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1.  Association of MAPT haplotypes with Alzheimer’s disease risk and MAPT brain gene expression levels 
MAPT encodes for tau, the predominant component of neurofibrillary tangles that are neuropathological hallmarks of Alzheimer’s disease (AD). Genetic association of MAPT variants with late-onset AD (LOAD) risk has been inconsistent, although insufficient power and incomplete assessment of MAPT haplotypes may account for this.
We examined the association of MAPT haplotypes with LOAD risk in more than 20,000 subjects (n-cases = 9,814, n-controls = 11,550) from Mayo Clinic (n-cases = 2,052, n-controls = 3,406) and the Alzheimer’s Disease Genetics Consortium (ADGC, n-cases = 7,762, n-controls = 8,144). We also assessed associations with brain MAPT gene expression levels measured in the cerebellum (n = 197) and temporal cortex (n = 202) of LOAD subjects. Six single nucleotide polymorphisms (SNPs) which tag MAPT haplotypes with frequencies greater than 1% were evaluated.
H2-haplotype tagging rs8070723-G allele associated with reduced risk of LOAD (odds ratio, OR = 0.90, 95% confidence interval, CI = 0.85-0.95, p = 5.2E-05) with consistent results in the Mayo (OR = 0.81, p = 7.0E-04) and ADGC (OR = 0.89, p = 1.26E-04) cohorts. rs3785883-A allele was also nominally significantly associated with LOAD risk (OR = 1.06, 95% CI = 1.01-1.13, p = 0.034). Haplotype analysis revealed significant global association with LOAD risk in the combined cohort (p = 0.033), with significant association of the H2 haplotype with reduced risk of LOAD as expected (p = 1.53E-04) and suggestive association with additional haplotypes. MAPT SNPs and haplotypes also associated with brain MAPT levels in the cerebellum and temporal cortex of AD subjects with the strongest associations observed for the H2 haplotype and reduced brain MAPT levels (β = -0.16 to -0.20, p = 1.0E-03 to 3.0E-03).
These results confirm the previously reported MAPT H2 associations with LOAD risk in two large series, that this haplotype has the strongest effect on brain MAPT expression amongst those tested and identify additional haplotypes with suggestive associations, which require replication in independent series. These biologically congruent results provide compelling evidence to screen the MAPT region for regulatory variants which confer LOAD risk by influencing its brain gene expression.
PMCID: PMC4198935  PMID: 25324900
2.  NGAL, MIC-1 and CA19-9 in Pancreatic Juice: Pathobiological Implications in Diagnosing Benign and Malignant Disease of the Pancreas 
Pancreas  2013;42(3):494-501.
Pancreatic diseases pose significant diagnostic challenge as signs and symptoms often overlap. We investigated the potential of pancreatic juice neutrophil gelatinase–associated lipocalin (NGAL), macrophage inhibitory cytokine-1 (MIC-1), and carbohydrate antigen 19-9 (CA19-9) to aid in the diagnosis of patients with symptoms suggestive of pancreatic diseases.
105 chronic pancreatitis (CP), pancreatic cancer (PC), and non-pancreatic non-healthy (NPNH, patients with symptoms mimicking pancreatic disease but found to be free of pancreatic pathology) patients underwent endoscopic pancreatic juice collection following secretin-stimulation. NGAL and MIC-1 levels were measured by enzyme-linked immunosorbent assay while CA19-9 was measured by radioimmunoassay.
NGAL, MIC-1, and CA19-9 were significantly elevated in the pancreatic juice of CP and PC patients as compared to NPNH controls (p<0.034). NGAL appeared most promising in differentiating diseased versus non-diseased pancreata (AUCs=0.88–0.91) while MIC-1 was found to be higher in PC than CP patients (p=0.043). Interestingly, MIC-1 levels in diabetic PC patients were higher than in non-diabetic PC (p=0.030) and diabetic CP patients (p=0.087). CA19-9 showed the least ability to distinguish patient groups (AUCs=0.61–0.76).
Pancreatic juice NGAL shows potential utility in establishing pancreatic etiology in the context of non-specific symptoms while MIC-1 may aid in differentiating PC from CP.
PMCID: PMC3578014  PMID: 23146921
Pancreatic Disease; NGAL; MIC-1; CA19-9; Pancreatic Juice
3.  Effects of Amitriptyline on Gastric Sensorimotor Function and Postprandial Symptoms in Healthy Individuals: A Randomized, Double-Blind, Placebo-Controlled Trial 
Low-dose tricyclic antidepressants have been used to treat chronic somatic and gastrointestinal pain disorders, including refractory functional dyspepsia. However, there are only limited data on the effects of these drugs on upper gastrointestinal function.
To compare the effects of two doses of amitriptyline (AMT) and placebo on gastric accommodation, emptying, satiation, and postprandial symptoms in healthy volunteers.
Using a parallel-group, double-blind, placebo-controlled design, 41 healthy volunteers were randomized to AMT 25 mg, AMT 50 mg, or placebo for 2 wk. During the final 3 days of therapy, the following end points were assessed: fasting and postprandial gastric volumes, 2- and 4-h gastric emptying, time and volume to maximum satiation using a nutrient drink test, and postprandial symptoms 30 min later using 10-cm visual analog scales. AMT and metabolite levels were measured.
