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1.  Expression of SORL1 and a novel SORL1 splice variant in normal and Alzheimers disease brain 
Variations in sortilin-related receptor (SORL1) expression and function have been implicated in Alzheimers Disease (AD). Here, to gain insights into SORL1, we evaluated SORL1 expression and splicing as a function of AD and AD neuropathology, neural gene expression and a candidate single nucleotide polymorphism (SNP).
To identify SORL1 splice variants, we scanned each of the 46 internal SORL1 exons in human brain RNA samples and readily found SORL1 isoforms that lack exon 2 or exon 19. Quantification in a case-control series of the more abundant isoform lacking exon 2 (delta-2-SORL1), as well as the "full-length" SORL1 (FL-SORL1) isoform containing exon 2 showed that expression of FL-SORL1 was reduced in AD individuals. Moreover, FL-SORL1 was reduced in cognitively intact individuals with significant AD-like neuropathology. In contrast, the expression of the delta-2-SORL1 isoform was similar in AD and non-AD brains. The expression of FL-SORL1 was significantly associated with synaptophysin expression while delta-2-SORL1 was modestly enriched in white matter. Lastly, FL-SORL1 expression was associated with rs661057, a SORL1 intron one SNP that has been associated with AD risk. A linear regression analysis found that rs661057, synaptophysin expression and AD neuropathology were each associated with FL-SORL1 expression.
These results confirm that FL-SORL1 expression declines in AD and with AD-associated neuropathology, suggest that FL-SORL1 declines in cognitively-intact individuals with AD-associated neuropathology, identify a novel SORL1 splice variant that is expressed similarly in AD and non-AD individuals, and provide evidence that an AD-associated SNP is associated with SORL1 expression. Overall, these results contribute to our understanding of SORL1 expression in the human brain.
PMCID: PMC2776013  PMID: 19889229
2.  The Alzheimer’s associated 5′ region of the SORL1 gene cis-regulates SORL1 transcripts expression 
Neurobiology of Aging  2010;33(7):1485.e1-1485.e8.
SORL1 has been identified as a major contributor to Late-Onset Alzheimer’s disease (LOAD). We test whether genetic variability in the 5′of SORL1 gene modulates the risk to develop LOAD via regulation of SORL1-mRNA expression and splicing. Two brain structures, differentially vulnerable to LOAD pathology, were examined in 144 brain samples from 92 neurologically normal individuals. The temporal cortex, which is more susceptible to Alzheimer’s pathology, demonstrated ~2-fold increase in SORL1-mRNAs levels in carriers of the minor alleles at SNPs, rs7945931 and rs2298525, compared to non-carriers. No genetic effect on total-SORL1-mRNA levels was detected in the frontal-cortex. However, rs11600875 minor allele was associated with significantly increased levels of exon-2 skipping, but only in frontal cortex. No correlation of SORL1-mRNAs expression was found between frontal and temporal cortexes. Collectively, indicating the brain-region specificity of the genetic regulation of SORL1 expression. Our results suggest that genetic regulation of SORL1 expression plays a role in disease risk and maybe responsible for the reported LOAD-associations. Further studies to detect the actual pathogenic variant/s are necessary.
PMCID: PMC3117021  PMID: 21185108
SORL1; Alzheimer’s disease; mRNA; splicing; correlation to gene expression
3.  Polymorphisms affecting gene transcription and mRNA processing in pharmacogenetic candidate genes: detection through allelic expression imbalance in human target tissues 
Pharmacogenetics and genomics  2008;18(9):781-791.
Genetic variation in mRNA expression plays a critical role in human phenotypic diversity, but it has proven difficult to detect regulatory polymorphisms - mostly single nucleotide polymorphisms (rSNPs). Additionally, variants in the transcribed region, termed here ‘structural RNA SNPs’ (srSNPs), can affect mRNA processing and turnover. Both rSNPs and srSNPs cause allelic mRNA expression imbalance (AEI) in heterozygous individuals. We have applied a rapid and accurate AEI methodology for testing 42 genes implicated in human diseases and drug response, specifically cardiovascular and CNS diseases, and affecting drug metabolism and transport. Each gene was analyzed in physiologically relevant human autopsy tissues, including brain, heart, liver, intestines, and lymphocytes. Substantial AEI was observed in ∼55% of the surveyed genes. Focusing on cardiovascular candidate genes in human hearts, AEI analysis revealed frequent cis-acting regulatory factors in SOD2 and ACE mRNA expression, having potential clinical significance. SNP scanning to locate regulatory polymorphisms in a number of genes failed to support several previously proposed promoter SNPs discovered with use of reporter gene assays in heterologous tissues, while srSNPs appear more frequent than expected. Computational analysis of mRNA folding indicates that ∼90% of srSNPs affects mRNA folding, and hence potentially function. Our results indicate that both rSNPs and srSNPs represent a still largely untapped reservoir of variants that contribute to human phenotypic diversity.
PMCID: PMC2779843  PMID: 18698231
4.  Impact of SORL1 Single Nucleotide Polymorphisms on Alzheimer's Disease Cerebrospinal Fluid Markers 
Recently, genetic variants of the neuronal sortilin-related receptor with A-type repeats (SORL1, also called LR11 or sorLA) have emerged as risk factors for the development of Alzheimer's disease (AD).
In this study, SORL1 gene polymorphisms, which have been shown to be related to AD, were analyzed for associations with cerebrospinal fluid (CSF) amyloid beta1–42 (Aβ1–42), phosphorylated tau181, and total tau levels in a non-Hispanic Caucasian sample, which encompassed 100 cognitively healthy elderly individuals, 166 patients with mild cognitive impairment, and 87 patients with probable AD. The data were obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database ( Moreover, the impact of gene-gene interactions between SORL1 single nucleotide polymorphisms (SNPs) and the apolipoprotein E (APOE) ∊4 allele, the major genetic risk factor for sporadic AD, on Aβ1–42 concentrations was investigated.
