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1.  L-DOPS corrects neurochemical abnormalities in a Menkes disease mouse model 
Annals of neurology  2012;73(2):259-265.
Menkes disease is a lethal neurodegenerative disorder of infancy caused by mutations in a copper-transporting ATPase gene, ATP7A. Among its multiple cellular tasks, ATP7A transfers copper to dopamine-beta-hydroxylase (DBH) within the lumen of the Golgi network or secretory granules, catalyzing the conversion of dopamine to norepinephrine. In a well-established mouse model of Menkes disease, mottled-brindled, we tested whether systemic administration of L-threo-dihydroxyphenylserine (L-DOPS), a drug used successfully to treat autosomal recessive norepinephrine deficiency, would improve brain neurochemical abnormalities and neuropathology.
At 8, 10, and 12 days of age, wild type and mo-br mice received intraperi-toneal injections of 200μg/g body weight of L-DOPS, or mock solution. Five hours after the final injection, the mice were euthanized and brains removed. We measured catecholamine metabolites affected by DBH via high-performance liquid chromatography with electrochemical detection, and assessed brain histopathology.
Compared to mock-treated controls, mo-br mice that received intraperitoneal L-DOPS showed significant increases in brain norepinephrine (P<0.001) and its deaminated metabolite, dihydroxyphenylglycol (DHPG, P<0.05). The ratio of a non-beta-hydroxylated metabolite in the catecholamine biosynthetic pathway, dihydroxyphenylacetic acid, to the beta-hydroxylated metabolite, dihydroxyphenylglycol, improved equivalently to results obtained previously with brain-directed ATP7A gene therapy (P<0.01). However, L-DOPS treatment did not arrest global brain pathology or improve somatic growth, as gene therapy had.
We conclude that 1) L-DOPS crosses the blood-brain barrier in mo-br mice and corrects brain neurochemical abnormalities, 2) norepinephrine deficiency is not the cause of neurodegeneration in mo-br mice, and 3) L-DOPS treatment may ameliorate noradrenergic hypofunction in Menkes disease.
PMCID: PMC3597755  PMID: 23224983
2.  Potential applications of nanotechnology for the diagnosis and treatment of pancreatic cancer 
Despite improvements in our understanding of pancreatic cancer and the emerging concept of personalized medicine for the treatment of this disease, it is still the fourth most common cause of cancer death in the western world. It is established that pancreatic cancer is a highly heterogeneous disease with a complex tumor microenvironment. Indeed the extensive stroma surrounding the cancer cells has been shown to be important in promoting tumor growth and metastases, as well as sequestering chemotherapeutic agents consequently decreasing delivery to the tumor cells. Nanotechnology has come to the forefront in the areas of medical diagnostics, imaging, and therapeutic drug delivery. This review will focus on the potential applications of nanotechnology for diagnosis, imaging, and delivery of therapeutic agents for the treatment of pancreatic cancer.
PMCID: PMC3900771  PMID: 24478715
pancreatic cancer; nanotechnology; nano-diagnostics; molecular imaging; therapeutic drug delivery; tumor stroma
3.  Single nucleotide polymorphism upstream of interleukin 28B associated with phase 1 and phase 2 of early viral kinetics in patients infected with HCV genotype 1 
Journal of hepatology  2011;56(3):10.1016/j.jhep.2011.10.004.
Background & Aims
We studied the relationship between IL28B gene-related SNP rs12979860 and early viral kinetics (day 0–28) during peginterferon and ribavirin treatment, in 173 African Americans (AA) and 188 Caucasian Americans (CA) with HCV genotype 1.
We studied the relationship between IL28B 16 gene-related SNP rs12979860 and early viral kinetics (day 0–28) 17 during peginterferon and ribavirin treatment, in 171 African 18 Americans (AA) and 188 Caucasian Americans (CA) with HCV 19 genotype 1.
Compared to non-C/C genotypes, C/C was associated with greater declines in serum HCV RNA during phase 1 (day 0–2), phase 2 (day 7–28), and day 0–28 and higher response (undetected HCV RNA) rates at weeks 4 and 12 in AA and CA. A static phase and increases in HCV RNA from day 2 to 7 were more common in patients with non-C/C genotypes. C/C was also associated with higher week 24, 48, and 72 response rates in CA (p <0.01) but not in AA. At baseline, SNP genotype was the only independent predictor of phase 1; SNP genotype and phase 1 were independent predictors of phase 2 (p<0.001). There were no racial differences in HCV RNA declines during phase 1, day 2–7, phase 2, and day 0–28 with the same SNP genotype. AA with C/C and C/T genotypes had lower week 24, 48, and 72 (SVR) rates than did CA (p = 0.03). SNP C/C predicted higher SVR rates in AA and CA with high baseline HCV RNA (≥ 600,000 IU/ml), and in CA with ≥ 1 log10 IU/ml decrease in HCV RNA from day 0 to 28.