AMT slowed gastric emptying at 2 h (median 75% for placebo, 57% for AMT 25 mg, 67% for AMT 50 mg; P = 0.037) and 4 h (median 98% for placebo, 96% for AMT 25 mg, 92% for AMT 50 mg; P = 0.003). AMT did not affect gastric volumes or satiation volume, but it did reduce nausea scores at 30 min in a dose-dependent manner (median 2.1 for placebo, 0.9 for AMT 25 mg, and 0.0 for AMT 50 mg; P = 0.009).
In healthy volunteers, AMT slows gastric emptying of solids, but it does not significantly affect gastric volumes or satiation. AMT reduces nausea after challenge with a high calorie liquid load.
PMCID: PMC3897125  PMID: 18803000
4.  Shorter peripheral blood telomeres are a potential biomarker for patients with advanced colorectal adenomas 
The International journal of biological markers  2012;27(4):10.5301/JBM.2012.9347.
Colorectal cancer (CRC) can be prevented by the early detection and removal of advanced adenomas (AAs) by colonoscopy. Our aim was to evaluate peripheral blood leukocyte (PBL) telomere length as a potential biomarker for the presence of AAs.
PBL telomere length was measured in patients with AAs (n = 35), in a control group of similar-aged patients who had a normal colonoscopy (n = 145) and in a separate population group with no history of cancer, again similarly aged (n = 495). Telomere measurements were performed using a quantitative PCR assay and reported a ratio of telomere and single copy gene measurements.
Telomere lengths tended to be lower in the patients with AAs than in patients in the normal colonoscopy group (p < 0.001) as well as those in the population group (p = 0.011). A telomere/single copy gene ratio of 0.5 was found to have an estimated 94% sensitivity and 56% specificity for AAs; a combination of sensitivity and specificity for which a value of >0.5 would reduce the odds of a patient having AAs by a factor of 0.11 (the negative likelihood ratio). Thirty three percent of individuals in the population group tested above this cut off and could be considered at low risk for AAs.
PBL telomeres are shortened in patients with colorectal neoplasia suggesting that PBL telomere length could be a promising non-invasive blood biomarker to pre-screen for risk of AAs prior to colonoscopy.
PMCID: PMC3841073  PMID: 22865299
Telomeres; Colorectal polyps; Colorectal adenomas
5.  Maternal Transmission of Alzheimer Disease 
Some propose maternal Alzheimer disease (1) inheritance. We compared dementia family histories in AD cases and cognitively normal controls. We expected more mothers to have AD in both groups. If maternal risk was not only due to female longevity more AD cases’ than controls’ mothers should be demented. We matched 196 AD cases to 200 controls by gender and age. We obtained parent dementia status and age of death for 348 AD and 319 control parents. 24 (12%) controls’ fathers, 26 (13%) AD patient fathers, 58 (29%) controls’ mothers and 55 (28%) AD mothers had memory difficulty. More mothers than fathers had memory problems in both groups and the statistical significance persisted after adjusting for parent age at death and APOE for controls (OR=2.40, p=0.004) but not cases (OR=1.63, p=0.14), although results are qualitatively similar. There was no evidence of a real difference between the two groups in interaction analysis (p=0.41). Mothers of both cases and controls were more often affected than fathers, even after adjusting for age. Cases’ mothers were no more often demented than controls’ mothers, which does not support the maternal AD transmission. Rather, the increased number of affected mothers relates, at least in part, to female longevity.
PMCID: PMC3371291  PMID: 22273801
Alzheimer disease; Inheritance; Genetics; Maternal
6.  Novel late-onset Alzheimer disease loci variants associate with brain gene expression 
Neurology  2012;79(3):221-228.
Recent genome-wide association studies (GWAS) of late-onset Alzheimer disease (LOAD) identified 9 novel risk loci. Discovery of functional variants within genes at these loci is required to confirm their role in Alzheimer disease (AD). Single nucleotide polymorphisms that influence gene expression (eSNPs) constitute an important class of functional variants. We therefore investigated the influence of the novel LOAD risk loci on human brain gene expression.
We measured gene expression levels in the cerebellum and temporal cortex of autopsied AD subjects and those with other brain pathologies (∼400 total subjects). To determine whether any of the novel LOAD risk variants are eSNPs, we tested their cis-association with expression of 6 nearby LOAD candidate genes detectable in human brain (ABCA7, BIN1, CLU, MS4A4A, MS4A6A, PICALM) and an additional 13 genes ±100 kb of these SNPs. To identify additional eSNPs that influence brain gene expression levels of the novel candidate LOAD genes, we identified SNPs ±100 kb of their location and tested for cis-associations.
CLU rs11136000 (p = 7.81 × 10−4) and MS4A4A rs2304933/rs2304935 (p = 1.48 × 10−4–1.86 × 10−4) significantly influence temporal cortex expression levels of these genes. The LOAD-protective CLU and risky MS4A4A locus alleles associate with higher brain levels of these genes. There are other cis-variants that significantly influence brain expression of CLU and ABCA7 (p = 4.01 × 10−5–9.09 × 10−9), some of which also associate with AD risk (p = 2.64 × 10−2–6.25 × 10−5).
CLU and MS4A4A eSNPs may at least partly explain the LOAD risk association at these loci. CLU and ABCA7 may harbor additional strong eSNPs. These results have implications in the search for functional variants at the novel LOAD risk loci.
PMCID: PMC3398432  PMID: 22722634
7.  LRRTM3 Interacts with APP and BACE1 and Has Variants Associating with Late-Onset Alzheimer’s Disease (LOAD) 
PLoS ONE  2013;8(6):e64164.