Significant associations between CSF Aβ1–42 levels and the SORL1 SNPs 23 (rs3824968) and 24 (rs2282649) were detected in the AD group. The latter association became marginally statistically insignificant after Bonferroni correction for multiple comparisons. Carriers of the SORL1 SNP24 T allele and the SNP23 A allele both had lower CSF Aβ1–42 concentrations than non-carriers of these alleles. The analysis of the impact of interactions between APOE ∊4 allele and SORL1 SNPs on CSF Aβ1–42 levels unraveled significant influences of APOE.
Our findings provide further support for the notion that SORL1 genetic variants are related to AD pathology, probably by regulating the amyloid cascade.
PMCID: PMC3696367  PMID: 21997402
Dementia; Mild cognitive impairment; Healthy aging; Amyloid cascade; Association
5.  Dopamine Transporter Gene Variant Affecting Expression in Human Brain is Associated with Bipolar Disorder 
Neuropsychopharmacology  2011;36(8):1644-1655.
The gene encoding the dopamine transporter (DAT) has been implicated in CNS disorders, but the responsible polymorphisms remain uncertain. To search for regulatory polymorphisms, we measured allelic DAT mRNA expression in substantia nigra of human autopsy brain tissues, using two marker SNPs (rs6347 in exon 9 and rs27072 in the 3′-UTR). Allelic mRNA expression imbalance (AEI), an indicator of cis-acting regulatory polymorphisms, was observed in all tissues heterozygous for either of the two marker SNPs. SNP scanning of the DAT locus with AEI ratios as the phenotype, followed by in vitro molecular genetics studies, demonstrated that rs27072 C>T affects mRNA expression and translation. Expression of the minor T allele was dynamically regulated in transfected cell cultures, possibly involving microRNA interactions. Both rs6347 and rs3836790 (intron8 5/6 VNTR) also seemed to affect DAT expression, but not the commonly tested 9/10 VNTR in the 3′UTR (rs28363170). All four polymorphisms (rs6347, intron8 5/6 VNTR, rs27072 and 3′UTR 9/10 VNTR) were genotyped in clinical cohorts, representing schizophrenia, bipolar disorder, depression, and controls. Only rs27072 was significantly associated with bipolar disorder (OR=2.1, p=0.03). This result was replicated in a second bipolar/control population (OR=1.65, p=0.01), supporting a critical role for DAT regulation in bipolar disorder.
PMCID: PMC3138671  PMID: 21525861
dopamine transporter; bipolar disorder; allelic expression imbalance; SLC6A3; rs27072; Dopamine; Depression; Unipolar/Bipolar; Pharmacogenetics/Pharmacogenomics; Neurogenetics; Allelic expression imbalance; Dopamine transporter; SLC6A3
6.  Meta-analysis of the Association Between Variants in SORL1 and Alzheimer Disease 
Archives of neurology  2011;68(1):99-106.
To reexamine the association between the neuronal sortilin-related receptor gene (SORL1) and Alzheimer disease (AD).
Comprehensive and unbiased meta-analysis of all published and unpublished data from case-control studies for the SORL1 single-nucleotide polymorphisms (SNPs) that had been repeatedly assessed across studies.
Academic research institutions in the United States, the Netherlands, Canada, Belgium, the United Kingdom, Singapore, Japan, Sweden, Germany, France, and Italy.
All published white and Asian case-control data sets, which included a total of 12 464 cases and 17 929 controls.
Main Outcome Measures
Alzheimer disease according to the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) and the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (now known as the Alzheimer’s Association).
In the white data sets, several markers were associated with AD after correction for multiple testing, including previously reported SNPs 8, 9, and 10 (P<.001). In addition, the C-G-C haplotype at SNPs 8 through 10 was associated with AD risk (P<.001). In the combined Asian data sets, SNPs 19 and 23 through 25 were associated with AD risk (P<.001). The disease-associated alleles at SNPs 8, 9, and 10 (120 873 131-120 886 175 base pairs [bp]; C-G-C alleles), at SNP 19 (120 953 300 bp; G allele), and at SNPs 24 through 25 (120 988 611 bp; T and C alleles) were the same previously reported alleles. The SNPs 4 through 5, 8 through 10, 12, and 19 through 25 belong to distinct linkage disequilibrium blocks. The same alleles at SNPs 8 through 10 (C-G-C), 19 (G), and 24 and 25 (T and C) have also been associated with AD endophenotypes, including white matter hyperintensities and hippocampal atrophy on magnetic resonance imaging, cerebrospinal fluid measures of amyloid β-peptide 42, and full-length SORL1 expression in the human brain.
This comprehensive meta-analysis provides confirmatory evidence that multiple SORL1 variants in distinct linkage disequilibrium blocks are associated with AD.
PMCID: PMC3086666  PMID: 21220680
7.  Analyzing allele specific RNA expression using mixture models 
BMC Genomics  2015;16(1):566.
Measuring allele-specific RNA expression provides valuable insights into cis-acting genetic and epigenetic regulation of gene expression. Widespread adoption of high-throughput sequencing technologies for studying RNA expression (RNA-Seq) permits measurement of allelic RNA expression imbalance (AEI) at heterozygous single nucleotide polymorphisms (SNPs) across the entire transcriptome, and this approach has become especially popular with the emergence of large databases, such as GTEx. However, the existing binomial-type methods used to model allelic expression from RNA-seq assume a strong negative correlation between reference and variant allele reads, which may not be reasonable biologically.