SNP rs12979860 is strongly associated with both phase 1 and phase 2 HCV RNA kinetics in AA and CA with HCV genotype 1.
PMCID: PMC3884806  PMID: 22027585
IL28B gene; Hepatitis C virus; Viral kinetics; Single nucleotide polymorphism
4.  HGV2011: Personalized Genomic Medicine Meets the Incidentalome 
Human mutation  2012;33(3):10.1002/humu.22008.
The 12th International Meeting on Human Genome Variation and Complex Genome Analysis (HGV2011: Berkeley, California, USA, 8th–10th September 2011) was a stimulating workshop where researchers from academia and industry explored the latest progress, challenges, and opportunities in genome variation research. Key themes included progress beyond GWAS, variation in human populations, use of sequence data in medical settings, large-scale sequencing data analysis, and bioinformatics approaches to large datasets.
PMCID: PMC3867005  PMID: 22170622
human variation; GWAS; SNP; medical genomics
5.  Vesicular Uptake Blockade Generates the Toxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde in PC12 Cells: Relevance to the Pathogenesis of Parkinson Disease 
Journal of neurochemistry  2012;123(6):932-943.
Parkinson disease entails profound loss of nigrostriatal dopaminergic terminals, decreased vesicular uptake of intra-neuronal catecholamines, and relatively increased putamen tissue concentrations of the toxic dopamine metabolite, 3,4-dihydroxyphenylacetaldehyde (DOPAL). The objective of this study was to test whether vesicular uptake blockade augments endogenous DOPAL production. We also examined whether intracellular DOPAL contributes to apoptosis and, since alpha-synuclein oligomers may be pathogenetic in Parkinson disease, oligomerizes alpha-synuclein. Catechols were assayed in PC12 cells after reserpine to block vesicular uptake, with or without inhibition of enzymes metabolizing DOPAL—daidzein for aldehyde dehydrogenase and AL1576 for aldehyde reductase. Vesicular uptake was quantified by a method based on 6F- or 13C-dopamine incubation; DOPAL toxicity by apoptosis responses to exogenous dopamine, with or without daidzein+AL1576; and DOPAL-induced synuclein oligomerization by synuclein dimer production during DOPA incubation, with or without inhibition of L-aromatic-amino-acid decarboxylase or monoamine oxidase. Reserpine inhibited vesicular uptake by 95–97% and rapidly increased cell DOPAL content (p=0.0008). Daidzein+AL1576 augmented DOPAL responses to reserpine (p=0.004). Intracellular DOPAL contributed to dopamine-evoked apoptosis and DOPA-evoked synuclein dimerization. The findings fit with the “catecholaldehyde hypothesis,” according to which decreased vesicular sequestration of cytosolic catecholamines and impaired catecholaldehyde detoxification contribute to the catecholaminergic denervation that characterizes Parkinson disease.
PMCID: PMC3514596  PMID: 22906103
Dihydroxyphenylacetaldehyde; DOPAL; aldehyde dehydrogenase; reserpine; Parkinson disease; monoamine oxidase
6.  IL28B polymorphisms are associated with histological recurrence and treatment response following liver transplantation in patients with HCV Infection 
Hepatology (Baltimore, Md.)  2011;53(1):10.1002/hep.24074.
Polymorphism in the IL28B gene region, encoding interferon-lambda(λ)-3, is strongly predictive of response to antiviral treatment in the non-transplant setting. We sought to determine the prevalence and impact on clinical outcomes of donor and recipient IL28B genotypes among liver transplant recipients.
Cohort study including 189 consecutive hepatitis C virus (HCV) patients who underwent liver transplantation between 1-1-1995 and 1-1-2005 in the Mayo Clinic, Rochester. Genotyping of the polymorphism rs12979860 was performed on DNA collected from all donors and recipients in the cohort. 65 patients received IFN-based antiviral therapy.
The CC IL28B variant was less common in the CHC recipients than in non-HCV donor livers (33% vs 47%, P=0.03). IL28B recipient genotype was significantly predictive of fibrosis stage, with TT genotype being associated with more rapid fibrosis (Pearson Chi-square p=0.024 for the comparison G vs A). Donor and recipient IL28B genotype were independently associated with SVR (P<0.005). The presence of IL28B CC variant in either the recipient (R) or donor (D) liver was associated with increased rate of SVR (D-non-CC / R-non-CC = 3/19 (16%) vs D-CC / R-non-CC=11/22 (50%) vs D-non-CC / R-CC=5/12 (42 %) vs R-CC / D-CC=6/7 (86%), P=0.0095). IL28B genotype was not significantly associated with survival (overall / liver related).
Recipient IL28B genotype is associated with more severe histological recurrence of HCV. Recipient and donor liver IL28B genotype are strongly and independently associated with IFN-based treatment response in patients post-OLT. The data suggest that CC donor livers might be preferentially allocated to patients with HCV infection.