Leucine rich repeat transmembrane protein 3 (LRRTM3) is member of a synaptic protein family. LRRTM3 is a nested gene within α-T catenin (CTNNA3) and resides at the linkage peak for late-onset Alzheimer’s disease (LOAD) risk and plasma amyloid β (Aβ) levels. In-vitro knock-down of LRRTM3 was previously shown to decrease secreted Aβ, although the mechanism of this is unclear. In SH-SY5Y cells overexpressing APP and transiently transfected with LRRTM3 alone or with BACE1, we showed that LRRTM3 co-localizes with both APP and BACE1 in early endosomes, where BACE1 processing of APP occurs. Additionally, LRRTM3 co-localizes with APP in primary neuronal cultures from Tg2576 mice transduced with LRRTM3-expressing adeno-associated virus. Moreover, LRRTM3 co-immunoprecipitates with both endogenous APP and overexpressed BACE1, in HEK293T cells transfected with LRRTM3. SH-SY5Y cells with knock-down of LRRTM3 had lower BACE1 and higher CTNNA3 mRNA levels, but no change in APP. Brain mRNA levels of LRRTM3 showed significant correlations with BACE1, CTNNA3 and APP in ∼400 humans, but not in LRRTM3 knock-out mice. Finally, we assessed 69 single nucleotide polymorphisms (SNPs) within and flanking LRRTM3 in 1,567 LOADs and 2,082 controls and identified 8 SNPs within a linkage disequilibrium block encompassing 5′UTR-Intron 1 of LRRTM3 that formed multilocus genotypes (MLG) with suggestive global association with LOAD risk (p = 0.06), and significant individual MLGs. These 8 SNPs were genotyped in an independent series (1,258 LOADs and 718 controls) and had significant global and individual MLG associations in the combined dataset (p = 0.02–0.05). Collectively, these results suggest that protein interactions between LRRTM3, APP and BACE1, as well as complex associations between mRNA levels of LRRTM3, CTNNA3, APP and BACE1 in humans might influence APP metabolism and ultimately risk of AD.
PMCID: PMC3672107  PMID: 23750206
8.  Replication of BIN1 association with Alzheimer’s disease and evaluation of genetic interactions 
The most recent late-onset Alzheimer’s disease (LOAD) genome-wide association study revealed genome-wide significant association of two new loci: rs744373 near BIN1 (p=1.6×10−11) and rs597668 near EXOC3L2/BLOC1S3/MARK4 (p=6.5×10−9). We have genotyped these variants in a large (3,287 LOAD, 4,396 controls), independent dataset comprising eleven case-control series from the USA and Europe. We performed meta-analyses of the association of these variants with LOAD and also tested for association using logistic regression adjusted by age-at-diagnosis, sex and APOE ε4 status. Meta-analysis results showed no evidence of series heterogeneity and logistic regression analysis successfully replicated the association of BIN1 (rs744373) with LOAD with an odds ratio (OR=1.17, p=1.1×10−4) comparable to that previously reported (OR=1.15). The variant near EXOC3L2 (rs597668) showed only suggestive association with LOAD (p=0.09) after correcting for the presence of the APOE ε4 allele. Addition of our follow-up data to the results previously reported increased the strength of evidence for association with BIN1 (11,825 LOAD, 32,570 controls, rs744373 Fisher combined p=3.8×10−20). We also tested for epistatic interaction between these variants and APOEε4 as well as with the previously replicated LOAD GWAS genes (CLU: rs11136000, CR1; rs3818361, and PICALM: rs3851179). No significant interactions between these genes were detected. In summary, we provide additional evidence for the variant near BIN1 (rs744373) as a LOAD risk modifier, but our results indicate that the effect of EXOC3L2 independent of APOE ε4 should be studied further.
PMCID: PMC3489170  PMID: 21321396
Alzheimer Disease; Late Onset; Heterogeneity; Meta-Analysis; Case-Control Studies
9.  Hippocampal sclerosis in the elderly: genetic and pathologic findings, some mimicking Alzheimer disease clinically 
Hippocampal sclerosis (HpScl) in the elderly is often associated with neurodegeneration.
We studied the clinical and pathologic features of HpScl in 205 consecutive patients with dementia who came to autopsy from 1997 to 2008, focusing on associations with TDP-43 pathology and allelic variants in the progranulin (GRN) and apolipoprotein E (APOE).
Of the 205 dementia patients, 28 had HpScl (14%). TDP-43 pathology was more frequent in cases with HpScl compared to those without HpScl (89% vs. 24%). GRN rs5848 T-allele but not APOE ε4 was associated with HpScl. In cases of HpScl with TDP-43 pathology and age of onset after 75 (n=11), 8 had AD-like amnestic syndrome, but most (6/8) had pathology not consistent with AD (Braak stage III or less), including 4 with frontotemporal lobar degeneration (FTLD-TDP), 1 with diffuse Lewy body disease and 1 with “pure HpScl.”
HpScl is common in an elderly cohort with dementia, occurring in 14% of the cases in this series, and 89% have TDP-43 pathology, often associated with a risk variant in GRN. Patients with HpScl who present after age 75 often have presentations consistent with AD, but at autopsy have non-Alzheimer pathologies. Elderly patients with HpScl may be mistaken for AD.
PMCID: PMC3107353  PMID: 21346515
10.  Brain Expression Genome-Wide Association Study (eGWAS) Identifies Human Disease-Associated Variants 
PLoS Genetics  2012;8(6):e1002707.
