Lina A. Shehadeh

A Genomic Pathway Approach to a Complex Disease: Axon Guidance and Parkinson Disease

While major inroads have been made in identifying the genetic causes of rare Mendelian disorders, little progress has been made in the discovery of common gene variations that predispose to complex diseases. The single gene variants that have been shown to associate reproducibly with complex diseases typically have small effect sizes or attributable risks. However, the joint actions of common gene variants within pathways may play a major role in predisposing to complex diseases (the paradigm of complex genetics). The goal of this study was to determine whether polymorphism in a candidate pathway (axon guidance) predisposed to a complex disease (Parkinson disease [PD]). We mined a whole-genome association dataset and identified single nucleotide polymorphisms (SNPs) that were within axon-guidance pathway genes. We then constructed models of axon-guidance pathway SNPs that predicted three outcomes: PD susceptibility (odds ratio = 90.8, p = 4.64 × 10−38), survival free of PD (hazards ratio = 19.0, p = 5.43 × 10−48), and PD age at onset (R 2 = 0.68, p = 1.68 × 10−51). By contrast, models constructed from thousands of random selections of genomic SNPs predicted the three PD outcomes poorly. Mining of a second whole-genome association dataset and mining of an expression profiling dataset also supported a role for many axon-guidance pathway genes in PD. These findings could have important implications regarding the pathogenesis of PD. This genomic pathway approach may also offer insights into other complex diseases such as Alzheimer disease, diabetes mellitus, nicotine and alcohol dependence, and several cancers.

Age-related molecular genetic changes of murine bone marrow mesenchymal stem cells.

BackgroundMesenchymal stem cells (MSC) are pluripotent cells, present in the bone marrow and other tissues that can differentiate into cells of all germ layers and may be involved in tissue maintenance and repair in adult organisms. Because of their plasticity and accessibility these cells are also prime candidates for regenerative medicine. The contribution of stem cell aging to organismal aging is under debate and one theory is that reparative processes deteriorate as a consequence of stem cell aging and/or decrease in number. Age has been linked with changes in osteogenic and adipogenic potential of MSCs.ResultsHere we report on changes in global gene expression of cultured MSCs isolated from the bone marrow of mice at ages 2, 8, and 26-months. Microarray analyses revealed significant changes in the expression of more than 8000 genes with stage-specific changes of multiple differentiation, cell cycle and growth factor genes. Key markers of adipogenesis including lipoprotein lipase, FABP4, and Itm2a displayed age-dependent declines. Expression of the master cell cycle regulators p53 and p21 and growth factors HGF and VEGF also declined significantly at 26 months. These changes were evident despite multiple cell divisions in vitro after bone marrow isolation.ConclusionsThe results suggest that MSCs are subject to molecular genetic changes during aging that are conserved during passage in culture. These changes may affect the physiological functions and the potential of autologous MSCs for stem cell therapy.

Quantitative Control of Adaptive Cardiac Hypertrophy by Acetyltransferase p300

Background— Acetyltransferase p300 is essential for cardiac development and is thought to be involved in cardiac myocyte growth through MEF2- and GATA4-dependent transcription. However, the importance of p300 in the modulation of cardiac growth in vivo is unknown. Methods and Results— Pressure overload induced by transverse aortic coarctation, postnatal physiological growth, and human heart failure were associated with large increases in p300. Minimal transgenic overexpression of p300 (1.5- to 3.5-fold) induced striking myocyte and cardiac hypertrophy. Both mortality and cardiac mass were directly related to p300 protein dosage. Heterozygous loss of a single p300 allele reduced pressure overload–induced hypertrophy by ≈50% and rescued the hypertrophic phenotype of p300 overexpressers. Increased p300 expression had no effect on total histone deacetylase activity but was associated with proportional increases in p300 acetyltransferase activity and acetylation of the p300 substrates histone 3 and GATA-4. Remarkably, a doubling of p300 levels was associated with the de novo acetylation of MEF2. Consistent with this, genes specifically upregulated in p300 transgenic hearts were highly enriched for MEF2 binding sites. Conclusions— Small increments in p300 are necessary and sufficient to drive myocardial hypertrophy, possibly through acetylation of MEF2 and upstream of signals promoting phosphorylation or nuclear export of histone deacetylases. We propose that induction of myocardial p300 content is a primary rate-limiting event in the response to hemodynamic loading in vivo and that p300 availability drives and constrains adaptive myocardial growth. Specific reduction of p300 content or activity may diminish stress-induced hypertrophy and forestall the development of heart failure.

