Michelle Apperson

Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood

BackgroundGene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization.MethodsWhole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT), 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS) and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms.ResultsReference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder).ConclusionThe reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

Gene Expression in Peripheral Blood Differs after Cardioembolic Compared with Large-Vessel Atherosclerotic Stroke: Biomarkers for the Etiology of Ischemic Stroke

There are no biomarkers that differentiate cardioembolic from large-vessel atherosclerotic stroke, although the treatments differ for each and ~30% of strokes and transient ischemic attacks have undetermined etiologies using current clinical criteria. We aimed to define gene expression profiles in blood that differentiate cardioembolic from large-vessel atherosclerotic stroke. Peripheral blood samples were obtained from healthy controls and acute ischemic stroke patients (< 3, 5, and 24 h). RNA was purified, labeled, and applied to Affymetrix Human U133 Plus 2.0 Arrays. Expression profiles in the blood of cardioembolic stroke patients are distinctive from those of large-vessel atherosclerotic stroke patients. Seventy-seven genes differ at least 1.5-fold between them, and a minimum number of 23 genes differentiate the two types of stroke with at least 95.2% specificity and 95.2% sensitivity for each. Genes regulated in large-vessel atherosclerotic stroke are expressed in platelets and monocytes and modulate hemostasis. Genes regulated in cardioembolic stroke are expressed in neutrophils and modulate immune responses to infectious stimuli. This new method can be used to predict whether a stroke of unknown etiology was because of cardioembolism or large-vessel atherosclerosis that would lead to different therapy. These results have wide ranging implications for similar disorders.

Levetiracetam monotherapy for elderly patients with epilepsy

We retrospectively identified 14 elderly patients with a history of partial seizures who received levetiracetam (LEV) monotherapy. Patients began LEV either as first line therapy (n=5) or were converted to LEV monotherapy (n=9) after failing prior antiepileptic medications (AEDs). Thirteen patients continued on LEV monotherapy for at least 6 months. One patient was lost to follow-up. Eight patients (61.5%) became seizure free. Four patients who began LEV as a first line therapy became seizure free, whereas the remaining four patients who converted to LEV after they failed their previous AEDs became seizure free. Four patients (30.7%) had more than a 50% seizure reduction of seizures. Only one patient had no significant change in seizure frequency after started on LEV. The total dosages used to control seizures were 500-3000 mg/day, (mean 1839.2 mg/day). LEV monotherapy can be effective and well tolerated in this group of patients. A prospective, larger, double blind monotherapy study is needed to confirm this finding.

FMR1 premutation in females diagnosed with multiple sclerosis.

Fragile X associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that affects individuals with premutation alleles of the fragile X mental retardation ( FMR1 ) gene. The clinical features of FXTAS include intention tremor, ataxia, parkinsonism, peripheral neuropathy, autonomic dysfunction and cognitive impairment.1 Such symptoms are accompanied by characteristic MRI focal areas of increased T2 signal in the middle cerebellar peduncles (MCP).1 In this report, we describe two female patients with FXTAS who were initially diagnosed with multiple sclerosis (MS) and treated accordingly but who also met diagnostic criteria for FXTAS. Their clinical features and MRI appearance are compared with other female FXTAS cases previously reported without a diagnosis of MS. A recent report of a female who died of MS and who also had FXTAS inclusions suggests that these two disorders can occur together.2 ### Patient No 1 Case 1 is a 48-year-old female premutation carrier with a history of MS which began at the age of 33 years. Initially, her handwriting quality declined, and she subsequently became ataxic. A combination of weakness, clumsiness and ataxia forced her to use a wheelchair beginning in her late thirties. During that same period, she was diagnosed with MS following a CSF study which showed elevated IgG (9.8 mg/dl; reference range 0.5–6.1 mg/dl) without oligoclonal bands or myelin basic protein. Her course was consistent with relapsing–remitting MS with early secondary progression, and she was treated …

Genomic Profiles of Stroke in Blood

These studies show that gene expression changes in most patients by 2 to 3 hours after ischemic stroke, and in all patients studied by 24 hours.