PMCID: PMC3835596  PMID: 21254179
7.  Long term control of a maxillary sinus mucoepidermoid carcinoma with low dose radiation therapy: a case report 
Mucoepidermoid carcinoma of the maxillary sinus is a rare malignancy of the head and neck. The location of this tumour near vital structures and its large size at presentation makes surgical resection with negative margins challenging. In incurable cases, relief from symptoms such as epistaxis may be achieved with radiation therapy. We present a case of mucoepidermoid carcinoma of the maxillary sinus that was effectively palliated with a short course of radiation therapy, achieving complete cessation of bleeding, decrease in tumour size, and long term control. We surveyed the literature on mucoepidermoid carcinomas and propose that some tumours may be particularly radiosensitive, benefiting from even short courses of radiation therapy.
PMCID: PMC3829377  PMID: 24165756
Mucoepidermoid carcinoma; Radiotherapy; Head and neck; Palliation
8.  Fusion of Large-Scale Genomic Knowledge and Frequency Data Computationally Prioritizes Variants in Epilepsy 
PLoS Genetics  2013;9(9):e1003797.
Curation and interpretation of copy number variants identified by genome-wide testing is challenged by the large number of events harbored in each personal genome. Conventional determination of phenotypic relevance relies on patterns of higher frequency in affected individuals versus controls; however, an increasing amount of ascertained variation is rare or private to clans. Consequently, frequency data have less utility to resolve pathogenic from benign. One solution is disease-specific algorithms that leverage gene knowledge together with variant frequency to aid prioritization. We used large-scale resources including Gene Ontology, protein-protein interactions and other annotation systems together with a broad set of 83 genes with known associations to epilepsy to construct a pathogenicity score for the phenotype. We evaluated the score for all annotated human genes and applied Bayesian methods to combine the derived pathogenicity score with frequency information from our diagnostic laboratory. Analysis determined Bayes factors and posterior distributions for each gene. We applied our method to subjects with abnormal chromosomal microarray results and confirmed epilepsy diagnoses gathered by electronic medical record review. Genes deleted in our subjects with epilepsy had significantly higher pathogenicity scores and Bayes factors compared to subjects referred for non-neurologic indications. We also applied our scores to identify a recently validated epilepsy gene in a complex genomic region and to reveal candidate genes for epilepsy. We propose a potential use in clinical decision support for our results in the context of genome-wide screening. Our approach demonstrates the utility of integrative data in medical genomics.
Author Summary
Improvements in sequencing and microarray technologies have increased the resolution and scope of genetic testing. As a result, millions of variations are identified in each personal genome of unrelated individuals. In the context of testing for genetic diseases, identifying the variant or variants contributing to illness among such a large number of candidates is difficult. Conventional studies to identify causative variants have relied on patterns of higher frequency in affected patients compared with individuals that are well. However, it is often the rarest variations that cause human disease, making frequency information alone less useful. Many groups have turned to computational analysis to aid in interpretation of genetic variants. Epilepsy is a disease where such tools would be useful, as only a fraction of patients with suspected genetic epilepsy have a specific genetic diagnosis. To help improve variant interpretation in epilepsy, we used computational analysis to combine knowledge about genes from large cloud information sources with mutation frequency from our diagnostic laboratory to score all genes as to how likely they are to be associated with epilepsy. We use these scores to identify possible candidate genes in epilepsy, and explore other downstream applications.
PMCID: PMC3784560  PMID: 24086149
9.  Association of cognitive dysfunction with neurocirculatory abnormalities in early Parkinson disease 
Neurology  2012;79(13):1323-1331.
Cognitive impairment and neurocirculatory abnormalities such as orthostatic hypotension (OH), supine hypertension (SH), and failure to decrease blood pressure at night (nondipping) occur relatively commonly in Parkinson disease (PD); however, whether cognitive dysfunction in early PD is related to neurocirculatory abnormalities has not been established. Cognitive dysfunction in PD is associated with white matter hyperintensities on MRI. We report results of an analysis of neuropsychological and hemodynamic parameters in patients with early PD.
Among 87 patients, 25 had normal cognition, 48 had mild cognitive impairment, and 14 had dementia, based on comprehensive neuropsychological tests. Orthostatic vital signs and ambulatory 24-hour blood pressure monitoring were recorded, and brain magnetic resonance scans were obtained for all patients.
Cognitive impairment was associated with OH, SH, and white matter hyperintensities but not with nondipping. Dementia and white matter hyperintensities were common in SH. Of 13 patients with OH + SH, every one had mild cognitive impairment or dementia.
Cognitive dysfunction is related to neurocirculatory abnormalities, especially OH + SH, in early PD, raising the possibility that early detection and effective treatment of those abnormalities might slow the rate of cognitive decline.
PMCID: PMC3448741  PMID: 22972639
10.  In utero copper treatment for Menkes disease associated with a severe ATP7A mutation 
Molecular genetics and metabolism  2012;107(1-2):222-228.