SRRM2, a Potential Blood Biomarker Revealing High Alternative Splicing in Parkinson's Disease

Background Parkinsons disease (PD) is a progressive neurodegenerative disorder that affects about five million people worldwide. Diagnosis remains clinical, based on phenotypic patterns. The discovery of laboratory markers that will enhance diagnostic accuracy, allow pre-clinical detection and tracking of disease progression is critically needed. These biomarkers may include transcripts with different isoforms. Methodology/Principal Findings We performed extensive analysis on 3 PD microarray experiments available through GEO and found that the RNA splicing gene SRRM2 (or SRm300), sereine/arginine repetitive matrix 2, was the only gene differentially upregulated among all the three PD experiments. SRRM2 expression was not changed in the blood of other neurological diseased patients versus the healthy controls. Using real-time PCR, we report that the shorter transcript of SRRM2 was 1.7 fold (p = 0.008) upregulated in the substantia nigra of PDs vs controls while the longer transcript was 0.4 downregulated in both the substantia nigra (p = 0.03) and amygdala (p = 0.003). To validate our results and test for the possibility of alternative splicing in PD, we performed independent microarray scans, using Affymetrix Exon_ST1 arrays, from peripheral blood of 28 individuals (17 PDs and 11 Ctrls) and found a significant upregulation of the upstream (5′) exons of SRRM2 and a downregulation of the downstream exons, causing a total of 0.7 fold down regulation (p = 0.04) of the long isoform. In addition, we report novel information about hundreds of genes with significant alternative splicing (differential exonic expression) in PD blood versus controls. Conclusions/Significance The consistent dysregulation of the RNA splicing factor SRRM2 in two different PD neuronal sources and in PD blood but not in blood of other neurologically diseased patients makes SRRM2 a strong candidate gene for PD and draws attention to the role of RNA splicing in the disease.

Beyond Parkinson disease: amyotrophic lateral sclerosis and the axon guidance pathway.

Background We recently described a genomic pathway approach to study complex diseases. We demonstrated that models constructed using single nucleotide polymorphisms (SNPs) within axon guidance pathway genes were highly predictive of Parkinson disease (PD) susceptibility, survival free of PD, and age at onset of PD within two independent whole-genome association datasets. We also demonstrated that several axon guidance pathway genes represented by SNPs within our final models were differentially expressed in PD. Methodology/Principal Findings Here we employed our genomic pathway approach to analyze data from a whole-genome association dataset of amyotrophic lateral sclerosis (ALS); and demonstrated that models constructed using SNPs within axon guidance pathway genes were highly predictive of ALS susceptibility (odds ratio = 1739.73, p = 2.92×10−60), survival free of ALS (hazards ratio = 149.80, p = 1.25×10−74), and age at onset of ALS (R2 = 0.86, p = 5.96×10−66). We also extended our analyses of a whole-genome association dataset of PD, which shared 320,202 genomic SNPs in common with the whole-genome association dataset of ALS. We compared for ALS and PD the genes represented by SNPs in the final models for susceptibility, survival free of disease, and age at onset of disease and noted that 52.2%, 37.8%, and 34.9% of the genes were shared respectively. Conclusions/Significance Our findings for the axon guidance pathway and ALS have prior biological plausibility, overlap partially with PD, and may provide important insight into the causes of these and related neurodegenerative disorders.

Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.

Expression of Lewy body protein septin 4 in postmortem brain of Parkinson's disease and control subjects.