Psychogenic nonepileptic seizures are associated with an increased risk of obesity

Psychogenic nonepileptic seizures (NES) are somatic manifestations of psychological distress. There is some evidence that weight problems are more common in patients with psychiatric illness. We have observed that patients admitted for video-EEG monitoring who we diagnosed with NES commonly have a larger body habitus than patients with epilepsy. The goal of this study was to test our hypothesis that there was a significant difference in body mass index (BMI) in patients with nonepileptic seizures compared with their epileptic counterparts. We compared the BMIs of 46 NES patients and 46 age- and gender-matched epileptic controls and found that the NES patients had significantly higher BMIs (30.5 vs 26.1, P=0.006) than controls. This remained true after controlling for weight-gain properties of antiepileptic drugs. These results are compared with the prevalence of overweight and obesity in the general population. Possible explanations of the findings and limitations of the study are discussed.

Differences in exon expression and alternatively spliced genes in blood of multiple sclerosis compared to healthy control subjects

Using whole genome exon microarrays 120 exons were differentially expressed between medication-free multiple sclerosis (MS) subjects in remission and healthy control subjects (HS) (p<0.001, fold change>|1.2|). These exons differentiated MS from HS using cluster analyses, principal components analyses (PCAs) and cross-validation. In addition, 340 genes (transcripts) were predicted to be alternatively spliced in MS compared to HS. These findings may provide insight into the pathophysiology of MS and potentially provide prognostic and diagnostic biomarkers. However, given that multiple comparisons were performed on a very small sample, these preliminary findings require confirmation using a much larger independent cohort.

Genomics of brain and blood: Progress and pitfalls

Summary:  Gene expression profiles in brain and blood of animals and humans can be useful for diagnosis, prognosis, and treatment of epilepsy. This article reviews recent progress and prospects for the future.

Gene expression in blood of subjects with Duchenne muscular dystrophy

The objective of this study was to examine RNA expression in blood of subjects with Duchenne muscular dystrophy (DMD). Whole blood was collected into PAX gene tubes and RNA was isolated for 3- to 20-year-old males with DMD (n = 34) and for age- and gender-matched normal healthy controls (n = 21). DMD was confirmed by genetic testing in all subjects. RNA expression was measured on Affymetrix whole-genome human U133 Plus 2.0 GeneChips. Using a Benjamini–Hochberg false discovery rate of 0.05 to correct for multiple comparisons, an unpaired t test for DMD versus controls yielded 10,763 regulated probes with no fold change cutoff, 1,467 probes with >|1.5|-fold change, 191 probes with >|2.0|-fold change, and 59 probes with a >|2.5|-fold change. These genes (probes) separated DMD from controls using cluster analyses. Almost all of the genes regulated in peripheral blood were different from the genes reported to be regulated in diseased muscle of subjects with DMD. It is proposed that the genes regulated in blood of subjects with Duchenne muscular dystrophy are indicative, at least in part, of the immune response to the diseased DMD muscle. The regulated genes might be used to monitor therapy or provide novel targets for immune-directed therapy for DMD.

Genome wide differences of gene expression associated with HLA-DRB1 genotype in multiple sclerosis: a pilot study.

Using two microarray platforms, we identify HLA-DRB5 as the most highly expressed gene in MS compared to healthy subjects. As expected, HLA-DRB5 expression was associated with the HLA-DRB1*1501 MS susceptibility allele. Besides HLA-DRB5, there were 1219 differentially expressed exons (p<0.01, |fold change (FC)|>1.2) that differed between HLA-DRB1*1501 Positive multiple sclerosis subjects (MSP) compared to HLA-DRB1*1501 negative multiple sclerosis subjects (MSN). Analysis of the regulated genes revealed significantly different immune signaling pathways including IL-4 and IL-17 in these two MS genotypes. Different risk alleles appear to be associated with different patterns of gene expression that may reflect differences in pathophysiology of these two MS subtypes. These preliminary data will need to be confirmed in future studies.