Here we propose a new strategy for AEI analysis using RNA-seq data. Under the null hypothesis of no AEI, a group of SNPs (possibly across multiple genes) is considered comparable if their respective total sums of the allelic reads are of similar magnitude. Within each group of “comparable” SNPs, we identify SNPs with AEI signal by fitting a mixture of folded Skellam distributions to the absolute values of read differences. By applying this methodology to RNA-Seq data from human autopsy brain tissues, we identified numerous instances of moderate to strong imbalanced allelic RNA expression at heterozygous SNPs. Findings with SLC1A3 mRNA exhibiting known expression differences are discussed as examples.
The folded Skellam mixture model searches for SNPs with significant difference between reference and variant allele reads (adjusted for different library sizes), using information from a group of “comparable” SNPs across multiple genes. This model is particularly suitable for performing AEI analysis on genes with few heterozygous SNPs available from RNA-seq, and it can fit over-dispersed read counts without specifying the direction of the correlation between reference and variant alleles.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1749-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4521363  PMID: 26231172
Allelic RNA expression imbalance (AEI); Allele-specific expression (ASE); RNA-seq; Poisson mixture; Folded Skellam mixture; Human brain
8.  Independent and epistatic effects of variants in VPS10-d receptors on Alzheimer disease risk and processing of the amyloid precursor protein (APP) 
Translational Psychiatry  2013;3(5):e256-.
Genetic variants in the sortilin-related receptor (SORL1) and the sortilin-related vacuolar protein sorting 10 (VPS10) domain-containing receptor 1 (SORCS1) are associated with increased risk of Alzheimer's disease (AD), declining cognitive function and altered amyloid precursor protein (APP) processing. We explored whether other members of the (VPS10) domain-containing receptor protein family (the sortilin-related VPS10 domain-containing receptors 2 and 3 (SORCS2 and SORCS3) and sortilin (SORT1)) would have similar effects either independently or together. We conducted the analyses in a large Caucasian case control data set (n=11 840 cases, 10 931 controls) to determine the associations between single nucleotide polymorphisms (SNPs) in all the five homologous genes and AD risk. Evidence for interactions between SNPs in the five VPS10 domain receptor family genes was determined in epistatic statistical models. We also compared expression levels of SORCS2, SORCS3 and SORT1 in AD and control brains using microarray gene expression analyses and assessed the effects of these genes on γ-secretase processing of APP. Several SNPs in SORL1, SORCS1, SORCS2 and SORCS3 were associated with AD. In addition, four specific linkage disequilibrium blocks in SORCS1, SORCS2 and SORCS3 showed additive epistatic effects on the risk of AD (P⩽0.0006). SORCS3, but not SORCS2 or SORT1, showed reduced expression in AD compared with control brains, but knockdown of all the three genes using short hairpin RNAs in HEK293 cells caused a significant threefold increase in APP processing (from P<0.001 to P<0.05). These findings indicate that in addition to SORL1 and SORCS1, variants in other members of the VPS10 domain receptor family (that is, SORCS1, SORCS2, SORCS3) are associated with AD risk and alter APP processing. More importantly, the results indicate that variants within these genes have epistatic effects on AD risk.
PMCID: PMC3669917  PMID: 23673467
Alzheimer's disease; SORCS2; SORCS3; SORT1
9.  Next-generation DNA sequencing-based assay for measuring allelic expression imbalance (AEI) of candidate neuropsychiatric disorder genes in human brain 
BMC Genomics  2011;12:518.
Common genetic variants that regulate gene expression are widely suspected to contribute to the etiology and phenotypic variability of complex diseases. Although high-throughput, microarray-based assays have been developed to measure differences in mRNA expression among independent samples, these assays often lack the sensitivity to detect rare mRNAs and the reproducibility to quantify small changes in mRNA expression. By contrast, PCR-based allelic expression imbalance (AEI) assays, which use a "marker" single nucleotide polymorphism (mSNP) in the mRNA to distinguish expression from pairs of genetic alleles in individual samples, have high sensitivity and accuracy, allowing differences in mRNA expression greater than 1.2-fold to be quantified with high reproducibility. In this paper, we describe the use of an efficient PCR/next-generation DNA sequencing-based assay to analyze allele-specific differences in mRNA expression for candidate neuropsychiatric disorder genes in human brain.
Using our assay, we successfully analyzed AEI for 70 candidate neuropsychiatric disorder genes in 52 independent human brain samples. Among these genes, 62/70 (89%) showed AEI ratios greater than 1 ± 0.2 in at least one sample and 8/70 (11%) showed no AEI. Arranging log2AEI ratios in increasing order from negative-to-positive values revealed highly reproducible distributions of log2AEI ratios that are distinct for each gene/marker SNP combination. Mathematical modeling suggests that these log2AEI distributions can provide important clues concerning the number, location and contributions of cis-acting regulatory variants to mRNA expression.
We have developed a highly sensitive and reproducible method for quantifying AEI of mRNA expressed in human brain. Importantly, this assay allowed quantification of differential mRNA expression for many candidate disease genes entirely missed in previously published microarray-based studies of mRNA expression in human brain. Given the ability of next-generation sequencing technology to generate large numbers of independent sequencing reads, our method should be suitable for analyzing from 100- to 200-candidate genes in 100 samples in a single experiment. We believe that this is the appropriate scale for investigating variation in mRNA expression for defined sets candidate disorder genes, allowing, for example, comprehensive coverage of genes that function within biological pathways implicated in specific disorders. The combination of AEI measurements and mathematical modeling described in this study can assist in identifying SNPs that correlate with mRNA expression. Alleles of these SNPs (individually or as sets) that accurately predict high- or low-mRNA expression should be useful as markers in genetic association studies aimed at linking candidate genes to specific neuropsychiatric disorders.