Menkes disease is a lethal X-linked recessive neurodegenerative disorder of copper transport caused by mutations in ATP7A, which encodes a copper-transporting ATPase. Early postnatal treatment with copper injections often improves clinical outcomes in affected infants. While Menkes disease newborns appear normal neurologically, analyses of fetal tissues including placenta indicate abnormal copper distribution and suggest a prenatal onset of the metal transport defect. In an affected fetus whose parents found termination unacceptable and who understood the associated risks, we began in utero copper histidine treatment at 31.5 weeks gestational age. Copper histidine (900 μg per dose) was administered directly to the fetus by intramuscular injection (fetal quadriceps or gluteus) under ultrasound guidance. Percutaneous umbilical blood sampling enabled serial measurement of fetal copper and ceruloplasmin levels that were used to guide therapy over a four-week period. Fetal copper levels rose from 17 μg/dL prior to treatment to 45 μg/dL, and ceruloplasmin levels from 39 mg/L to 122 mg/L. After pulmonary maturity was confirmed biochemically, the baby was delivered at 35.5 weeks and daily copper histidine therapy (250 μg sc b.i.d.) was begun. Despite this very early intervention with copper, the infant showed hypotonia, developmental delay, and electroencephalographic abnormalities and died of respiratory failure at 5.5 months of age. The patient’s ATP7A mutation, which severely disrupted mRNA splicing, resulted in complete absence of ATP7A protein on Western blots. These investigations suggest that prenatally initiated copper replacement is inadequate to correct Menkes disease caused by severe loss-of-function mutations, and that postnatal ATP7A gene addition represents a rational approach in such circumstances.
PMCID: PMC3444639  PMID: 22695177
11.  Clinical and molecular characterization of HER2 amplified-pancreatic cancer 
Genome Medicine  2013;5(8):78.
Pancreatic cancer is one of the most lethal and molecularly diverse malignancies. Repurposing of therapeutics that target specific molecular mechanisms in different disease types offers potential for rapid improvements in outcome. Although HER2 amplification occurs in pancreatic cancer, it is inadequately characterized to exploit the potential of anti-HER2 therapies.
HER2 amplification was detected and further analyzed using multiple genomic sequencing approaches. Standardized reference laboratory assays defined HER2 amplification in a large cohort of patients (n = 469) with pancreatic ductal adenocarcinoma (PDAC).
An amplified inversion event (1 MB) was identified at the HER2 locus in a patient with PDAC. Using standardized laboratory assays, we established diagnostic criteria for HER2 amplification in PDAC, and observed a prevalence of 2%. Clinically, HER2- amplified PDAC was characterized by a lack of liver metastases, and a preponderance of lung and brain metastases. Excluding breast and gastric cancer, the incidence of HER2-amplified cancers in the USA is >22,000 per annum.
HER2 amplification occurs in 2% of PDAC, and has distinct features with implications for clinical practice. The molecular heterogeneity of PDAC implies that even an incidence of 2% represents an attractive target for anti-HER2 therapies, as options for PDAC are limited. Recruiting patients based on HER2 amplification, rather than organ of origin, could make trials of anti-HER2 therapies feasible in less common cancer types.
PMCID: PMC3978667  PMID: 24004612
12.  Genic Intolerance to Functional Variation and the Interpretation of Personal Genomes 
PLoS Genetics  2013;9(8):e1003709.
A central challenge in interpreting personal genomes is determining which mutations most likely influence disease. Although progress has been made in scoring the functional impact of individual mutations, the characteristics of the genes in which those mutations are found remain largely unexplored. For example, genes known to carry few common functional variants in healthy individuals may be judged more likely to cause certain kinds of disease than genes known to carry many such variants. Until now, however, it has not been possible to develop a quantitative assessment of how well genes tolerate functional genetic variation on a genome-wide scale. Here we describe an effort that uses sequence data from 6503 whole exome sequences made available by the NHLBI Exome Sequencing Project (ESP). Specifically, we develop an intolerance scoring system that assesses whether genes have relatively more or less functional genetic variation than expected based on the apparently neutral variation found in the gene. To illustrate the utility of this intolerance score, we show that genes responsible for Mendelian diseases are significantly more intolerant to functional genetic variation than genes that do not cause any known disease, but with striking variation in intolerance among genes causing different classes of genetic disease. We conclude by showing that use of an intolerance ranking system can aid in interpreting personal genomes and identifying pathogenic mutations.