Genetic variants that modify brain gene expression may also influence risk for human diseases. We measured expression levels of 24,526 transcripts in brain samples from the cerebellum and temporal cortex of autopsied subjects with Alzheimer's disease (AD, cerebellar n = 197, temporal cortex n = 202) and with other brain pathologies (non–AD, cerebellar n = 177, temporal cortex n = 197). We conducted an expression genome-wide association study (eGWAS) using 213,528 cisSNPs within ±100 kb of the tested transcripts. We identified 2,980 cerebellar cisSNP/transcript level associations (2,596 unique cisSNPs) significant in both ADs and non–ADs (q<0.05, p = 7.70×10−5–1.67×10−82). Of these, 2,089 were also significant in the temporal cortex (p = 1.85×10−5–1.70×10−141). The top cerebellar cisSNPs had 2.4-fold enrichment for human disease-associated variants (p<10−6). We identified novel cisSNP/transcript associations for human disease-associated variants, including progressive supranuclear palsy SLCO1A2/rs11568563, Parkinson's disease (PD) MMRN1/rs6532197, Paget's disease OPTN/rs1561570; and we confirmed others, including PD MAPT/rs242557, systemic lupus erythematosus and ulcerative colitis IRF5/rs4728142, and type 1 diabetes mellitus RPS26/rs1701704. In our eGWAS, there was 2.9–3.3 fold enrichment (p<10−6) of significant cisSNPs with suggestive AD–risk association (p<10−3) in the Alzheimer's Disease Genetics Consortium GWAS. These results demonstrate the significant contributions of genetic factors to human brain gene expression, which are reliably detected across different brain regions and pathologies. The significant enrichment of brain cisSNPs among disease-associated variants advocates gene expression changes as a mechanism for many central nervous system (CNS) and non–CNS diseases. Combined assessment of expression and disease GWAS may provide complementary information in discovery of human disease variants with functional implications. Our findings have implications for the design and interpretation of eGWAS in general and the use of brain expression quantitative trait loci in the study of human disease genetics.
Author Summary
Genetic variants that regulate gene expression levels can also influence human disease risk. Discovery of genomic loci that alter brain gene expression levels (brain expression quantitative trait loci = eQTLs) can be instrumental in the identification of genetic risk underlying both central nervous system (CNS) and non–CNS diseases. To systematically assess the role of brain eQTLs in human disease and to evaluate the influence of brain region and pathology in eQTL mapping, we performed an expression genome-wide association study (eGWAS) in 773 brain samples from the cerebellum and temporal cortex of ∼200 autopsied subjects with Alzheimer's disease (AD) and ∼200 with other brain pathologies (non–AD). We identified ∼3,000 significant associations between cisSNPs near ∼700 genes and their cerebellar transcript levels, which replicate in ADs and non–ADs. More than 2,000 of these associations were reproducible in the temporal cortex. The top cisSNPs are enriched for both CNS and non–CNS disease-associated variants. We identified novel and confirmed previous cisSNP/transcript associations for many disease loci, suggesting gene expression regulation as their mechanism of action. These findings demonstrate the reproducibility of the eQTL approach across different brain regions and pathologies, and advocate the combined use of gene expression and disease GWAS for identification and functional characterization of human disease-associated variants.
PMCID: PMC3369937  PMID: 22685416
11.  Glutathione S-transferase omega genes in Alzheimer and Parkinson disease risk, age-at-diagnosis and brain gene expression: an association study with mechanistic implications 
Glutathione S-transferase omega-1 and 2 genes (GSTO1, GSTO2), residing within an Alzheimer and Parkinson disease (AD and PD) linkage region, have diverse functions including mitigation of oxidative stress and may underlie the pathophysiology of both diseases. GSTO polymorphisms were previously reported to associate with risk and age-at-onset of these diseases, although inconsistent follow-up study designs make interpretation of results difficult. We assessed two previously reported SNPs, GSTO1 rs4925 and GSTO2 rs156697, in AD (3,493 ADs vs. 4,617 controls) and PD (678 PDs vs. 712 controls) for association with disease risk (case-controls), age-at-diagnosis (cases) and brain gene expression levels (autopsied subjects).
We found that rs156697 minor allele associates with significantly increased risk (odds ratio = 1.14, p = 0.038) in the older ADs with age-at-diagnosis > 80 years. The minor allele of GSTO1 rs4925 associates with decreased risk in familial PD (odds ratio = 0.78, p = 0.034). There was no other association with disease risk or age-at-diagnosis. The minor alleles of both GSTO SNPs associate with lower brain levels of GSTO2 (p = 4.7 × 10-11-1.9 × 10-27), but not GSTO1. Pathway analysis of significant genes in our brain expression GWAS, identified significant enrichment for glutathione metabolism genes (p = 0.003).
These results suggest that GSTO locus variants may lower brain GSTO2 levels and consequently confer AD risk in older age. Other glutathione metabolism genes should be assessed for their effects on AD and other chronic, neurologic diseases.
PMCID: PMC3393625  PMID: 22494505
GSTO genes; Disease risk; Gene expression; Association
12.  Association of common KIBRA variants with episodic memory and AD risk 
Neurobiology of aging  2010;32(3):557.e1-557.e9.
