2011;69(3):560-569.
Recently, genome-wide association studies have identified three new susceptibility loci for Alzheimer’s disease (AD), CLU, CR1, and PICALM. We leveraged available neuropsychological and autopsy data from two cohort studies to investigate whether these loci are associated with cognitive decline and AD neuropathology.
The Religious Orders Study (ROS) and Rush Memory and Aging Project (MAP) are longitudinal studies that enroll non-demented subjects and include annual clinical evaluations and brain donation at death. We evaluated CR1 (rs6656401), CLU (rs11136000) and PICALM (rs7110631) in 1666 subjects. We evaluated associations between genotypes and rate of change in cognitive function as well as AD-related pathology. Lastly, we used pathway analysis to determine if relationships between SNPs and cognitive decline were mediated through AD pathology.
Among our study cohort, the mean years of follow-up was 7.8 for ROS and 4.3 for MAP. Only the CR1 locus was associated with both global cognitive decline (p=0.011) and global AD pathology (p=0.025). More specifically, the locus affects the deposition of neuritic amyloid plaque (p=0.009). In a mediation analysis, controlling for amyloid pathology strongly attenuated the effect of the CR1 locus on cognitive decline.
We found that common variation at the CR1 locus has a broad impact on cognition and that this effect is largely mediated by an individual’s amyloid plaque burden. We therefore highlight one functional consequence of the CR1 susceptibility allele and generalize the role of this locus to cognitive aging in the general population.
PMCID: PMC3066288  PMID: 21391232
NeuroImage  2010;54(3):1896-1902.
In a genome-wide association study (GWAS) of late-onset Alzheimer's disease (AD), we found an association between common haplotypes of the GAB2 gene and AD risk in carriers of the apolipoprotein E (APOE) ε4 allele, the major late-onset AD susceptibility gene. We previously proposed the use of fluorodeoxyglucose positron emission tomography (FDG-PET) measurements as a quantitative presymptomatic endophenotype, more closely related to disease risk than the clinical syndrome itself, to help evaluate putative genetic and non-genetic modifiers of AD risk. In this study, we examined the relationship between the presence or absence of the relatively protective GAB2 haplotype and PET measurements of regional-to-whole brain FDG uptake in several AD-affected brain regions in 158 cognitively normal late-middle-aged APOEε4 homozygotes, heterozygotes, and non-carriers. GAB2 haplotypes were characterized using Affymetrix Genome-Wide Human SNP 6.0 Array data from each of these subjects. As predicted, the possibly protective GAB2 haplotype was associated with higher regional-to-whole brain FDG uptake in AD-affected brain regions in APOEε4 carriers. While additional studies are needed, this study supports the association between the possibly protective GAB2 haplotype and the risk of late-onset AD in APOEε4 carriers. It also supports the use of brain-imaging endophenotypes to help assess possible modifiers of AD risk.
PMCID: PMC3010232  PMID: 20888920
Alzheimer's disease; fluorodeoxyglucose positron emission tomography
Nature methods  2008;5(10):887-893.
We developed a generalized framework for multiplexed resequencing of targeted regions of the human genome on the Illumina Genome Analyzer using degenerate indexed DNA sequence barcodes ligated to fragmented DNA prior to sequencing. Using this method, the DNA of multiple HapMap individuals was simultaneously sequenced at several ENCODE (ENCyclopedia of DNA Elements) regions. We then evaluated the use of Bayes factors for discovering and genotyping polymorphisms from aligned sequenced reads. If we required that predicted polymorphisms be either previously identified by dbSNP or be visually evident upon reinspection of archived ENCODE traces, we observed a false-positive rate of 11.3% using strict thresholds (Ks>1,000) for predicting variants and 69.6% for lax thresholds (Ks>10). Conversely, false-negative rates ranged from 10.8% to 90.8%, with those at stricter cut-offs occurring at lower coverage (< 10 aligned reads). These results suggest that >90% of genetic variants are discoverable using multiplexed sequencing provided sufficient coverage at the polymorphic base.
PMCID: PMC3171277  PMID: 18794863
22.  Association of CR1, CLU and PICALM with Alzheimer's disease in a cohort of clinically characterized and neuropathologically verified individuals 
Human Molecular Genetics  2010;19(16):3295-3301.
In this study, we assess 34 of the most replicated genetic associations for Alzheimer's disease (AD) using data generated on Affymetrix SNP 6.0 arrays and imputed at over 5.7 million markers from a unique cohort of over 1600 neuropathologically defined AD cases and controls (1019 cases and 591 controls). Testing the top genes from the AlzGene meta-analysis, we confirm the well-known association with APOE single nucleotide polymorphisms (SNPs), the CLU, PICALM and CR1 SNPs recently implicated in unusually large data sets, and previously implicated CST3 and ACE SNPs. In the cases of CLU, PICALM and CR1, as well as in APOE, the odds ratios we find are slightly larger than those previously reported in clinical samples, consistent with what we believe to be more accurate classification of disease in the clinically characterized and neuropathologically confirmed AD cases and controls.
