Genetic analysis of Parkinson's disease using Next-generation sequencing

Neurological diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Epilepsy and Multiple Sclerosis are included in the Global burden of disease study as these disorders have a high impact on public health. Lack of effective treatment has motivated the researchers to perform early diagnostics, by identifying new gene mutations, which can improve the therapies. The aim of this thesis was a genetic analysis of PD using next-generation sequencing data. In this thesis, whole genome sequencing (WGS) and whole exome sequencing (WES) using DNA from familial PD patients and healthy individuals was performed in order to identify the PD causal genes. A large repository of sporadic PD WES data and a genotyping array was used to replicate our findings. The PD patients from Germany were stratified for clinical trials on the basis of mitochondrial endo-phenotype by performing risk profiling of associated Single Nucleotide Polymorphisms (SNPs) using exome genotyping array. The sporadic PD WES and genotyping array data from International Parkinson’s disease Genomics Consortium was used to perform association tests, to determine the burden of rare variants in candidate genes of interest. Furthermore, mRNA sequencing of all the genes under the PD GWAS loci after knockdown with short hairpin RNAs was performed, to identify the actual genes contributing to PD risk and the novel pathways involved in PD. Finally, an epistatic interaction of a Mendelian PD gene and associated locus was performed to understand the joint contribution to PD risk. Taking everything into account, we identified pathogenic variants in known and some novel genes causing PD in families. On the basis of risk profiling some of the German PD patients will undergo clinical trials with coenzyme Q10 and vitamin K2. The association tests using sporadic PD data helped to identify some novel genes significantly associated with PD risk. The knockdown experiments facilitated the identification of genes contributing to PD risk in some of the PD GWAS loci

PLA2G6 Mutations Related to Distinct Phenotypes: A New Case with Early-onset Parkinsonism

Background: PLA2G6-associated neurodegeneration (PLAN) is a recessive neurodegenerative disorder characterized by three distinct phenotypes: infantile neuroaxonal dystrophy (INAD), atypical neuroaxonal dystrophy (atypical NAD), and PLA2G6-related dystonia–parkinsonism. Methods: A consanguineous index case from Turkey was diagnosed with early-onset Parkinsonism at the Istanbul Faculty of Medicine. She and her unaffected brother were subjected to whole-genome sequencing. Results: In this report, we describe a 33-year-old index case with parental consanguinity and early-onset Parkinsonism. Whole-genome sequencing of this individual revealed that a homozygous p.R747W mutation in PLA2G6 segregates with the disease in this family Discussion: This result supports the importance of prioritizing this gene in mutational analysis of autosomal recessive Parkinsonism, and confirms the clinical heterogeneity of PLAN

Single-cell analysis of patient-derived PDAC organoids reveals cell state heterogeneity and a conserved developmental hierarchy

Pancreatic tumors are frequently divided into basal and classical subtypes. Here, the authors use single cell sequencing to investigate organoids derived from pancreatic cancer tissue and find a hierarchy of distinct cell states, and classical and basal cells existing within the same tumor. Pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer mortality by 2030. Bulk transcriptomic analyses have distinguished 'classical' from 'basal-like' tumors with more aggressive clinical behavior. We derive PDAC organoids from 18 primary tumors and two matched liver metastases, and show that 'classical' and 'basal-like' cells coexist in individual organoids. By single-cell transcriptome analysis of PDAC organoids and primary PDAC, we identify distinct tumor cell states shared across patients, including a cycling progenitor cell state and a differentiated secretory state. Cell states are connected by a differentiation hierarchy, with 'classical' cells concentrated at the endpoint. In an imaging-based drug screen, expression of 'classical' subtype genes correlates with better drug response. Our results thus uncover a functional hierarchy of PDAC cell states linked to transcriptional tumor subtypes, and support the use of PDAC organoids as a clinically relevant model for in vitro studies of tumor heterogeneity

Lack of evidence for a role of genetic variation in TMEM230 in the risk for Parkinson's disease in the Caucasian population