PMCID: PMC3228908  PMID: 22013986
10.  The SORL1 Gene and Convergent Neural Risk for Alzheimer's Disease Across the Human Lifespan 
Molecular psychiatry  2013;19(10):1125-1132.
Prior to intervention trials in individuals genetically at-risk for late-onset Alzheimer's disease, critical first steps are identifying where (neuroanatomic effects), when (timepoint in the lifespan) and how (gene expression and neuropathology) Alzheimer's risk genes impact the brain. We hypothesized that variants in the sortilin-like receptor (SORL1) gene would affect multiple Alzheimer's phenotypes before the clinical onset of symptoms. Four independent samples were analyzed to determine effects of SORL1 genetic risk variants across the lifespan at multiple phenotypic levels: 1) microstructural integrity of white matter using diffusion tensor imaging in two healthy control samples (n=118, age 18-86, and n=68, age 8-40); 2) gene expression using the Braincloud postmortem healthy control sample (n=269, age 0-92); and 3) Alzheimer's neuropathology (amyloid plaques and tau tangles) using a postmortem sample of healthy, mild cognitive impairment (MCI), and Alzheimer's individuals (n=710, age 66-108). SORL1 risk variants predicted lower white matter fractional anisotropy in an age-independent manner, in fronto-temporal white matter tracts in both samples at 5% FWE-corrected thresholds. SORL1 risk variants also predicted decreased SORL1 mRNA expression, most prominently during childhood and adolescence, and significantly predicted increases in amyloid pathology in postmortem brain. Importantly, the effects of SORL1 variation on both white matter microstructure and gene expression were observed during neurodevelopmental phases of the human lifespan. Further, the neuropathological mechanism of risk appears to primarily involve amyloidogenic pathways. Interventions targeted toward the SORL1 amyloid risk pathway may be of greatest value during early phases of the lifespan.
PMCID: PMC4004725  PMID: 24166411
Alzheimer's disease; neuroimaging; genetics; SORL1; amyloid; gene expression
11.  Allelic expression analysis of the osteoarthritis susceptibility gene COL11A1 in human joint tissues 
The single nucleotide polymorphism (SNP) rs2615977 is associated with osteoarthritis (OA) and is located in intron 31 of COL11A1, a strong candidate gene for this degenerative musculoskeletal disease. Furthermore, the common non-synonymous COL11A1 SNP rs1676486 is associated with another degenerative musculoskeletal disease, lumbar disc herniation (LDH). rs1676486 is a C-T transition mediating its affect on LDH susceptibility by modulating COL11A1 expression. The risk T-allele of rs1676486 leads to reduced expression of the COL11A1 transcript, a phenomenon known as allelic expression imbalance (AEI). We were keen therefore to assess whether the effect that rs1676486 has on COL11A1 expression in LDH is also observed in OA and whether the rs2615977 association to OA also marked AEI.
Using RNA from OA cartilage, we assessed whether either SNP correlated with COL11A1 AEI by 1) measuring COL11A1 expression and stratifying the data by genotype at each SNP; and 2) quantifying the mRNA transcribed from each allele of the two SNPs. We also assessed whether rs1676486 was associated with OA susceptibility using a case–control cohort of over 18,000 individuals.
We observed significant AEI at rs1676486 (p < 0.0001) with the T-allele correlating with reduced COL11A1 expression. This corresponded with observations in LDH but the SNP was not associated with OA. We did not observe AEI at rs2615977.
COL11A1 is subject to AEI in OA cartilage. AEI at rs1676486 is a risk factor for LDH, but not for OA. These two diseases therefore share a common functional phenotype, namely AEI of COL11A1, but this appears to be a disease risk only in LDH. Other functional effects on COL11A1 presumably account for the OA susceptibility that maps to this gene.
PMCID: PMC3599795  PMID: 23497244
Osteoarthritis; Lumbar disc herniation; Genetics; Susceptibility; COL11A1; Allelic expression
12.  An exploratory study of the association between SORL1 polymorphisms and sporadic Alzheimer’s disease in the Han Chinese population 
In previous studies, we reported that the sortilin-related receptor, L (DLR class) A repeats containing (SORL1) gene single nucleotide polymorphisms (SNPs) are associated with the risk of sporadic Alzheimer’s disease (SAD) in the Han Chinese population. To further explore the relationships between SORL1 genetic variants and SAD, we conducted a two-step study. Sequencing analysis in 50 case samples identified 14 SNPs within the promoter and untranslated region of the SORL1 gene. Subsequent genotyping analysis in 106 patients with SAD and 179 healthy controls detected a significant association between the “G” allele of SNP rs1133174 in the 3′ untranslated region of the SORL1 gene and SAD risk (odds ratio =1.92, 95% confidence interval [95% CI] =1.28–2.90, adjusted P=0.028). In addition, “G” allele carriers of rs1133174 (GA + GG) have a 2.15-fold increased risk of SAD compared to noncarriers (AA) (adjusted P=0.042). However, no significant positive associations were observed in the other 13 SNPs within the SORL1 gene. These preliminary findings suggest that the SORL1 SNP rs1133174 may be a potential risk locus for SAD in the Han Chinese population.
PMCID: PMC4472075  PMID: 26109858
SORL1; Alzheimer’s disease; polymorphism; Han Chinese; association
13.  The Association Between Genetic Variants in SORL1 and Alzheimer’s Disease in an Urban, Multiethnic, Community-Based Cohort 
Archives of neurology  2007;64(4):501-506.