KIBRA SNP rs17070145 was identified in a GWAS of memory performance, with some but not all follow-up studies confirming association of its T allele with enhanced memory. This allele was associated with reduced Alzheimer's disease (AD) risk in one study, which also found overexpression of KIBRA in memory-related brain regions of ADs. We genotyped rs17070145 and 14 additional SNPs in 2571 LOADs vs. 2842 controls, including African-Americans. We found significantly reduced risk for rs17070145 T allele in the older African-American subjects (p=0.007) and a suggestive effect in the older Caucasian series. Meta-analysis of this allele in >8000 subjects from our and published series showed a suggestive protective effect (p=0.07). Analysis of episodic memory in control subjects did not identify associations with rs17070145, though other SNPs showed significant associations in one series. KIBRA showed evidence of overexpression in the AD temporal cortex (p=0.06) but not cerebellum. These results suggest a modest role for KIBRA as a cognition and AD risk gene, and also highlight the multifactorial complexity of its genetic associations.
PMCID: PMC3065956  PMID: 21185624
Alzheimer's disease; Association studies in genetics; Case control studies
13.  Altered microRNA expression in frontotemporal lobar degeneration with TDP-43 pathology caused by progranulin mutations 
BMC Genomics  2011;12:527.
Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative disorder that can be triggered through genetic or sporadic mechanisms. MicroRNAs (miRNAs) have become a major therapeutic focus as their pervasive expression and powerful regulatory roles in disease pathogenesis become increasingly apparent. Here we examine the role of miRNAs in FTLD patients with TAR DNA-binding protein 43 pathology (FTLD-TDP) caused by genetic mutations in the progranulin (PGRN) gene.
Using miRNA array profiling, we identified the 20 miRNAs that showed greatest evidence (unadjusted P < 0.05) of dysregulation in frontal cortex of eight FTLD-TDP patients carrying PGRN mutations when compared to 32 FTLD-TDP patients with no apparent genetic abnormalities. Quantitative real-time PCR (qRT-PCR) analyses provided technical validation of the differential expression for 9 of the 20 miRNAs in frontal cortex. Additional qRT-PCR analyses showed that 5 out of 9 miRNAs (miR-922, miR-516a-3p, miR-571, miR-548b-5p, and miR-548c-5p) were also significantly dysregulated (unadjusted P < 0.05) in cerebellar tissue samples of PGRN mutation carriers, consistent with a systemic reduction in PGRN levels. We developed a list of gene targets for the 5 candidate miRNAs and found 18 genes dysregulated in a reported FTLD mRNA study to exhibit anti-correlated miRNA-mRNA patterns in affected cortex and cerebellar tissue. Among the targets is brain-specific angiogenesis inhibitor 3, which was recently identified as an important player in synapse biology.
Our study suggests that miRNAs may contribute to the pathogenesis of FTLD-TDP caused by PGRN mutations and provides new insight into potential future therapeutic options.
PMCID: PMC3229715  PMID: 22032330
Frontotemporal lobar degeneration; TDP-43; microRNA; progranulin
14.  Exploring the optimal fluorescein dose in probe-based confocal laser endomicroscopy for colonic imaging 
Probe-based confocal laser endomicroscopy (pCLE) is an emerging method for in-vivo imaging of the gastrointestinal tract and requires a contrast agent. Fluorescein is the most commonly used agent. The optimal dose of fluorescein for pCLE in colon is unknown.
Exploration of optimal dose of fluorescein for pCLE in colon.
Comparative, prospective pilot trail.
Tertiary-care center.
18 participants underwent colonoscopy without complications.
pCLE videos were recorded in normal cecum, using 10% fluorescein intravenously.
Main Outcome Measurements
For subjective analysis, pCLE videos were scored for quality, by 2 observers, independently and blinded to fluorescein dose. For objective analysis, signal-to-noise ratios (SNR) were calculated for each video by an expert.
6 fluorescein doses were used, including 0.5 mL, 1 mL, 2.5 mL, 5 mL, 7.5 mL and 10 mL and each dose was used in three patients. For each dose, median image quality score was 2.5, 2.0, 3.25, 4.0, 4.0 and 3.5 by first observer and 2.0, 3.0, 4.0, 5.0, 4.0 and 4.0 by second observer, respectively. The subjective quality scores increased from 0.5 mL to 5.0 mL, with no evidence of further improved quality at 7.5 mL and 10 mL doses. SNR were not significantly different between doses but trended higher for higher doses.
Small sample size. The results can not be applied to other parts of gastrointestinal tract i.e. duodenum, esophagus with different blood supply.
This preliminary study suggests that the optimal dose of fluorescein for high quality pCLE imaging in colon is approximately 5.0 mL.
PMCID: PMC3350888  PMID: 22586530
confocal; colon; fluorescein dose; contrast
15.  Replication of CLU, CR1 and PICALM associations with Alzheimer’s disease 
Archives of neurology  2010;67(8):961-964.
Harold et al. and Lambert et al. recently published two large genome-wide association studies of late onset Alzheimer’s disease (LOAD) in which CLU, CR1, and PICALM were identified as novel LOAD genes.
To test for replication of the association between variants in the CLU, CR1 and PICALM genes with Alzheimer’s disease.
Case-control association study
Community-based ascertainment of patients seen at the Mayo Clinic Jacksonville, FL and Rochester, MN, and autopsy-confirmed cases and controls whose pathology was evaluated at the Mayo Clinic Jacksonville. Additional samples were obtained from the National Cell Repository for Alzheimer’s Disease (NCRAD).