Author Summary
This work uses empirical single nucleotide variant data from the NHLBI Exome Sequencing Project to introduce a genome-wide scoring system that ranks human genes in terms of their intolerance to standing functional genetic variation in the human population. It is often inferred that genes carrying relatively fewer or relatively more common functional variants in healthy individuals may be judged respectively more or less likely to cause certain kinds of disease. We show that this intolerance score correlates remarkably well with genes already known to cause Mendelian diseases (P<10−26). Equally striking, however, are the differences in the relationship between standing genetic variation and disease causing genes for different disease types. Considering disorder classes defined by Goh et al (2007) human disease network, we show a nearly opposite pattern for genes linked to developmental disorders and those linked to immunological disorders, with the former being preferentially caused by genes that do not tolerate functional variation and the latter caused by genes with an excess of common functional variation. We conclude by showing that use of an intolerance ranking system can facilitate interpreting personal genomes and can facilitate identifying high impact mutations through the gene in which they occur.
PMCID: PMC3749936  PMID: 23990802
13.  Adrenomedullary Response to Glucagon in Patients with Primary Sjögren’s Syndrome 
Several studies showed signs of autonomic dysfunction in patients with primary Sjögren's syndrome (pSS). Adrenomedullary function might be of importance for pSS pathogenesis by affecting salivary gland functions and modulating immune responses. The aim of the study was to evaluate the adrenomedullary hormonal system in patients with pSS.
The glucagon test (1 mg i.v.) was performed in 18 pSS patients and 13 control subjects. During the testing each patient had electrocardiographic and impedance cardiographic monitoring. Plasma epinephrine and norepinephrine were assayed by liquid chromatography with electrochemical detection after batch alumina extraction.
Baseline concentrations of epinephrine and norepinephrine were comparable between pSS and controls. Glucagon administration induced a significant increase in systolic blood pressure, diastolic blood pressure, heart rate, cardiac output (p < 0.01), stroke volume; however the changes were comparable between pSS and controls. Epinephrine levels increased (p < 0.01) in response to glucagon administration while norepinephrine concentration did not change. There was no significant difference in neurochemical responses to glucagon between pSS and controls. In conclusion, the present results suggest normal adrenomedullary function in pSS.
PMCID: PMC3378756  PMID: 22350211
Primary Sjögren’s syndrome; epinephrine; adrenal medulla; norepinephrine
14.  Genetic Determinants of Variable Metabolism Have Little Impact on the Clinical Use of Leading Antipsychotics in the CATIE study 
To evaluate systematically in real clinical settings whether functional genetic variations in drug metabolizing enzymes influence optimized doses, efficacy, and safety of antipsychotic medications.
DNA was collected from 750 patients with chronic schizophrenia treated with five antipsychotic drugs (olanzapine, quetiapine, risperidone, ziprasidone and perphenazine) as part of the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) study. Doses for each of the medicines were optimized to 1, 2, 3, or 4x units in identically-appearing capsules in a double blind design. We analyzed 25 known functional genetic variants in the major and minor metabolizing enzymes for each medication. These variants were tested for association with optimized dose and other relevant clinical outcomes.
None of the tested variants showed a nominally significant main effect in association with any of the tested phenotypes in European-Americans, African-Americans or all patients. Even after accounting for potential covariates no genetic variant was found to be associated with dosing, efficacy, overall tolerability, or tardive dyskinesia.
There are no strong associations between common functional genetic variants in drug metabolizing enzymes and dosing, safety or efficacy of leading antipsychotics, strongly suggesting merely modest effects on the use of these medicines in most patients in typical clinical settings.
PMCID: PMC3697113  PMID: 18813134
Pharmacogenetics; CYP 450; Drug Metabolizing Enzymes; Antipsychotics; Personalized Medicine
15.  Cardiovascular dysautonomia in Parkinson Disease: From pathophysiology to pathogenesis 
Neurobiology of Disease  2011;46(3):572-580.
Signs or symptoms of impaired autonomic regulation of the circulation often attend Parkinson disease (PD). This review covers biomarkers and mechanisms of autonomic cardiovascular abnormalities in PD and related alpha-synucleinopathies. The clearest clinical laboratory correlate of dysautonomia in PD is loss of myocardial noradrenergic innervation, detected by cardiac sympathetic neuroimaging. About 30–40% of PD patients have orthostatic hypotension (OH), defined as a persistent, consistent fall in systolic blood pressure of at least 20 mm Hg or diastolic blood pressure of at least 10 mm Hg within three minutes of change in position from supine to standing. Neuroimaging evidence of cardiac sympathetic denervation is universal in PD with OH (PD+OH). In PD without OH about half the patients have diffuse left ventricular myocardial sympathetic denervation, a substantial minority have partial denervation confined to the inferolateral or apical walls, and a small number have normal innervation. Among patients with partial denervation the neuronal loss invariably progresses over time, and in those with normal innervation at least some loss eventually becomes evident. Thus, cardiac sympathetic denervation in PD occurs independently of the movement disorder. PD+OH also entails extra-cardiac noradrenergic denervation, but this is not as severe as in pure autonomic failure. PD+OH patients have failure of both the parasympathetic and sympathetic components of the arterial baroreflex. OH in PD therefore seems to reflect a “triple whammy” of cardiac and extra-cardiac noradrenergic denervation and baroreflex failure. In contrast, most patients with multiple system atrophy, which can resemble PD+OH clinically, do not have evidence for cardiac or extra-cardiac noradrenergic denervation. Catecholamines in the neuronal cytoplasm are potentially toxic, via spontaneous and enzyme-catalyzed oxidation. Normally cytoplasmic catecholamines are efficiently taken up into vesicles via the vesicular monoamine transporter. The recent finding of decreased vesicular uptake in Lewy body diseases therefore suggests a pathogenetic mechanism for loss of catecholaminergic neurons in the periphery and brain.