Variants in 3′ and 5′ regions of SORL1, the neuronal sorting protein-related receptor, were recently found to be associated with late onset familial and sporadic Alzheimer’s disease in several datasets that were selected for familial aggregation or were ethnically diverse or clinic-based selected series.
To investigate the association between Alzheimer’s disease and variant alleles in SORL1 using a series of single nucleotide polymorphisms (SNPs) in an urban, multiethnic community-based population.
Design & Setting
We used a nested case-control analysis in a population-based, prospective study of aging and dementia in Medicare recipients, 65 years and older, residing in northern Manhattan.
There were 296 patients with probable Alzheimer’s disease and 428 healthy elderly controls. The participants were of African American (34%), Caribbean Hispanic (51%) or non-Hispanic whites (15%).
Main Outcome Measures
We genotyped all 29 SNPs in SORL1 that were examined in the earlier report. We assessed allelic association with AD using standard case-control methods which included APOE genotype as a covariate.
Several individual SNPs and SNP haplotypes were significantly associated with AD in this prospectively collected community-based cohort, confirming the previously reported positive association of SORL1 with Alzheimer’s disease. SNP 12 near the 5′ region was associated with AD in African-Americans and Hispanics. Two SNPs in the 3′ region were also associated with AD in African-Americans (SNP 26) and Whites (SNP 20). A single haplotype in the 3′ region was associated with AD in Hispanics. However, several different haplotypes were associated with AD in the African-Americans and Whites, including the “TTC” haplotypes at SNPs 23–25 (p=0.035) that was significantly associated with AD in the North European Whites in the previous report.
This study confirms the association between genetic variants in SORL1 and AD. While the associations observed in these datasets overlap with those previously reported, the finding of novel SNP and haplotype associations suggest that there may be extensive allelic heterogeneity in SORL1. Broad regions of the SORL1 gene will therefore need to be scrutinized for functional pathogenic variants.
PMCID: PMC2639214  PMID: 17420311
SORL1; Alzheimer’s disease; sporadic; African American; Caribbean Hispanic
14.  An intronic PICALM polymorphism, rs588076, is associated with allelic expression of a PICALM isoform 
Although genome wide studies have associated single nucleotide polymorphisms (SNP)s near PICALM with Alzheimer’s disease (AD), the mechanism underlying this association is unclear. PICALM is involved in clathrin-mediated endocytosis and modulates Aß clearance in vitro. Comparing allelic expression provides the means to detect cis-acting regulatory polymorphisms. Thus, we evaluated whether PICALM showed allele expression imbalance (AEI) and whether this imbalance was associated with the AD-associated polymorphism, rs3851179.
We measured PICALM allelic expression in 42 human brain samples by using next-generation sequencing. Overall, PICALM demonstrated equal allelic expression with no detectable influence by rs3851179. A single sample demonstrated robust global PICALM allelic expression imbalance (AEI), i.e., each of the measured isoforms showed AEI. Moreover, the PICALM isoform lacking exons 18 and 19 (D18-19 PICALM) showed significant AEI in a subset of individuals. Sequencing these individuals and subsequent genotyping revealed that rs588076, located in PICALM intron 17, was robustly associated with this imbalance in D18-19 PICALM allelic expression (p = 9.54 x 10-5). This polymorphism has been associated previously with systolic blood pressure response to calcium channel blocking agents. To evaluate whether this polymorphism was associated with AD, we genotyped 3269 individuals and found that rs588076 was modestly associated with AD. However, when both the primary AD SNP rs3851179 was added to the logistic regression model, only rs3851179 was significantly associated with AD.
PICALM expression shows no evidence of AEI associated with rs3851179. Robust global AEI was detected in one sample, suggesting the existence of a rare SNP that strongly modulates PICALM expression. AEI was detected for the D18-19 PICALM isoform, and rs588076 was associated with this AEI pattern. Conditional on rs3851179, rs588076 was not associated with AD risk, suggesting that D18-19 PICALM is not critical in AD. In summary, this analysis of PICALM allelic expression provides novel insights into the genetics of PICALM expression and AD risk.
PMCID: PMC4150683  PMID: 25169757
PICALM; Alzheimer’s disease; Next-generation sequencing; Allelic expression imbalance; Single nucleotide polymorphism
15.  Association between Genetic Variants in Sortilin-Related Receptor 1 (SORL1) and Alzheimer's Disease in Adults with Down syndrome 
Neuroscience letters  2007;425(2):105-109.
Recent reports have suggested that variants in the sortilin-related receptor gene (SORL1) increase the risk of late onset Alzheimer's disease (AD) in Northern European, Hispanic, African-American and Isreali-Arab populations. SORL1 directs trafficking of amyloid precursor protein (APP) and under-expression of SORL1 may lead to over-expression of β amyloid peptides. Adults with Down syndrome (DS) over-express APP and have early onset and high risk for AD. We investigated the relation of seven variants in the gene for SORL1 to age at onset and risk for AD among 208 adults with DS, 45–70 years of age at baseline. Participants were ascertained through the New York State developmental disability service system and followed at 18-month intervals. Information from cognitive assessments, caregiver interviews, medical record review and neurological examination was used to establish the diagnosis of dementia. Homozygosity for the minor T allele in rs556349 and for the minor C allele in rs536360 was associated with later age at onset and reduced risk of AD (HR= 0.26, 95% CI: 0.08–0.86; and HR= 0.40, 95% CI: 0.16–0.98, respectively). Mean age at onset was approximately four years later in individuals who were homozygous for those alleles compared with those who had at least one major allele. These findings indicate a modest association of variants in SORL1 with AD. In addition, we did not observe the same alleles to be associated with AD compared with earlier studies, suggesting that these SNPs are in linkage disequilibrium (LD) with the putative functional variants or that expression of the SORL1 gene and hence its interaction with APP might be modified by the extremely high levels of APP characteristic of Down syndrome. Thus, further studies are needed to identify functional variants that influence risk for AD in this uniquely vulnerable population.