LOAD case-control series of European descent consisting of 1,829 LOAD cases and 2,576 controls
Main Outcome Measure
Clinical or pathology-confirmed diagnosis of LOAD
In our follow-up study of 1,829 LOAD cases and 2,576 controls, the most significant SNPs in CLU (rs11136000), CR1 (rs3818361), and PICALM (rs3851179) were tested for allelic association and gave ORs of 0.82, 1.15, and 0.80 respectively that were comparable in direction and magnitude to those originally reported with p values of 8.6×10−5, 0.014, and 1.3×10−5 that were significant even after Bonferroni correction for 3 SNPs tested.
These results showing near perfect replication provide the first additional evidence that CLU, CR1, and PICALM are LOAD genes.
PMCID: PMC2919638  PMID: 20554627
16.  Replication of EPHA1 and CD33 associations with late-onset Alzheimer's disease: a multi-centre case-control study 
A recently published genome-wide association study (GWAS) of late-onset Alzheimer's disease (LOAD) revealed genome-wide significant association of variants in or near MS4A4A, CD2AP, EPHA1 and CD33. Meta-analyses of this and a previously published GWAS revealed significant association at ABCA7 and MS4A, independent evidence for association of CD2AP, CD33 and EPHA1 and an opposing yet significant association of a variant near ARID5B. In this study, we genotyped five variants (in or near CD2AP, EPHA1, ARID5B, and CD33) in a large (2,634 LOAD, 4,201 controls), independent dataset comprising six case-control series from the USA and Europe. We performed meta-analyses of the association of these variants with LOAD and tested for association using logistic regression adjusted by age-at-diagnosis, gender, and APOE ε4 dosage.
We found no significant evidence of series heterogeneity. Associations with LOAD were successfully replicated for EPHA1 (rs11767557; OR = 0.87, p = 5 × 10-4) and CD33 (rs3865444; OR = 0.92, p = 0.049), with odds ratios comparable to those previously reported. Although the two ARID5B variants (rs2588969 and rs494288) showed significant association with LOAD in meta-analysis of our dataset (p = 0.046 and 0.008, respectively), the associations did not survive adjustment for covariates (p = 0.30 and 0.11, respectively). We had insufficient evidence in our data to support the association of the CD2AP variant (rs9349407, p = 0.56).
Our data overwhelmingly support the association of EPHA1 and CD33 variants with LOAD risk: addition of our data to the results previously reported (total n > 42,000) increased the strength of evidence for these variants, providing impressive p-values of 2.1 × 10-15 (EPHA1) and 1.8 × 10-13 (CD33).
PMCID: PMC3157442  PMID: 21798052
17.  Preliminary accuracy and interobserver agreement for the detection of intraepithelial neoplasia in Barrett’s esophagus with probe-based confocal laser endomicroscopy 
Gastrointestinal endoscopy  2010;72(1):19-24.
Confocal laser endomicroscopy (CLE) is a rapidly emerging method for in vivo imaging of the GI tract.
To determine the preliminary evaluation accuracy and interobserver agreement of probe-based CLE (pCLE) in Barrett’s esophagus (BE).
Prospective, double-blind review of pCLE images of 40 sites of BE tissue by using matching biopsies as the reference standard. A training set of 20 images with known histology was first reviewed to standardize image interpretation, followed by blinded review of 20 unknown images.
Eleven experts in BE imaging from 4 different endoscopy centers from the United States and Europe evaluated the images.
This study involved nonconsecutive patients undergoing BE surveillance or evaluation of high-grade intraepithelial neoplasia or early adenocarcinoma.
Intravenous fluorescein pCLE imaging of each site within the BE segment, followed by matching biopsy.
Main Outcome Measurements
Sensitivity, specificity, and agreement for the pCLE diagnosis of high-grade intraepithelial neoplasia or carcinoma.
In the validation set (n = 20), 11 cases had high-grade intraepithelial neoplasia or invasive carcinoma. The sensitivity for the diagnosis of neoplasia for the 11 endoscopists was 88% (range 6 of 11 to 11 of 11), and the specificity was 96% (range 7 of 9 to 9 of 9). There was substantial agreement on the pCLE diagnosis (86%, kappa 0.72; 95% confidence interval, 0.58–0.86). Endomicroscopists with prior pCLE experience had an overall sensitivity of 91% (all 10 of 11), specificity of 100% (all 9 of 9), and almost perfect agreement (92%, kappa 0.83; 95% confidence interval, 0.64–1.0).
Small sample size and use of offline video sequences.
Results suggest that pCLE for the diagnosis of neoplasia in BE has very high accuracy and reliability.
PMCID: PMC3144146  PMID: 20381042
18.  Genetic variation in PCDH11X is associated with susceptibility to late onset Alzheimer's disease 
Nature genetics  2009;41(2):192-198.
By analyzing late onset Alzheimer's disease (LOAD) in a genome wide association study (313,504 SNPs, 3 series, 844 cases/1,255 controls) and evaluating the 25 SNPs with most significant allelic association in 4 additional series (1,547 cases/1,209 controls), we identified a SNP (rs5984894) on Xq21.3 in PCDH11X that is strongly associated with LOAD in American Caucasians. Analysis of rs5984894 by multivariable logistic regression adjusted for sex gave global P values of 5.7×10-5 in stage I, 4.8×10-6 in stage II, and 3.9×10-12 in the combined data. Odds ratios were 1.75 (95% CI 1.42-2.16) for female homozygotes (P=2.0×10-7) and 1.26 (95% CI 1.05-1.51) for female heterozygotes (P=0.01) compared to female non-carriers. For male hemizygotes (P=0.07) compared to male non-carriers the odds ratio was 1.18 (95% CI 0.99-1.41).