PMCID: PMC3299874  PMID: 22094370
16.  Cerebrospinal fluid biomarkers of central catecholamine deficiency in Parkinson’s disease and other synucleinopathies 
Brain  2012;135(6):1900-1913.
Central catecholamine deficiency characterizes α-synucleinopathies such as Parkinson’s disease. We hypothesized that cerebrospinal fluid levels of neuronal metabolites of catecholamines provide neurochemical biomarkers of these disorders. To test this hypothesis we measured cerebrospinal fluid levels of catechols including dopamine, norepinephrine and their main respective neuronal metabolites dihydroxyphenylacetic acid and dihydroxyphenylglycol in Parkinson’s disease and two other synucleinopathies, multiple system atrophy and pure autonomic failure. Cerebrospinal fluid catechols were assayed in 146 subjects—108 synucleinopathy patients (34 Parkinson’s disease, 54 multiple system atrophy, 20 pure autonomic failure) and 38 controls. In 14 patients cerebrospinal fluid was obtained before or within 2 years after the onset of parkinsonism. The Parkinson’s disease, multiple system atrophy and pure autonomic failure groups all had lower cerebrospinal fluid dihydroxyphenylacetic acid [0.86 ± 0.09 (SEM), 1.00 ± 0.09, 1.32 ± 0.12 nmol/l] than controls (2.15 ± 0.18 nmol/l; P < 0.0001; P < 0.0001; P = 0.0002). Dihydroxyphenylglycol was also lower in the three synucleinopathies (8.82 ± 0.44, 7.75 ± 0.42, 5.82 ± 0.65 nmol/l) than controls (11.0 ± 0.62 nmol/l; P = 0.009, P < 0.0001, P < 0.0001). Dihydroxyphenylacetic acid was lower and dihydroxyphenylglycol higher in Parkinson’s disease than in pure autonomic failure. Dihydroxyphenylacetic acid was 100% sensitive at 89% specificity in separating patients with recent onset of parkinsonism from controls but was of no value in differentiating Parkinson’s disease from multiple system atrophy. Synucleinopathies feature cerebrospinal fluid neurochemical evidence for central dopamine and norepinephrine deficiency. Parkinson’s disease and pure autonomic failure involve differential dopaminergic versus noradrenergic lesions. Cerebrospinal fluid dihydroxyphenylacetic acid seems to provide a sensitive means to identify even early Parkinson’s disease.
PMCID: PMC3359749  PMID: 22451506
Parkinson's; dopamine; norepinephrine; DHPG; DOPAC; biomarker
17.  Novel mutation in VCP gene causes atypical amyotrophic lateral sclerosis 
Neurology  2012;79(22):2201-2208.
To identify the genetic variant that causes autosomal dominantly inherited motor neuron disease in a 4-generation Israeli-Arab family using genetic linkage and whole exome sequencing.
Genetic linkage analysis was performed in this family using Illumina single nucleotide polymorphism chips. Whole exome sequencing was then undertaken on DNA samples from 2 affected family members using an Illumina 2000 HiSeq platform in pursuit of potentially pathogenic genetic variants that comigrate with the disease in this pedigree. Variants meeting these criteria were then screened in all affected individuals.
A novel mutation (p.R191G) in the valosin-containing protein (VCP) gene was identified in the index family. Direct sequencing of the VCP gene in a panel of DNA from 274 unrelated individuals with familial amyotrophic lateral sclerosis (FALS) revealed 5 additional mutations. Among them, 2 were previously identified in pedigrees with a constellation of inclusion body myopathy with Paget disease of the bone and frontotemporal dementia (IBMPFD) and in FALS, and 2 other mutations (p.R159C and p.R155C) in IBMPFD alone. We did not detect VCP gene mutations in DNA from 178 cases of sporadic amyotrophic lateral sclerosis.
We report a novel VCP mutation identified in an amyotrophic lateral sclerosis family (p.R191G) with atypical clinical features. In our experience, VCP mutations arise in approximately 1.5% of FALS cases. Our study supports the view that motor neuron disease is part of the clinical spectrum of VCP-associated disease.
PMCID: PMC3570818  PMID: 23152587
18.  IL28B genotype is associated with differential expression of intrahepatic interferon-stimulated genes in chronic hepatitis C patients 
Hepatology (Baltimore, Md.)  2010;52(6):1888-1896.