PMCID: PMC2131721  PMID: 17826910
SORL1; Down syndrome; Alzheimer’s disease
16.  SORCS1 Alters Amyloid Precursor Protein Processing and Variants May Increase Alzheimer’s Disease Risk 
Annals of neurology  2011;69(1):47-64.
Sorting mechanisms that cause the amyloid precursor protein (APP) and the β-secretases and γ-secretases to colocalize in the same compartment play an important role in the regulation of Aβ production in Alzheimer’s disease (AD). We and others have reported that genetic variants in the Sortilin-related receptor (SORL1) increased the risk of AD, that SORL1 is involved in trafficking of APP, and that under expression of SORL1 leads to overproduction of Aβ. Here we explored the role of one of its homologs, the sortilin-related VPS10 domain containing receptor 1 (SORCS1), in AD.
We analyzed the genetic associations between AD and 16 SORCS1–single nucleotide polymorphisms (SNPs) in 6 independent data sets (2,809 cases and 3,482 controls). In addition, we compared SorCS1 expression levels of affected and unaffected brain regions in AD and control brains in microarray gene expression and real-time polymerase chain reaction (RT-PCR) sets, explored the effects of significant SORCS1-SNPs on SorCS1 brain expression levels, and explored the effect of suppression and overexpression of the common SorCS1 isoforms on APP processing and Aβ generation.
Inherited variants in SORCS1 were associated with AD in all datasets (0.001 < p < 0.049). In addition, SorCS1 influenced APP processing. While overexpression of SorCS1 reduced γ-secretase activity and Aβ levels, the suppression of SorCS1 increased γ-secretase processing of APP and the levels of Aβ.
These data suggest that inherited or acquired changes in SORCS1 expression or function may play a role in the pathogenesis of AD.
PMCID: PMC3086759  PMID: 21280075
17.  An intronic ncRNA-dependent regulation of SORL1 expression affecting Aβ formation is upregulated in post-mortem Alzheimer's disease brain samples 
Disease Models & Mechanisms  2012;6(2):424-433.
Recent studies indicated that sortilin-related receptor 1 (SORL1) is a risk gene for late-onset Alzheimer's disease (AD), although its role in the aetiology and/or progression of this disorder is not fully understood. Here, we report the finding of a non-coding (nc) RNA (hereafter referred to as 51A) that maps in antisense configuration to intron 1 of the SORL1 gene. 51A expression drives a splicing shift of SORL1 from the synthesis of the canonical long protein variant A to an alternatively spliced protein form. This process, resulting in a decreased synthesis of SORL1 variant A, is associated with impaired processing of amyloid precursor protein (APP), leading to increased Aβ formation. Interestingly, we found that 51A is expressed in human brains, being frequently upregulated in cerebral cortices from individuals with Alzheimer's disease. Altogether, these findings document a novel ncRNA-dependent regulatory pathway that might have relevant implications in neurodegeneration.
PMCID: PMC3597024  PMID: 22996644
18.  Association of Distinct Variants in SORL1 With Cerebrovascular and Neurodegenerative Changes Related to Alzheimer Disease 
Archives of neurology  2008;65(12):1640-1648.
Single-nucleotide polymorphisms (SNPs) in 2 distinct regions of the gene for the sortilin-related receptor (SORL1) (bounded by consecutively numbered SNPs 8−10 and 22−25) were shown to be associated with Alzheimer disease (AD) in multiple ethnically diverse samples.
To test the hypothesis that SORL1 is associated with brain magnetic resonance imaging (MRI) measurements of atrophy and/or vascular disease.
Design, Setting, and Patients
We evaluated the association of 30 SNPs spanning SORL1 with MRI measures of general cerebral atrophy, hippocampal atrophy, white matter hyperintensities, and overall cerebrovascular disease in 44 African American and 182 white sibships from the MIRAGE Study. We performed single-and 3-SNP haplotype association analyses using family-based tests. Haplotypes found to be significantly associated with at least 1 MRI trait were tested for association with 6 pathological traits in a separate sample of 69 white patients with autopsy-confirmed AD.
In white patients, white matter hyperintensities were associated with multiple markers in the region encompassing SNPs 6 to 10, whereas cerebral and hippocampal atrophy were associated with markers from the region including SNPs 21 to 26. Examination of specific 3-SNP haplotypes from these 2 regions in the autopsy-confirmed cases of AD revealed association of white matter disease with SNPs 8 to 10 and association of hippocampal atrophy with SNPs 22 to 26. The haplotype CGC at SNPs 8 to 10 was associated with fewer white matter changes in the clinical (P<.001) and autopsy (P=.02) samples.
Variants of SORL1 previously associated with AD are also associated with MRI and neuropathological measures of neurodegenerative and cerebrovascular disease. These findings not only support the hypothesis that multiple areas in SORL1 are of functional importance but also raise the possibility that multiple SORL1 variants influence amyloid precursor protein or endothelial lipoprotein processing or both in different regions of the brain.
PMCID: PMC2719762  PMID: 19064752
19.  The Neuronal Sortilin-Related Receptor Gene SORL1 and Late-Onset Alzheimer's Disease 
Recent studies indicate that two clusters of single nucleotide polymorphisms in the neuronal sortilin-related receptor gene (SORL1) are causally associated with late-onset Alzheimer's disease (AD). At the cellular level, SORL1 is thought to be involved in intracellular trafficking of amyloid precursor protein. When this gene is suppressed, toxic amyloid β production is increased, and high levels of amyloid β are associated with a higher AD risk. Extending the cellular findings, gene expression studies show that SORL1 is differentially expressed in AD patients compared with controls. Furthermore, several genetic studies have identified allelic and haplotypic SORL1 variants associated with late-onset AD, and these variants confer small to modest risk of AD. Taken together, the evidence for SORL1 as a causative gene is compelling. However, putative variants have not yet been identified. Further research is necessary to determine its utility as a diagnostic marker of AD or as a target for new therapeutic approaches.