PMCID: PMC2873177  PMID: 19136949
19.  Botulinum A Toxin/Dimethyl Sulfoxide Bladder Instillations for Women With Refractory Idiopathic Detrusor Overactivity: A Phase 1/2 Study 
Mayo Clinic Proceedings  2009;84(8):702-706.
We completed a phase 1/2 trial to evaluate the safety and potential efficacy of direct intravesical instillation of a botulinum type A toxin/dimethyl sulfoxide (DMSO) solution for treatment of idiopathic detrusor overactivity in women. Twenty-five women with medication-resistant, urodynamic-confirmed idiopathic detrusor overactivity were enrolled. A total of 9 patients were treated in phase 1 of the study. Three patients were given a 66% dosing of solution; 22 patients received the full 300 units of botulinum toxin and 50 mL of DMSO (50% concentration). Adverse effects, 24-hour pad weights, episodes of incontinence, postvoid residuals, and scores on the Blaivas-Groutz anti-incontinence scale, Indevus Urgency Severity Scale, Incontinence Impact Questionnaire-short form, and Urogenital Distress Inventory (6 items) questionnaire were recorded at baseline, 1 month, and 3 months after instillation. No serious adverse effects or clinically important increases in postvoid residual occurred. Among the 21 women who completed phase 2 of the study, the median number of incontinent episodes decreased from 4 at baseline to 2 at 1 month (P=.004) and increased to 4 at 3 months (P=.81). Median scores improved from baseline to 1 month on the Incontinence Impact Questionnaire (from 13 to 7; P=.007) and Urogenital Distress Inventory (from 10 to 5; P=.003). Although 11 women (52%) reported severe urgency based on the Indevus Urgency Severity Scale at baseline, only 1 (5%; P<.001) and 3 (14%; P=.004) women reported severe scores at 1 and 3 months, respectively. Direct instillation of botulinum toxin/DMSO solution is safe. Its promising clinical effect warrants further evaluation in a randomized, placebo-controlled phase 3 setting.
PMCID: PMC2719523  PMID: 19648387
20.  Is Incidental Lewy Body Disease Related to Parkinson Disease? Comparison of Risk Factor Profiles 
Archives of neurology  2009;66(9):1114.
To explore whether associations of potential risk factors for incidental Lewy Body Disease (iLBD) may be similar to Parkinson Disease (PD).
Design, Setting, and Patients
We identified brain-autopsied residents of Olmsted County, MN and immediate vicinity(1988–2004), age>60, without evidence of neurodegenerative disease or tremor, and evaluated by at least one physician within one year of death. Analysis for “incidental” Lewy pathology was done blinded to clinical abstraction.
Main Outcome Measures
Whether risk factors previously associated with PD in Olmsted County, MN are also associated with iLBD.
Of 235 subjects, 34 had iLBD(14.5%). The overall risk factor profiles for iLBD and PD were fairly similar between the two sets of OR estimates, with 11/16 ORs in the same direction. Prior Olmsted County studies documented 7 risk factors with statistically significant associations with PD; for two of these, the ORs for iLBD were in the same direction and statistically significant (physician, caffeine), whereas for three, they were in the same direction but not significant (education, head injury, number-of-children); they were in the opposite direction but not statistically significant for 2 (depression, anxiety). ILBD was not associated with various end-of-life conditions or causes-of-death, although they were slightly older and more likely cachectic.
Based on this exploratory study, iLBD and PD appear to have similar risk factor profiles. Thus, at least some cases of ILBD might represent preclinical PD, arrested PD or a partial syndrome due to a lesser burden of causative factors. ILBD is not explained by non-specific end-of-life brain insults.
PMCID: PMC2813519  PMID: 19752300
21.  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
22.  Sex-dependent Association of a Common Low Density Lipoprotein Receptor Polymorphism with RNA Splicing Efficiency in the Brain and Alzheimers Disease 
Human molecular genetics  2007;17(7):929-935.
Since apoE allele status is the predominant Alzheimers disease (AD) genetic risk factor, functional single nucleotide polymorphisms (SNP)s in brain apoE receptors represent excellent candidates for association with AD. Recently, we identified a SNP, rs688, as modulating the splicing efficiency of low-density lipoprotein receptor (LDLR) exon 12 in the female human liver and in minigene transfected HepG2 cells. Moreover, the rs688T minor allele associated with significantly higher LDL and total cholesterol in women in the Framingham Offspring Study. Since LDLR is a major apoE receptor in the brain, we hypothesized that rs688 modulates LDLR splicing in neural tissues and associates with AD. To evaluate this hypothesis, we first transfected LDLR minigenes into SH-SY5Y neuroblastoma cells and found that rs688T reduces exon 12 inclusion in this neural model. We then evaluated rs688 association with exon 12 splicing efficiency in vivo by quantifying LDLR splicing in human anterior cingulate tissue obtained at autopsy; the rs688T allele associated with decreased LDLR exon 12 splicing efficiency in aged men but not women. Lastly, we evaluated whether rs688 associates with AD by genotyping DNA from 1,457 men and 2,055 women drawn from three case-control series. The rs688T/T genotype was associated with increased AD odds in males (recessive model, odds ratio (OR) of 1.49, 95% confidence interval (CI) of 1.13−1.97, uncorrected p=0.005), but not in females. In summary, these studies identify a functional apoE receptor SNP that is associated with AD in a sex-dependent fashion.