Genetic variation in the IL28B region has been associated with sustained virological response (SVR) rates in chronic hepatitis C (CHC) patients treated with peginterferon-α and ribavirin. We hypothesized that IL28B polymorphism is associated with intrahepatic expression of interferon-stimulated genes (ISGs), known to influence treatment outcome. IL28B genotyping (rs12979860) and whole-genome RNA expression were performed using liver biopsies from 61 North American CHC patients. After correction for multiple testing (false discovery rate < 0.10), 164 transcripts were found to be differentially expressed by IL28B-type. The interferon signaling pathway was the most enriched canonical pathway differentially expressed by IL28B-type (p < 10−5), with most genes showing higher expression in livers of individuals carrying the poor-response IL28B-type. In 25 patients for which treatment response data were available, IL28B-type was associated with SVR (p = 0.0054). ISG expression was also associated with SVR; however, this was not independent of IL28B-type. Analysis of miR-122 expression in liver biopsies showed reduced miR-122 levels associated with poorer treatment outcome, independently of IL28B-type. No association was observed between IL28B-type and levels of liver IL28B or IL28A mRNA expression. IL28B protein sequence variants associated with rs12979860 were therefore investigated in vitro: no differences in ISG induction or inhibition of HCV replication were observed in Huh7.5 cells.
The good response IL28B variant was strongly associated with lower level ISG expression. The results suggest that IL28B genotype may explain the relationship between hepatic ISG expression and HCV treatment outcome, and this is independent of miR-122 expression. IL28B-type was not associated with intrahepatic IL28B mRNA expression in vivo. Further investigation of the precise molecular mechanism(s) by which IL28B genetic variation influences HCV outcomes is warranted.
PMCID: PMC3653303  PMID: 20931559
interferon lambda; hepatitis C virus; gene expression
19.  A polymorphism in the HCP5 gene associated with HLA-B*5701 does not restrict HIV-1 in vitro 
AIDS (London, England)  2010;24(1):155-157.
A single-nucleotide polymorphism (rs2395029) in the HCP5 gene associated with HLA-B*5701 is correlated with lower HIV-1 viral set point. The two allelic forms of coding region were ectopically expressed in TZM-bl cells for an effect on HIV-1 replication. No significant HIV-1 restriction was observed in the cells with infectivity assays throughout HIV-1 life cycle, suggesting that the association of HCP5 variant with viral control is likely due to HLA-B*5701-related effect or other functional variants in the haplotype or both.
PMCID: PMC3652654  PMID: 19935381
20.  Medial Mandibulotomies: Is there sufficient space in the midline to allow a mandibulotomy without compromising the dentition? 
The objective of this study was to determine the frequency of complications in median and paramedian mandibulotomies. In addition, the interdental space in the median and paramedian region was calculated.
Study design
Retrospective study.
Tertiary care center.
A retrospective chart review was performed for all cases where a mandibulotomy was performed from 2002 to 2010. 117 charts (61 paramedian and 56 median) were identified. We included data on complications, which fell in the following 2 categories: plate and dental complications. For our second objective, we evaluated 40 different patients with base of tongue or tonsillar cancer treated with intensity modulated radiation therapy (IMRT). The interdental space between the lateral incisors and the canines was electronically calculated on the digital Panorex images.
Main outcome measures
Dental and plate complications were evaluated. We also assessed interdental space.
Patient characteristics were not significantly different. The median group had significantly more dental complications (p=0.0375, RD=0.19 and 95% CI (0.0139-0.3661)). The paramedian group had significantly more plate complications (p=0.0375, RD=0.082 and 95% CI (0.0131-0.1508). The distance between the central incisors was significantly less than the distance between the lateral incisors and canines both at the crestal and apical levels (p=0.0086 and p<0.001).
There are significantly more dental complications in the median approach. There were significantly more plate complications in the paramedian group. In addition, there is significantly less space in the between the median region as compared to the paramedian region. This is the first study that documents the advantage of the paramedian approach for dental complications.
PMCID: PMC3652788  PMID: 23663661
Mandibulotomy; Complications; Median; Paramedian; Dental; Medial
21.  Genome-wide association study of interferon-related cytopenia in chronic hepatitis C patients 
Journal of hepatology  2011;56(2):313-319.
Background & Aims
Interferon-alfa (IFN)-related cytopenias are common and may be dose-limiting. We performed a genome wide association study on a well-characterized genotype 1 HCV cohort to identify genetic determinants of peginterferon-α (peg-IFN)-related thrombocytopenia, neutropenia, and leukopenia.
1604/3070 patients in the IDEAL study consented to genetic testing. Trial inclusion criteria included a platelet (Pl) count ≥80 × 109/L and an absolute neutrophil count (ANC) ≥ 1500/mm3. Samples were genotyped using the Illumina Human610-quad BeadChip. The primary analyses focused on the genetic determinants of quantitative change in cell counts (Pl, ANC, lymphocytes, monocytes, eosinophils, and basophils) at week 4 in patients >80% adherent to therapy (n = 1294).