PMCID: PMC2694663  PMID: 18713574
20.  Allelic mRNA expression imbalance in C-type lectins reveals a frequent regulatory SNP in the human surfactant protein A (SP-A) gene 
Genes and immunity  2013;14(2):99-106.
Genetic variation in C-type lectins influences infectious disease susceptibility but remains poorly understood. We employed allelic mRNA expression imbalance (AEI) technology for SP-A1, SP-A2, SP-D, DC-SIGN, MRC1, and Dectin-1, expressed in human macrophages and/or lung tissues. Frequent AEI, an indicator of regulatory polymorphisms, was observed in SP-A2, SP-D, and DC-SIGN. AEI was measured for SP-A2 in 38 lung tissues using four marker SNPs and was confirmed by next generation sequencing of one lung RNA sample. Genomic DNA at the SP-A2 DNA locus was sequenced by Ion Torrent technology in 16 samples. Correlation analysis of genotypes with AEI identified a haplotype block, and, specifically, the intronic SNP rs1650232 (30% MAF); the only variant consistently associated with an approximately two-fold change in mRNA allelic expression. Previously shown to alter a NAGNAG splice acceptor site with likely effects on SP-A2 expression, rs1650232 generates an alternative splice variant with three additional bases at the start of exon 3. Validated as a regulatory variant, rs1650232 is in partial LD with known SP-A2 marker SNPs previously associated with risk for respiratory diseases including tuberculosis. Applying functional DNA variants in clinical association studies, rather than marker SNPs, will advance our understanding of genetic susceptibility to infectious diseases.
PMCID: PMC3594410  PMID: 23328842
C-type lectin; marker SNP; regulatory variant; SP-A2; allelic expression imbalance
21.  Risk alleles for chronic hepatitis B are associated with decreased mRNA expression of HLA-DPA1 and HLA-DPB1 in normal human liver 
Genes and Immunity  2011;12(6):428-433.
A genome-wide association study identified single nucleotide polymorphisms (SNPs) rs3077 and rs9277535 located in the 3′ untranslated regions of human leukocyte antigen (HLA) class II genes HLA-DPA1 and HLA-DPB1, respectively, as the independent variants most strongly associated with chronic hepatitis B. We examined whether these SNPs are associated with mRNA expression of HLA-DPA1 and HLA-DPB1. We identified gene expression-associated SNPs (eSNPs) in normal liver samples obtained from 651 individuals of European ancestry by integrating genotype (∼650 000 SNPs) and gene expression (>39 000 transcripts) data from each sample. We used the Kruskal–Wallis test to determine associations between gene expression and genotype. To confirm findings, we measured allelic expression imbalance (AEI) of complementary DNA compared with DNA in liver specimens from subjects who were heterozygous for rs3077 and rs9277535. On a genome-wide basis, rs3077 was the SNP most strongly associated with HLA-DPA1 expression (p=10−48), and rs9277535 was strongly associated with HLA-DPB1 expression (p=10−15). Consistent with these gene expression associations, we observed AEI for both rs3077 (p=3.0 × 10−7; 17 samples) and rs9277535 (p=0.001; 17 samples). We conclude that the variants previously associated with chronic hepatitis B are also strongly associated with mRNA expression of HLA-DPA1 and HLA-DPB1, suggesting that expression of these genes is important in control of HBV.
PMCID: PMC3169805  PMID: 21346778
chronic hepatitis B; HLA; gene expression; genetics; genomics
22.  The rs1024611 Regulatory Region Polymorphism Is Associated with CCL2 Allelic Expression Imbalance 
PLoS ONE  2012;7(11):e49498.
CC chemokine ligand 2 (CCL2) is the most potent monocyte chemoattractant and inter-individual differences in its expression level have been associated with genetic variants mapping to the cis-regulatory regions of the gene. An A to G polymorphism in the CCL2 enhancer region at position –2578 (rs1024611; A>G), was found in most studies to be associated with higher serum CCL2 levels and increased susceptibility to a variety of diseases such as HIV-1 associated neurological disorders, tuberculosis, and atherosclerosis. However, the precise mechanism by which rs1024611influences CCL2 expression is not known. To address this knowledge gap, we tested the hypothesis that rs1024611G polymorphism is associated with allelic expression imbalance (AEI) of CCL2. We used haplotype analysis and identified a transcribed SNP in the 3′UTR (rs13900; C>T) can serve as a proxy for the rs1024611 and demonstrated that the rs1024611G allele displayed a perfect linkage disequilibrium with rs13900T allele. Allele-specific transcript quantification in lipopolysaccharide treated PBMCs obtained from heterozygous donors showed that rs13900T allele were expressed at higher levels when compared to rs13900C allele in all the donors examined suggesting that CCL2 is subjected to AEI and that that the allele containing rs1024611G is preferentially transcribed. We also found that AEI of CCL2 is a stable trait and could be detected in newly synthesized RNA. In contrast to these in vivo findings, in vitro assays with haplotype-specific reporter constructs indicated that the haplotype bearing rs1024611G had a lower or similar transcriptional activity when compared to the haplotype containing rs1024611A. This discordance between the in vivo and in vitro expression studies suggests that the CCL2 regulatory region polymorphisms may be functioning in a complex and context-dependent manner. In summary, our studies provide strong functional evidence and a rational explanation for the phenotypic effects of the CCL2 rs1024611G allele.
