PRKN (arkin RBR E3 ubiquitin protein ligase )

PARK2 (also known as Parkin RBR E3 ubiquitin protein ligase) is one of the largest genes in our genome. It undergoes an extensive alternative splicing both at transcript and protein level, producing multiple transcript variants and distinct protein isoforms. The precise function of PARK2 is still not clear; however, the encoded protein is a component of a multiprotein E3 ubiquitin ligase complex that mediates the targeting of substrates for proteasomal degradation. Mutations in this gene cause Parkinson disease and autosomal recessive juvenile Parkinson disease. Further molecular defects have been linked to other human malignancies. Here, we review some major data on PARK2, concerning the genetic structure, the transcription regulation, the encoded protein and functions, and its implication in human disease

Genetics of Parkinson’s Disease: The Role of Copy Number Variations

Parkinson’s disease (PD), the second most common progressive neurodegenerative disorder, was long believed to be a non-genetic sporadic origin syndrome. The identification of distinct genetic loci responsible for rare Mendelian forms of PD has represented a revolutionary breakthrough, allowing to discover novel mechanisms underlying this debilitating still incurable condition. Along with single-nucleotide polymorphisms (SNPs), other kinds of DNA molecular defects have emerged as significant disease-causing mutations, including large chromosomic structural rearrangements and copy number variations (CNVs). Due to their size variability and to the different sensitivity and resolution of detection methodologies, CNVs constitute a particular challenge in genetic studies and the pathogenetic or susceptibility impact of specific CNVs on PD is currently under debate. In this chapter, we will review the current literature and bioinformatic data describing the involvement of CNVs on PD pathobiology. We will discuss the recently highlighted role of PARK2 heterozygous CNVs, the possible common founder effects of PD gene rearrangements and the importance to map genetic breakpoints. We will also add a summary about the current available molecular methods and bioinformatics web resources to detect and interpret CNVs. Assessing the global genome-wide burden of large CNVs and elucidating the role of de novo rare structural variants on PD may reveal new candidate genes and consequently ameliorate diagnosis and counselling of mutations carriers

A Diagnostic Gene-Expression Signature in Fibroblasts of Amyotrophic Lateral Sclerosis

myotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease with limited treatment options. Diagnosis can be difficult due to the heterogeneity and non-specific nature of the initial symptoms, resulting in delays that compromise prompt access to effective therapeutic strategies. Transcriptome profiling of patient-derived peripheral cells represents a valuable benchmark in overcoming such challenges, providing the opportunity to identify molecular diagnostic signatures. In this study, we characterized transcriptome changes in skin fibroblasts of sporadic ALS patients (sALS) and controls and evaluated their utility as a molecular classifier for ALS diagnosis. Our analysis identified 277 differentially expressed transcripts predominantly involved in transcriptional regulation, synaptic transmission, and the inflammatory response. A support vector machine classifier based on this 277-gene signature was developed to discriminate patients with sALS from controls, showing significant predictive power in both the discovery dataset and in six independent publicly available gene expression datasets obtained from different sALS tissue/cell samples. Taken together, our findings support the utility of transcriptional signatures in peripheral cells as valuable biomarkers for the diagnosis of ALS

Expression of Parkin isoforms in human lymphomonocyte

Parkin (PARK2) is one of the largest gene in the human genome. Its mutations cause a form of autosomal recessive juvenile-onset of Parkinson disease (ARJPD) (1). To date, a repertoire of 21 parkin alternative splice variants has been identified. In the past, the role of the full-length parkin protein was extensively investigated and assessed also in human blood samples (2, 3). In contrast, less attention has been put on the other isoforms. In the present study, we investigated for the first time, the expression profile of parkin isoforms in three lymphomonocyte (LMN) subpopulations: T lymphocyte (CD2+), monocyte (CD14+) and B lymphocyte (CD19+). The expression of H1/H5 and H6 isoforms has been observed in total LMN homogenate, whereas H20 and H1/H5 variants were detected in all three LMN subpopulations by western blot analysis. The cellular distribution of parkin isoforms has been evaluated by immunofluorescence analysis. Although parkin is predominantly expressed in the cytoplasm, immunoreactivity has also been detected at nuclear and perinuclear level. Our data suggest that, the discovery of a specific expression profile of these isoforms into LMN of ARJP patients might allow developing new diagnostic tools for this neurodegenerative disease

Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

Friedreich ataxia is a multi-system autosomal recessive inherited disorder primarily caused by homozygous GAA repeat expansion mutations within intron 1 of the frataxin gene. The resulting deficiency of frataxin protein leads to progressive mitochondrial dysfunction, oxidative stress, and cell death, with the main affected sites being the large sensory neurons of the dorsal root ganglia and the dentate nucleus of the cerebellum. The GAA repeat expansions may be pure (GAA)n in sequence or may be interrupted with regions of non-GAA sequence. To our knowledge, there has been no large-scale study of FRDA patient DNA samples to determine the frequency of large interruptions in GAA repeat expansions. Therefore, we have investigated a panel of 245 Friedreich ataxia patient and carrier DNA samples using GAA repeat PCR amplification and MboII restriction enzyme digestion. We demonstrate that the vast majority (97.8%) of Friedreich ataxia GAA repeat expansion samples do not contain significant sequence changes that would result in abnormal MboII digestion profiles, indicating that they are primarily pure GAA repeats. These results show for the first time that large interruptions in the GAA repeats are very rare

Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

Friedreich ataxia is a multi-system autosomal recessive inherited disorder primarily caused by homozygous GAA repeat expansion mutations within intron 1 of the frataxin gene. The resulting deficiency of frataxin protein leads to progressive mitochondrial dysfunction, oxidative stress, and cell death, with the main affected sites being the large sensory neurons of the dorsal root ganglia and the dentate nucleus of the cerebellum. The GAA repeat expansions may be pure (GAA)n in sequence or may be interrupted with regions of non-GAA sequence. To our knowledge, there has been no large-scale study of FRDA patient DNA samples to determine the frequency of large interruptions in GAA repeat expansions. Therefore, we have investigated a panel of 245 Friedreich ataxia patient and carrier DNA samples using GAA repeat PCR amplification and MboII restriction enzyme digestion. We demonstrate that the vast majority (97.8%) of Friedreich ataxia GAA repeat expansion samples do not contain significant sequence changes that would result in abnormal MboII digestion profiles, indicating that they are primarily pure GAA repeats. These results show for the first time that large interruptions in the GAA repeats are very rare

A Comprehensive, Targeted NGS Approach to Assessing Molecular Diagnosis of Lysosomal Storage Diseases

With over 60 different disorders and a combined incidence occurring in 1:5000–7000 live births, lysosomal storage diseases (LSDs) represent a major public health problem and constitute an enormous burden for affected individuals and their families. Several reasons make the diagnosis of LSDs an arduous task for clinicians, including the phenotype and penetrance variability, the shared signs and symptoms, and the uncertainties related to biochemical enzymatic assay results. Developing a powerful diagnostic tool based on next generation sequencing (NGS) technology may help reduce the delayed diagnostic process for these families, leading to better outcomes for current therapies and providing the basis for more appropriate genetic counseling. Herein, we employed a targeted NGS-based panel to scan the coding regions of 65 LSD-causative genes. A reference group sample (n = 26) with previously known genetic mutations was used to test and validate the entire workflow. Our approach demonstrated elevated analytical accuracy, sensitivity, and specificity. We believe the adoption of comprehensive targeted sequencing strategies into a routine diagnostic route may accelerate both the identification and management of LSDs with overlapping clinical profiles, producing a significant reduction in delayed diagnostic response with beneficial results in the treatment outcome

Design and Validation of a Custom NGS Panel Targeting a Set of Lysosomal Storage Diseases Candidate for NBS Applications

Lysosomal storage diseases (LSDs) are a heterogeneous group of approximately 70 monogenic metabolic disorders whose diagnosis represents an arduous challenge for clinicians due to their variability in phenotype penetrance, clinical manifestations, and high allelic heterogeneity. In recent years, the approval of disease-specific therapies and the rapid emergence of novel rapid diagnostic methods has opened, for a set of selected LSDs, the possibility for inclusion in extensive national newborn screening (NBS) programs. Herein, we evaluated the clinical utility and diagnostic validity of a targeted next-generation sequencing (tNGS) panel (called NBS_LSDs), designed ad hoc to scan the coding regions of six genes (GBA, GAA, SMPD1, IDUA1, GLA, GALC) relevant for a group of LSDs candidate for inclusion in national NBS programs (MPSI, Pompe, Fabry, Krabbe, Niemann Pick A-B and Gaucher diseases). A standard group of 15 samples with previously known genetic mutations was used to test and validate the entire flowchart. Analytical accuracy, sensitivity, and specificity, as well as turnaround time and costs, were assessed. Results showed that the Ion AmpliSeq and Ion Chef System-based high-throughput NBS_LSDs tNGS panel is a fast, accurate, and cost-effective process. The introduction of this technology into routine NBS procedures as a second-tier test along with primary biochemical assays will allow facilitating the identification and management of selected LSDs and reducing diagnostic delay

EXONIC VARIATION IN PARKINSON'S DISEASE

Parkinson's disease (PD) is one of the most common movement disorders worldwide, characterized by a profound and selective loss of dopaminergic neurons in the substantia nigra pars compacta. Treatments aimed at compensating dopamine deficit can alleviate the major motor symptoms and enhance the patients quality of life, but finally are not able to halt or slow down disease progression. Therefore, there is an urgent need to better understand the molecular mechanisms underlying the physiopathology of PD and to identify new biomarkers and new therapeutic targets. The hypothesis addressed in this PhD thesis aims to decipher the structural variability of exonic regions in PD-linked genes and in their relative mRNA transcripts, in order to investigate if these perturbations have some effects on PD pathogenesis. Two major cellular events able to trigger exonic variations in both DNA and mRNA molecules will be examined: copy number variations and alternative splicing. Both mechanisms are well known to play a crucial role in PD onset and can modulate disease severity. An improved comprehension of exonic variability at both genomic and transcriptomic level may prompt new insights to understand the missing heritability and the variety of phenotypic outcomes in PD patients