PMCID: PMC2361153  PMID: 18065781
23.  Fine-mapping and candidate gene investigation within the PARK10 locus 
Herein, we investigate whether single-nucleotide polymorphisms (SNPs) across the PARK10 locus are associated with susceptibility to Parkinson's disease (PD) or age at onset (AAO) of disease. One hundred and eighty-eight SNPs were genotyped across the PARK10 locus in 180 PD patients and 180 controls from central Norway (stage 1). We then used the linkage disequilibrium (LD) structure from stage 1 to select 75 SNPs for genotyping in 186 patients and 186 controls from Ireland (stage 2). Nineteen SNPs were selected from this and previous studies for follow-up in an extended Norwegian series (530 patients and 1142 controls), the Irish series and a US series (221 patients and 221 controls) (stage 3). After correction for multiple testing, markers within ubiquitin specific peptidase 24 (USP24) are significantly associated with PD within Norwegian, Irish, and US series combined (rs13312: odds ratio (OR) 0.78, P<0.001; rs487230: OR 0.80, P=0.001). Independently, the association for rs13312 is strongest in the extended Norwegian series (OR 0.76, P=0.005), although not significant after correction for multiple testing (P≤0.003 is considered significant). ORs in the Irish series are almost identical, and a similar but a weaker effect was observed for the US series. No marker showed consistent association with AAO. Our data indicate that genetic variability in USP24 is associated with PD. Although our work extends and confirms a previous report, the observed effect size does not explain the PARK10 linkage peak.
PMCID: PMC2986168  PMID: 18854859
Parkinson's disease; linkage study; association study; risk factors; USP24
24.  Plasma progranulin levels predict progranulin mutation status in frontotemporal dementia patients and asymptomatic family members 
Brain  2009;132(3):583-591.
Mutations in the progranulin gene (GRN) are an important cause of frontotemporal lobar degeneration (FTLD) with ubiquitin and TAR DNA-binding protein 43 (TDP43)-positive pathology. The clinical presentation associated with GRN mutations is heterogeneous and may include clinical probable Alzheimer's disease. All GRN mutations identified thus far cause disease through a uniform disease mechanism, i.e. the loss of functional GRN or haploinsufficiency. To determine if expression of GRN in plasma could predict GRN mutation status and could be used as a biological marker, we optimized a GRN ELISA and studied plasma samples of a consecutive clinical FTLD series of 219 patients, 70 control individuals, 72 early-onset probable Alzheimer's disease patients and nine symptomatic and 18 asymptomatic relatives of GRN mutation families. All FTLD patients with GRN loss-of-function mutations showed significantly reduced levels of GRN in plasma to about one third of the levels observed in non-GRN carriers and control individuals (P < 0.001). No overlap in distributions of GRN levels was observed between the eight GRN loss-of-function mutation carriers (range: 53–94 ng/ml) and 191 non-GRN mutation carriers (range: 115–386 ng/ml). Similar low levels of GRN were identified in asymptomatic GRN mutation carriers. Importantly, ELISA analyses also identified one probable Alzheimer's disease patient (1.4%) carrying a loss-of-function mutation in GRN. Biochemical analyses further showed that the GRN ELISA only detects full-length GRN, no intermediate granulin fragments. This study demonstrates that using a GRN ELISA in plasma, pathogenic GRN mutations can be accurately detected in symptomatic and asymptomatic carriers. The ∼75% reduction in full-length GRN, suggests an unbalanced GRN metabolism in loss-of-function mutation carriers whereby more GRN is processed into granulins. We propose that plasma GRN levels could be used as a reliable and inexpensive tool to identify all GRN mutation carriers in early-onset dementia populations and asymptomatic at-risk individuals.
PMCID: PMC2664450  PMID: 19158106
Progranulin; ELISA; frontotemporal lobar degeneration; Alzheimer's disease
25.  Increased Incidence of Loco-Regional Recurrences Among African American Women with Terminal Stage Breast Cancer 
Clinical Medicine. Oncology  2008;2:547-550.
A prospective analysis of women with terminal breast cancer admitted to CHNE from November 2006–August 2007 evaluated anecdotal observations that African American (AA) women are likelier than Caucasian women to evidence loco-regional recurrences (LRR). Women with terminal breast cancer who were admitted to CHNE, a not-for-profit hospice serving over 90% of Northeast Florida hospice patients, were eligible for participation. 134 terminal breast cancer patients were assessed by hospice nurses for LRR presence via chest wall examination. 80% of them (107) were Caucasian, 17% (23) were AA and 3% (4) were of other ethnicities. Evidence of LRR were noted in 13% of the women (17/134). The proportion of patients with LRR was higher in AA women than Caucasian women (26% vs. 10%, 6/23 vs. 11/107, respectively), although this difference was not statistically significant (p = 0.08). The majority of Caucasian women with LRR consented to a medical record review, but a minority of AA women consented (8/11 vs. 2/6, respectively, p = 0.16).
Evaluating disparities in breast cancer care outcomes is possible by reviewing data from patients served by hospice programs that aid a majority of patients within a community. This pilot data suggests that AA women with breast cancer have a higher incidence of loco-regional failure as a component of their terminal breast cancer disease than Caucasian women. A smaller proportion of AA patients and families agreed to participate in a medical record review study than Caucasians. Larger studies are necessary to confirm these findings, to elucidate factors contributing to disparities and to develop potential solutions.
PMCID: PMC3161676  PMID: 21892330
oncology; LRR; loco-regional recurrences; breast cancer; African American women; terminal breast cancer; terminal stage breast cancer

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