In Parkinsons disease (PD) neuronal degeneration is associated with abnormal protein aggregation in various forms including Lewy bodies (LBs). A major component of LBs is α‐synuclein; septin 4 (SEPT4), a polymerizing GTP‐binding protein that serves as scaffold for diverse molecules has been found to colocalize with α‐synuclein in LBs. The central role of SEPT4 in the etiopathogenesis of PD has been suggested since SEPT4 also shows a physiological association with α‐synuclein and serves as a substrate for parkin. To this end, we studied the expression of septin 4 and α‐synuclein in postmortem human substantia nigra (SN) and amygdala from patients with PD and healthy controls. Twenty patients (14 men : 6 women, onset 63.0 ± 11.4 years, age 77.3 ± 7.6 years, Hoehn and Yahr 4.05/5) and 9 neurologically healthy controls (4 men/5 women, age at death 80.1 ± 8.6 years) were studied. Sporadic PD cases showed a statistically significant decrease of the fold change (FC) of SNCA (FC = 0.31, P = 0.00001) and SEPT4 (FC = 0.67, P = 0.054) gene expressions in the SN and the amygdala (SNCA: FC = 0.49, P = 0.02; SEPT4: FC = 0.32, P = 0.007) versus healthy controls. However, an increase of both proteins in PD versus control subjects was observed with immunoblotting. The semi‐quantitative protein ratio calculations revealed more than 10‐fold increases for both SEPT4 and α‐synuclein in PD versus control subjects. We present for the first time similar signal expression patterns and parallel accumulation of SEPT4 and α‐synuclein in well‐characterized postmortem PD brain. Considering the heterogeneous etiology of sporadic PD and the variability of individual human samples, SEPT4 accumulation may be regarded as one of the common pathological changes in PD and should therefore be further explored.

MicroRNAs: A new piece in the paediatric cardiovascular disease puzzle

Cardiovascular diseases in children comprise a large public health problem. The major goals of paediatric cardiologists and paediatric cardiovascular researchers are to identify the cause(s) of these diseases to improve treatment and preventive protocols. Recent studies show the involvement of microRNAs (miRs) in different aspects of heart development, function, and disease. Therefore, miR-based research in paediatric cardiovascular disorders is crucial for a better understanding of the underlying pathogenesis of the disease, and unravelling novel, efficient, preventive, and therapeutic means. The ultimate goal of such research is to secure normal cardiac development and hence decrease disabilities, improve clinical outcomes, and decrease the morbidity and mortality among children. This review focuses on the role of miRs in different paediatric cardiovascular conditions in an effort to encourage miR-based research in paediatric cardiovascular disorders.

Dynamic regulation of vascular myosin light chain (MYL9) with injury and aging.

Background Aging-associated changes in the cardiovascular system increase the risk for disease development and lead to profound alterations in vascular reactivity and stiffness. Elucidating the molecular response of arteries to injury and age will help understand the exaggerated remodeling of aging vessels. Methodology/Principal Findings We studied the gene expression profile in a model of mechanical vascular injury in the iliac artery of aging (22 months old) and young rats (4 months old). We investigated aging-related variations in gene expression at 30 min, 3 d and 7 d post injury. We found that the Myosin Light Chain gene (MYL9) was the only gene differentially expressed in the aged versus young injured arteries at all time points studied, peaking at day 3 after injury (4.6 fold upregulation (p<0.05) in the smooth muscle cell layers. We confirmed this finding on an aging aortic microarray experiment available through NCBIs GEO database. We found that Myl9 was consistently upregulated with age in healthy rat aortas. To determine the arterial localization of Myl9 with age and injury, we performed immunohistochemistry for Myl9 in rat iliac arteries and found that in healthy and injured (30 days post injury) arteries, Myl9 expression increased with age in the endothelial layers. Conclusions/Significance The consistent upregulation of the myosin light chain protein (Myl9) with age and injury in arterial tissue draws attention to the increased vascular permeability and to the age-caused predisposition to arterial constriction after balloon angioplasty.

Is the G2019S LRRK2 mutation common in all southern European populations

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene, especially the G2019S mutation, have been identified as a common cause of Parkinsons disease in southern European and other Mediterranean populations (Iberians, Ashkenazi Jews and North African Arabs). Owing to the geographic and historic vicinity of Greece with areas of high prevalence of LRRK2 mutations we studied the frequency of the G2019S mutation in a well characterized cohort of familial and sporadic Parkinsons disease patients of Greek origin from mainland Greece. The prevalence of the LRRK2 R1441C mutation and the G2385R Asian polymorphism was also determined. We identified no patients with any of the studied mutations/polymorphisms. Very low prevalence of the LRRK2 G2019S mutation has been reported in other southern European populations. LRRK2 mutations appear to be limited in certain populations and differing ancestry and founder effects may explain the reported variability. Accurate estimations of the frequency and penetrance of different LRRK2 mutations are essential for correct and cost-efficient use of genetic testing and proper genetic counseling of patients with Parkinsons disease.