6 SNPs on chromosome 20 were positively associated with Pl reduction (top SNP rs965469, p = 10−10). These tag SNPs are in high linkage disequilibrium with 2 functional variants in the ITPA gene, rs1127354 and rs7270101, that cause ITPase deficiency and protect against ribavirin (RBV)-induced hemolytic anemia (HA). rs1127354 and rs7270101 showed strong independent associations with Pl reduction (p = 10−12, p = 10−7) and entirely explained the genome-wide significant associations. We believe this is an example of an indirect genetic association due to a reactive thrombocytosis to RBV-induced anemia: Hb decline was inversely correlated with Pl reduction (r = −0.28, p = 10−17) and Hb change largely attenuated the association between the ITPA variants and Pl reduction in regression models. No common genetic variants were associated with pegIFN-induced neutropenia or leucopenia.
Two ITPA variants were associated with thrombocytopenia; this was largely explained by a thrombocytotic response to RBV-induced HA attenuating IFN-related thrombocytopenia. No genetic determinants of pegIFN-induced neutropenia were identified.
PMCID: PMC3634361  PMID: 21703177
GWAS; ITPA; Thrombocytopenia; Hepatitis C; Neutropenia; IL28B
22.  Modifier genetics in neuropsychiatric disease: challenges and opportunities 
Genome Biology  2012;13(3):150.
A new study focuses attention on multigenic interactions influencing the risk of autism spectrum disorders.
PMCID: PMC3439962  PMID: 22458452
23.  Mutations in the Profilin 1 Gene Cause Familial Amyotrophic Lateral Sclerosis 
Nature  2012;488(7412):499-503.
Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder resulting from motor neuron death. Approximately 10% of cases are familial (FALS), typically with a dominant inheritance mode. Despite numerous advances in recent years1-9, nearly 50% of FALS cases have unknown genetic etiology. Here we show that mutations within the profilin 1 (PFN1) gene can cause FALS. PFN1 is critical for monomeric (G)-actin conversion to filamentous (F)-actin. Exome sequencing of two large ALS families revealed different mutations within the PFN1 gene. Additional sequence analysis identified 4 mutations in 7 out of 274 FALS cases. Cells expressing PFN1 mutants contain ubiquitinated, insoluble aggregates that in many cases contain the ALS-associated protein TDP-43. PFN1 mutants also display decreased bound actin levels and can inhibit axon outgrowth. Furthermore, primary motor neurons expressing mutant PFN1 display smaller growth cones with a reduced F-/G-actin ratio. These observations further document that cytoskeletal pathway alterations contribute to ALS pathogenesis.
PMCID: PMC3575525  PMID: 22801503
24.  Variants in the ITPA Gene Protect Against Ribavirin-Induced Hemolytic Anemia in HIV/HCV-Coinfected Patients With All HCV Genotypes 
The Journal of Infectious Diseases  2011;205(3):376-383.
Background. A recent genome-wide association study reported a strong association with a single-nucleotide polymorphism (SNP) in the inosine triphosphate (ITPA) gene and hemolytic anemia in patients infected with hepatitis C virus (HCV) receiving pegylated interferon and ribavirin. We investigate these polymorphisms in a cohort of human immunodeficiency virus (HIV)/HCV–coinfected patients.
Methods. DNA was available for 161 patients with validated outcomes. We analyzed the association between the variants and week 4 hemoglobin reduction. Anemia over the course of therapy, ribavirin (RBV) dose reduction, serum RBV level, and rapid virological response (RVR) and sustained virological response (SVR) were also investigated. Using a candidate gene approach, ITPA variants rs1127354 and rs7270101 were tested using the ABI TaqMan kit. Multivariable models were used to identify predictors of anemia.
Results. A significant minority (33%) of patients were predicted to have reduced ITPase activity. The minor allele of each variant was associated with protection against week 4 anemia. In multivariable models only the genetic variants, creatinine, and zidovudine exposure remained significant. ITPase deficiency was not associated with RBV-dose reduction, RVR, or SVR.
Conclusions. This study confirms that polymorphisms in the ITPA gene are associated with protection from RBV-induced anemia in HIV/HCV-coinfected patients but not improved clinical outcomes.
PMCID: PMC3283113  PMID: 22158703
25.  Conditional Expression of Parkinson disease-related Mutant α-synuclein in the Midbrain Dopaminergic Neurons causes Progressive Neurodegeneration and Degradation of Transcription Factor Nuclear Receptor Related 1 
α-synuclein(α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson disease (PD). However, only a few studies on α-syn have been carried out in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We further systematically examined the subcellular abnormalities appeared in the mDA neurons of mutant mice, and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that over-expression of α-syn promoted a proteasome-dependent degradation of nuclear receptor related 1 protein (Nurr1); while inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.
PMCID: PMC3417246  PMID: 22764233

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