Analysis of Hunter Syndrome by RNA-Sequencing

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

QueryOR: a comprehensive web platform for genetic variant analysis and prioritization

Background: Whole genome and exome sequencing are contributing to the extraordinary progress in the study of human genetic variants. In this fast developing field, appropriate and easily accessible tools are required to facilitate data analysis. Results: Here we describe QueryOR, a web platform suitable for searching among known candidate genes as well as for finding novel gene-disease associations. QueryOR combines several innovative features that make it comprehensive, flexible and easy to use. Instead of being designed on specific datasets, it works on a general XML schema specifying formats and criteria of each data source. Thanks to this flexibility, new criteria can be easily added for future expansion. Currently, up to 70 user-selectable criteria are available, including a wide range of gene and variant features. Moreover, rather than progressively discarding variants taking one criterion at a time, the prioritization is achieved by a global positive selection process that considers all transcript isoforms, thus producing reliable results. QueryOR is easy to use and its intuitive interface allows to handle different kinds of inheritance as well as features related to sharing variants in different patients. QueryOR is suitable for investigating single patients, families or cohorts. Conclusions: QueryOR is a comprehensive and flexible web platform eligible for an easy user-driven variant prioritization. It is freely available for academic institutions at http://queryor.cribi.unipd.it/

Setup and Validation of a Targeted Next-Generation Sequencing Approach for the Diagnosis of Lysosomal Storage Disorders.

Lysosomal storage disorders (LSDs) are monogenic diseases, due to accumulation of specific undegraded substrates into lysosomes. LSD diagnosis could take several years because of both poor knowledge of these diseases and shared clinical features. The diagnostic approach includes clinical evaluations, biochemical tests, and genetic analysis of the suspected gene. In this study, we evaluated an LSD targeted sequencing panel as a tool capable to potentially reverse this classic diagnostic route. The panel includes 50 LSD genes and 230 intronic sequences conserved among 33 placental mammals. For the validation phase, 56 positive controls, 13 biochemically diagnosed patients, and nine undiagnosed patients were analyzed. Disease-causing variants were identified in 66% of the positive control alleles and in 62% of the biochemically diagnosed patients. Three undiagnosed patients were diagnosed. Eight patients undiagnosed by the panel were analyzed by whole exome sequencing: for two of them, the disease-causing variants were identified. Five patients, undiagnosed by both panel and exome analyses, were investigated through array comparative genomic hybridization: one of them was diagnosed. Conserved intronic fragment analysis, performed in cases unresolved by the first-level analysis, evidenced no candidate intronic variants. Targeted sequencing has low sequencing costs and short sequencing time. However, a coverage >60× to 80× must be ensured and/or Sanger validation should be performed. Moreover, it must be supported by a thorough clinical phenotyping

Molecular basis of mucopolysaccharidosis IVA (Morquio A syndrome) : a review and classification of GALNS gene variants and reporting of 68 novel variants

Mucopolysaccharidosis IVA (MPS IVA, Morquio A syndrome) is a rare autosomal recessive lysosomal storage disorder caused by mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. We collected, analyzed, and uniformly summarized all published GALNS gene variants, thus updating the previous mutation review (published in 2014). In addition, new variants were communicated by seven reference laboratories in Europe, the Middle East, Latin America, Asia, and the United States. All data were analyzed to determine common alleles, geographic distribution, level of homozygosity, and genotype-phenotype correlation. Moreover, variants were classified according to their pathogenicity as suggested by ACMG. Including those previously published, we assembled 446 unique variants, among which 68 were novel, from 1190 subjects (including newborn screening positive subjects). Variants' distribution was missense (65.0%), followed by nonsense (8.1%), splicing (7.2%), small frameshift deletions(del)/insertions(ins) (7.0%), intronic (4.0%), and large del/ins and complex rearrangements (3.8%). Half (50.4%) of the subjects were homozygous, 37.1% were compound heterozygous, and 10.7% had only one variant detected. The novel variants underwent in silico analysis to evaluate their pathogenicity. All variants were submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) to make them publicly available. Mutation updates are essential for the correct molecular diagnoses, genetic counseling, prenatal and preimplantation diagnosis, and disease management

Pandemic Phase-Adjusted Analysis of COVID-19 Outcomes Reveals Reduced Intrinsic Vulnerability and Substantial Vaccine Protection From Severe Acute Respiratory Syndrome Coronavirus 2 in Patients With Breast Cancer