Mutations in . TMEM230 have recently been associated to Parkinson's disease (PD). To further understand the role of this gene in the Caucasian population, we interrogated our large repository of next generation sequencing data from unrelated PD cases and controls, as well as multiplex families with autosomal dominant PD. We identified 2 heterozygous missense variants in 2 unrelated PD cases and not in our control database (p.Y106H and p.I162V), and a heterozygous missense variant in 2 PD cases from the same family (p.A163T). However, data presented herein is not sufficient to support the role of any of these variants in PD pathology. A series of unified sequence kernel association tests also failed to show a cumulative effect of rare variation in this gene on the risk of PD in the general Caucasian population. Further evaluation of genetic data from different populations is needed to understand the genetic role of . TMEM230 in PD etiology

Regulatory sites for splicing in human basal ganglia are enriched for disease-relevant information

Genome-wide association studies have generated an increasing number of common genetic variants associated with neurological and psychiatric disease risk. An improved understanding of the genetic control of gene expression in human brain is vital considering this is the likely modus operandum for many causal variants. However, human brain sampling complexities limit the explanatory power of brain-related expression quantitative trait loci (eQTL) and allele-specific expression (ASE) signals. We address this, using paired genomic and transcriptomic data from putamen and substantia nigra from 117 human brains, interrogating regulation at different RNA processing stages and uncovering novel transcripts. We identify disease-relevant regulatory loci, find that splicing eQTLs are enriched for regulatory information of neuron-specific genes, that ASEs provide cell-specific regulatory information with evidence for cellular specificity, and that incomplete annotation of the brain transcriptome limits interpretation of risk loci for neuropsychiatric disease. This resource of regulatory data is accessible through our web server, http://braineacv2.inf.um.es/

Subclavian Steal Syndrome (SSS)- A Rare Phenomenon

Subclavian Steal Syndrome (SSS) occurs when the first part of the subclavian artery is stenosed, proximal to the origin of vertebral artery. The first part of subclavian artery usually supplies the arm. Whenever the pressure is put in arms, flow reversal from the vertebral artery to the arms occurs leading to vertebrobasilar insufficiency (steals flow from brain). Atherosclerosis is the usual culprit so these patients are also prone to coronary artery disease and stroke

MicroRNAs Distinguish Cytogenetic Subgroups in Pediatric AML and Contribute to Complex Regulatory Networks in AML-Relevant Pathways

<div><h3>Background</h3><p>The role of microRNAs (miRNAs), important post-transcriptional regulators, in the pathogenesis of acute myeloid leukemia (AML) is just emerging and has been mainly studied in adults. First studies in children investigate single selected miRNAs, however, a comprehensive overview of miRNA expression and function in children and young adults is missing so far.</p> <h3>Methodology/Principal Findings</h3><p>We here globally identified differentially expressed miRNAs between AML subtypes in a survey of 102 children and adolescent. Pediatric samples with core-binding factor AML and promyelocytic leukemia could be distinguished from each other and from MLL-rearranged AML subtypes by differentially expressed miRNAs including miR-126, -146a, -181a/b, -100, and miR-125b. Subsequently, we established a newly devised immunoprecipitation assay followed by rapid microarray detection for the isolation of Argonaute proteins, the hallmark of miRNA targeting complexes, from cell line models resembling core-binding factor and promyelocytic leukemia. Applying this method, we were able to identify Ago-associated miRNAs and their targeted mRNAs.</p> <h3>Conclusions/Significance</h3><p>miRNAs as well as their mRNA-targets showed binding preferences for the different Argonaute proteins in a cell context-dependent manner. Bioinformatically-derived pathway analysis suggested a concerted action of all four Argonaute complexes in the regulation of AML-relevant pathways. For the first time, to our knowledge, a complete AML data set resulting from carefully devised biochemical isolation experiments and analysis of Ago-associated miRNAs and their target-mRNAs is now available.</p> </div