PMCID: PMC2908469  PMID: 20534741
23.  Reduced Posterior Cingulate Mitochondrial Activity in Expired Young Adult Carriers of the APOE ε4 Allele, the Major Late-Onset Alzheimer's Susceptibility Gene 
In vivoPET imaging studies of young-adult carriers of the apolipoprotein E ε4 allele (APOEε4), the major Alzheimer's disease (AD) susceptibility gene, have demonstrated declines in glucose metabolism in brain areas later vulnerable to AD, such as posterior cingulate cortex, decades before the possible onset of symptoms. We have previously shown in postmortem studies that such metabolic declines in AD are associated with brain regional mitochondrial dysfunction. To determine whether young adult at-risk individuals demonstrate similar mitochondrial functional decline, we histochemically assessed postmortem tissues from the posterior cingulate cortex of young-adult carriers and noncarriers of APOEε4. At-risk ε4 carriers had lower mitochondrial cytochrome oxidase activity than noncarriers in posterior cingulate cortex, particularly within the superficial cortical lamina, a pattern similar to that seen in AD patients. Except for one 34 year-old ε4 homozygote, the ε4 carriers did not have increased soluble amyloid-β, histologic amyloid-β, or tau pathology in this same region. This functional biomarker may prove useful in early detection and tracking of AD and indicates that mitochondrial mechanisms may contribute to the predisposition to AD before any evidence of amyloid or tau pathology.
PMCID: PMC3124564  PMID: 20847408
Alzheimer's etiology; bioenergetics; biomarkers; cytochrome c oxidase; differential vulnerability; neocortex
24.  Evidence for an association between KIBRA and late-onset Alzheimer’s disease 
Neurobiology of aging  2008;31(6):901-909.
We recently reported evidence for an association between the individual variation in normal human episodic memory and a common variant of the KIBRA gene, KIBRA rs17070145 (T-allele). Since memory impairment is a cardinal clinical feature of Alzheimer’s disease (AD), we investigated the possibility of an association between the KIBRA gene and AD using data from neuronal gene expression, brain imaging studies, and genetic association tests. KIBRA was significantly over-expressed and 3 of its 4 known binding partners under-expressed in AD-affected hippocampal, posterior cingulate and temporal cortex regions (p<0.010, corrected) in a study of laser capture microdissected neurons. Using positron emission tomography in a cohort of cognitively normal, late-middle-aged persons genotyped for KIBRA rs17070145, KIBRA T non-carriers exhibited lower glucose metabolism than did carriers in posterior cingulate and precuneus brain regions (P<0.001, uncorrected). Lastly, non-carriers of the KIBRA rs17070145 T-allele had increased risk of late-onset AD in an association study of 702 neuropathologically verified expired subjects (p=0.034; OR=1.29) and in a combined analysis of 1026 additional living and expired subjects (p=0.039; OR=1.26). Our findings suggest that KIBRA is associated with both individual variation in normal episodic memory and predisposition to AD.
PMCID: PMC2913703  PMID: 18789830
genetics; imaging; expression profiling; memory
25.  Hypometabolism in Alzheimer's-Affected Brain Regions in Cognitively Normal Latinos Carrying the Apolipoprotein E ε4 Allele 
Archives of neurology  2010;67(4):462-468.
The extent to which the apolipoprotein E (APOE) ε4 allele is a susceptibility gene for late-onset Alzheimer's disease (AD) in Latino individuals continues to be clarified. In this study, fluorodeoxyglucose positron emission tomography (PET) was used to investigate whether regional reductions in the cerebral metabolic rate for glucose (CMRgl) previously found in cognitively normal late-middle-aged APOE ε4 carriers extends to members of the Latino Mexican-American community.
A brain mapping algorithm (SPM5) was used to compare cross-sectional regional CMRgl in Latino APOE ε4 carriers versus noncarriers.
11 APOE ε4 carriers and 16 noncarriers from Arizona's Latino community (mean age 54.6±6.4 years) matched for sex, mean age and educational level, and who were predominantly of self-designated Mexican origin.
Participant groups had similar distributions for age, gender, education, family history of dementia, clinical ratings and neuropsychological test scores. Latino APOE ε4 carriers had lower CMRgl than the noncarriers in posterior cingulate, precuneus and parietal regions previously found to be preferentially affected in AD patients and cognitively normal non-Latino APOE ε4 carriers. Additionally, the Latino APOE ε4 carriers had lower CMRgl in middle and anterior cingulate cortex, hippocampus and thalamus.
This study provides support for the relationship between APOE ε4 and risk of AD in Latinos. It illustrates the role of PET as a presymptomatic endophenotype for the assessment of AD risk factors, and supports the inclusion of Latino APOE ε4 carriers in proof-of-concept studies using FDG PET to evaluate promising presymptomatic treatments in cognitively normal carriers of this common AD susceptibility gene.
PMCID: PMC2943432  PMID: 20385913

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