PURPOSE Although representing the majority of newly diagnosed cancers, patients with breast cancer appear less vulnerable to COVID-19 mortality compared with other malignancies. In the absence of patients on active cancer therapy included in vaccination trials, a contemporary real-world evaluation of outcomes during the various pandemic phases, as well as of the impact of vaccination, is needed to better inform clinical practice. METHODS We compared COVID-19 morbidity and mortality among patients with breast cancer across prevaccination (February 27, 2020-November 30, 2020), Alpha-Delta (December 1, 2020-December 14, 2021), and Omicron (December 15, 2021-January 31, 2022) phases using OnCovid registry participants (ClinicalTrials.gov identifier: NCT04393974). Twenty-eight-day case fatality rate (CFR28) and COVID-19 severity were compared in unvaccinated versus double-dosed/boosted patients (vaccinated) with inverse probability of treatment weighting models adjusted for country of origin, age, number of comorbidities, tumor stage, and receipt of systemic anticancer therapy within 1 month of COVID-19 diagnosis. RESULTS By the data lock of February 4, 2022, the registry counted 613 eligible patients with breast cancer: 60.1% (n = 312) hormone receptor-positive, 25.2% (n = 131) human epidermal growth factor receptor 2-positive, and 14.6% (n = 76) triple-negative. The majority (61%; n = 374) had localized/locally advanced disease. Median age was 62 years (interquartile range, 51-74 years). A total of 193 patients (31.5%) presented >= 2 comorbidities and 69% (n = 330) were never smokers. In total, 392 (63.9%), 164 (26.8%), and 57 (9.3%) were diagnosed during the prevaccination, Alpha-Delta, and Omicron phases, respectively. Analysis of CFR28 demonstrates comparable estimates of mortality across the three pandemic phases (13.9%, 12.2%, 5.3%, respectively; P = .182). Nevertheless, a significant improvement in outcome measures of COVID-19 severity across the three pandemic time periods was observed. Importantly, when reported separately, unvaccinated patients from the Alpha-Delta and Omicron phases achieved comparable outcomes to those from the prevaccination phase. Of 566 patients eligible for the vaccination analysis, 72 (12.7%) were fully vaccinated and 494 (87.3%) were unvaccinated. We confirmed with inverse probability of treatment weighting multivariable analysis and following a clustered robust correction for participating center that vaccinated patients achieved improved CFR28 (odds ratio [OR], 0.19; 95% CI, 0.09 to 0.40), hospitalization (OR, 0.28; 95% CI, 0.11 to 0.69), COVID-19 complications (OR, 0.16; 95% CI, 0.06 to 0.45), and reduced requirement of COVID-19-specific therapy (OR, 0.24; 95% CI, 0.09 to 0.63) and oxygen therapy (OR, 0.24; 95% CI, 0.09 to 0.67) compared with unvaccinated controls. CONCLUSION Our findings highlight a consistent reduction of COVID-19 severity in patients with breast cancer during the Omicron outbreak in Europe. We also demonstrate that even in this population, a complete severe acute respiratory syndrome coronavirus 2 vaccination course is a strong determinant of improved morbidity and mortality from COVID-19

Lunar Gravitational-Wave Antenna

Monitoring of vibrational eigenmodes of an elastic body excited by gravitational waves was one of the first concepts proposed for the detection of gravitational waves. At laboratory scale, these experiments became known as resonant-bar detectors first developed by Joseph Weber in the 1960s. Due to the dimensions of these bars, the targeted signal frequencies were in the kHz range. Weber also pointed out that monitoring of vibrations of Earth or Moon could reveal gravitational waves in the mHz band. His Lunar Surface Gravimeter experiment deployed on the Moon by the Apollo 17 crew had a technical failure rendering the data useless. In this article, we revisit the idea and propose a Lunar Gravitational-Wave Antenna (LGWA). We find that LGWA could become an important partner observatory for joint observations with the space-borne, laser-interferometric detector LISA, and at the same time contribute an independent science case due to LGWA's unique features. Technical challenges need to be overcome for the deployment of the experiment, and development of inertial vibration sensor technology lays out a future path for this exciting detector concept.Comment: 29 pages, 17 figure

SARS-CoV-2 omicron (B.1.1.529)-related COVID-19 sequelae in vaccinated and unvaccinated patients with cancer: results from the OnCovid registry