PMCID: PMC3500309  PMID: 23166687
23.  Diabetes-associated SorCS1 regulates Alzheimer’s amyloid-β metabolism: Evidence for involvement of SorL1 and the retromer complex 
The Journal of Neuroscience  2010;30(39):13110-13115.
SorCS1 and SorL1/SorLA/LR11 belong to the sortilin family of vacuolar protein sorting-10 (Vps10) domain-containing proteins. Both are genetically associated with Alzheimer’s disease (AD), and SORL1 expression is decreased in the brains of patients suffering from AD. SORCS1 is also genetically associated with types 1 and 2 diabetes mellitus (T1DM, T2DM). We have undertaken a study of the possible role(s) for SorCS1 in metabolism of the Alzheimer’s amyloid-β peptide (Aβ) and the Aβ precursor protein (APP), to test the hypothesis that Sorcs1-deficiency might be a common genetic risk factor underlying the predisposition to AD that is associated with T2DM. Overexpression of SorCS1Cβ-myc in cultured cells caused a reduction (p=0.002) in Aβ generation. Conversely, endogenous murine Aβ40 and Aβ42 levels were increased (Aβ40, p=0.044; Aβ42, p=0.007) in the brains of female Sorcs1 hypomorphic mice, possibly paralleling the sexual dimorphism that is characteristic of the genetic associations of SORCS1 with AD and DM. Since SorL1 directly interacts with Vps35 to modulate APP metabolism, we investigated the possibility that SorCS1Cβ-myc interacts with APP, SorL1, and/or Vps35. We readily recovered SorCS1:APP, SorCS1:SorL1, and SorCS1:Vps35 complexes from nontransgenic mouse brain. Notably, total Vps35 protein levels were decreased by 49% (p=0.009) and total SorL1 protein levels were decreased by 29% (p=0.003) in the brains of female Sorcs1-hypomorphic mice. From these data, we propose that dysfunction of SorCS1 may contribute to both the APP/Aβ disturbance underlying AD and the insulin/glucose disturbance underlying DM.
PMCID: PMC3274732  PMID: 20881129
AD; T1DM; T2DM; protein trafficking; APP; SorCS1; Vps10 domain; retromer
24.  Neuronal LR11 Expression Does Not Differentiate between Clinically-Defined Alzheimer's Disease and Control Brains 
PLoS ONE  2012;7(8):e40527.
Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Because the pathological changes underlying this disease can begin decades prior to the onset of cognitive impairment, identifying the earliest events in the AD pathological cascade has critical implications for both the diagnosis and treatment of this disease. We previously reported that compared to autopsy confirmed healthy control brain, expression of LR11 (or SorLA) is markedly reduced in AD brain as well as in a subset of people with mild cognitive impairment (MCI), a prodromal clinical stage of AD. Recent studies of the LR11 gene SORL1 have suggested that the association between SORL1 single nucleotide polymorphisms (SNPs) and AD risk may not be universal. Therefore, we sought to confirm our earlier findings in a population chosen solely based on clinical criteria, as in most genetic studies. Quantitative immunohistochemistry was used to measure LR11 expression in 43 cases from the Religious Orders Study that were chosen based on a final pre-mortem clinical diagnosis of MCI, mild/moderate AD or no cognitive impairment (NCI). LR11 expression was highly variable in all three diagnostic groups, with no significant group differences. Low LR11 cases were identified using the lowest tertile of LR11 expression observed across all cases as a threshold. Contrary to previous reports, low LR11 expression was found in only 29% of AD cases. A similar proportion of both the MCI and NCI cases also displayed low LR11 expression. AD-associated lesions were present in the majority of cases regardless of diagnostic group, although we found no association between LR11 levels and pathological variables. These findings suggest that the relationship between LR11 expression and the development of AD may be more complicated than originally believed.
PMCID: PMC3424248  PMID: 22927900
25.  Allelic expression imbalance at high-density lipoprotein cholesterol locus MMAB-MVK 
Human Molecular Genetics  2010;19(10):1921-1929.
Genome-wide association studies (GWAS) have identified numerous loci associated with various complex traits for which the underlying susceptibility gene(s) remain unknown. In a GWAS for high-density lipoprotein-cholesterol (HDL-C) level, one strongly associated locus contains at least two biologically compelling candidates, methylmalonic aciduria cblB type (MMAB) and mevalonate kinase (MVK). To detect evidence of cis-acting regulation at this locus, we measured relative allelic expression of transcribed SNPs in five genes using human hepatocyte samples heterozygous for the transcribed SNP. If an HDL-C-associated SNP allele differentially regulates mRNA level in cis, samples heterozygous both for a transcribed SNP and an HDL-C-associated SNP should display allelic expression imbalance (AEI) of the transcribed SNP. We designed statistical tests to detect AEI in a comprehensive set of linkage disequilibrium (LD) scenarios between the transcribed SNP and an HDL-C-associated SNP (rs7298565) in phase unknown samples. We observed significant AEI of 22% in MMAB (P = 1.4 × 10−13, transcribed SNP rs11067231), and the allele associated with lower HDL-C level was associated with greater MMAB transcript level. The same rs7298565 allele was also associated with higher MMAB mRNA level (P = 0.0081) and higher MMAB protein level (P = 0.0020). In contrast, MVK, UBE3B, KCTD10 and ACACB did not show significant AEI (P ≥ 0.05). These data suggest MMAB is the most likely gene influencing HDL-C levels at this locus and demonstrate that measuring AEI at loci containing more than one candidate gene can prioritize genes for functional studies.
PMCID: PMC2860891  PMID: 20159775

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