Hunter Syndrome (Mucopolysaccharidosis type II, MPS II) is a rare inherited metabolic disease due to an extremely reduced or total absent activity of the lysosomal enzyme iduronate 2-sulfatase (IDS), involved in the degradation of the mucopolysaccharides heparan- and dermatan-sulphate. This causes a progressive pathologic accumulation of the two macromolecules within cell lysosomes and in the extracellular matrix of most tissues and organs, leading to their general malfunctioning and finally to death. In fact, due to the housekeeping nature of IDS, most of the organ systems are involved in the pathology, including the central nervous system in the severe forms of the disease.
MPS II belongs to the group of Mucopolysaccharidoses (MPSs), a cluster of pathologies characterized by accumulation of mucopolysaccahrides (or glycosaminoglycans, GAG). They, in turn, represent a subgroup of the wider class of the Lysosmal Storage Disorders (LSDs), about fifty pathological conditions characterized by the progressive endo- and extra-cellular overstorage of several types of undegraded macromolecules.
LSDs, for long time poorly considered by the medical-scientific community, have received in the past few years an increasing attention due to their elevated overall incidence, up to 1:1500-1:7000 live newborns, dependently on the population analyzed. Although the enzyme or protein defect underlying each of these pathologies is known, almost unknown remains the complexity of the biochemical pathways involved or altered in the lysosomal storage in general, or in specific type of storage. Studies conducted in the last decade have separately highlighted alterations of signalling proteins, intracellular calcium homeostasis, oxidative stress, autophagy, intracellular trafficking, lipid biosynthesis and iron metabolism. However, no systematic and complete studies have been so far conducted for the analysis of the whole pathologic scenario. This would help to acquire a general overview of the lysosomal storage and would also help in defining new, potential therapeutic targets and/or biomarkers useful in the diagnosis, prognosis, progression of LSDs as well as in the evaluation of efficacy of the therapeutic strategies applied. Moreover, since LSDs share several pathological signs and symptoms, it appears evident that a deep analysis of some of them could be of great help in the understanding of others.
For the first time, this project evaluated, by an high throughput technology, the whole transcriptome profile of LSD cells by comparing skin fibroblasts obtained from Hunter patients and healthy controls, thus allowing a deep analysis of MPS II pathogenesis. The study, conducted by total RNA sequencing, was performed by using the SOLiD technology. Results have shown alterations in: 1) basic cellular processes as cell cycle, apoptosis, intercellular communication; 2) metabolic processes as proteoglycan metabolism, synthesis of lipids, aminoacids and nucleotides; 3) response to stimuli as oxidative stress, insulin, cytokines; 4) alteration of the developmental processes.
From the therapeutic point of view, as for MPS II the major treatment strategy used in the last 5-6 years is represented by the Enzyme Replacement Therapy (ERT), consisting in the weekly systemic administration of the active form of the enzyme, which is missing in the patients. Clinical monitoring of the patients under treatment, organized since 2005 by Shire HGT, has shown, among other results, an important subjectivity in the efficacy of ERT, as expected for a pathology presenting several degrees of severity and a high number of different mutations. Despite this, ERT is administered to all patients following the same therapeutic protocol. Therefore, it becomes necessary to perform a deep clinical and molecular evaluation to identify potential candidate biomarkers of efficacy allowing an adequate follow-up of the patients under ERT; this would allow the set up of a personalized therapeutic protocol.
Starting from these considerations, in this project an in vitro evaluation of ERT has been performed in Hunter primary fibroblasts treated with the therapeutic IDS enzyme and collected 24 and 144 h post-treatment. Their transcriptional profile has been studied to characterize the early cellular response to the enzyme supply. Such analysis allowed to highlight 20 candidate biomarkers of therapeutic efficacy. Some of these have been afterwards evaluated by using Real Time PCR, in blood samples obtained from Hunter patients under ERT. Finally, a correlation analysis was performed between clinical parameter obtained by the follow-up of the Hunter population and the gene expression profile of each gene. Such analysis has shown a good correlation for 8 pairs of gene/parameter evaluated. In particular, correlations were found for hearing impairment, seizures, hepatomegaly, splenomegaly, and other clinical parameters, with at least one gene. The analysis of the other candidate genes isolated from transcriptome analysis might indentify other potential biomarker