Background COVID-19 sequelae can affect about 15% of patients with cancer who survive the acute phase of SARS-CoV-2 infection and can substantially impair their survival and continuity of oncological care. We aimed to investigate whether previous immunisation affects long-term sequelae in the context of evolving variants of concern of SARS-CoV-2. Methods OnCovid is an active registry that includes patients aged 18 years or older from 37 institutions across Belgium, France, Germany, Italy, Spain, and the UK with a laboratory-confirmed diagnosis of COVID-19 and a history of solid or haematological malignancy, either active or in remission, followed up from COVID-19 diagnosis until death. We evaluated the prevalence of COVID-19 sequelae in patients who survived COVID-19 and underwent a formal clinical reassessment, categorising infection according to the date of diagnosis as the omicron (B.1.1.529) phase from Dec 15, 2021, to Jan 31, 2022; the alpha (B.1.1.7)-delta (B.1.617.2) phase from Dec 1, 2020, to Dec 14, 2021; and the pre-vaccination phase from Feb 27 to Nov 30, 2020. The prevalence of overall COVID-19 sequelae was compared according to SARS-CoV-2 immunisation status and in relation to post-COVID-19 survival and resumption of systemic anticancer therapy. This study is registered with ClinicalTrials.gov, NCT04393974. Findings At the follow-up update on June 20, 2022, 1909 eligible patients, evaluated after a median of 39 days (IQR 24-68) from COVID-19 diagnosis, were included (964 [ 50 center dot 7%] of 1902 patients with sex data were female and 938 [49 center dot 3%] were male). Overall, 317 (16 center dot 6%; 95% CI 14 center dot 8-18 center dot 5) of 1909 patients had at least one sequela from COVID-19 at the first oncological reassessment. The prevalence of COVID-19 sequelae was highest in the prevaccination phase (191 [19 center dot 1%; 95% CI 16 center dot 4-22 center dot 0] of 1000 patients). The prevalence was similar in the alpha-delta phase (110 [16 center dot 8%; 13 center dot 8- 20 center dot 3] of 653 patients, p=0 center dot 24), but significantly lower in the omicron phase (16 [6 center dot 2%; 3 center dot 5-10 center dot 2] of 256 patients, p<0 center dot 0001). In the alpha- delta phase, 84 (18 center dot 3%; 95% CI 14 center dot 6-22 center dot 7) of 458 unvaccinated patients and three (9 center dot 4%; 1 center dot 9- 27 center dot 3) of 32 unvaccinated patients in the omicron phase had sequelae. Patients who received a booster and those who received two vaccine doses had a significantly lower prevalence of overall COVID-19 sequelae than unvaccinated or partially vaccinated patients (ten [7 center dot 4%; 95% CI 3 center dot 5-13 center dot 5] of 136 boosted patients, 18 [9 center dot 8%; 5 center dot 8-15 center dot 5] of 183 patients who had two vaccine doses vs 277 [ 18 center dot 5%; 16 center dot 5-20 center dot 9] of 1489 unvaccinated patients, p=0 center dot 0001), respiratory sequelae (six [4 center dot 4%; 1 center dot 6-9 center dot 6], 11 [6 center dot 0%; 3 center dot 0-10 center dot 7] vs 148 [9 center dot 9%; 8 center dot 4- 11 center dot 6], p= 0 center dot 030), and prolonged fatigue (three [2 center dot 2%; 0 center dot 1-6 center dot 4], ten [5 center dot 4%; 2 center dot 6-10 center dot 0] vs 115 [7 center dot 7%; 6 center dot 3-9 center dot 3], p=0 center dot 037)

Analysis of Hunter Syndrome by RNA-Sequencing

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 biomarkersLa Sindrome di Hunter (o Mucopolisaccaridosi di tipo II, MPS II) è una malattia metabolica ereditaria rara, causata da un’attività estremamente ridotta o del tutto assente dell’enzima lisosomiale iduronato 2-solfatasi (IDS), deputato alla degradazione dei mucopolisaccaridi eparan- e dermatan-solfato. Tale deficit determina un progressivo accumulo patologico delle due macromolecole sia nei lisosomi cellulari che nella matrice extracellulare di quasi tutti i tessuti ed organi, conducendo progressivamente ad un malfunzionamento generale e, infine, alla morte. Infatti, essendo l’IDS un enzima ubiquitario, quasi tutti i distretti risultano compromessi, compreso il sistema nervoso centrale nelle forme severe della malattia. La MPS II appartiene al gruppo delle mucopolisaccaridosi (MPSs), un cluster di malattie caratterizzate proprio dall’accumulo di mucopolisaccaridi (o glicosaminoglicani, GAG. Esse rappresentano, a loro volta, un sottogruppo della più ampia classe delle malattie da accumulo lisososmiale (Lysosomal Storage Disorders, LSD), una cinquantina di patologie caratterizzate dall’accumulo endo- ed extra-cellulare di diversi tipi di macromolecole non degradate. Le LSD, a lungo trascurate dalla comunità medico-scientifica, negli ultimi anni hanno ricevuto una maggiore attenzione a causa della loro elevata incidenza complessiva, fino a 1:1500–1:7000 nati vivi, anche dipendentemente dalla popolazione analizzata. Nonostante il difetto enzimatico o comunque proteico alla base di ciascuna di queste patologie sia ormai noto, per lo più sconosciuta rimane la complessità dei pathways biochimici che risultano coinvolti o alterati nell’accumulo lisosomiale in generale, o in specifici tipi di accumulo. Gli studi condotti nell’ultima decade hanno separatamente evidenziato alterazioni a carico di proteine di segnale, dell’omeostasi del calcio endocellulare, dello stress ossidativo, dell’autofagia, del trafficking intracellulare, della biosintesi dei lipidi e del metabolismo del ferro. Nessuno studio è stato, tuttavia, condotto in modo sistematico e completo per l’analisi dell’intero quadro patologico. Ciò aiuterebbe non solo ad acquisire una visione complessiva del problema dell’accumulo lisosomiale, ma anche alla messa in luce di nuovi, potenziali target terapeutici e/o di biomarcatori utilizzabili nella diagnosi delle patologie, nella definizione della loro prognosi e progressione, nella valutazione di efficacia terapeutica dei trattamenti applicati. Inoltre, poiché le LSD condividono numerosi segni e sintomi patologici, è evidente come lo studio approfondito di alcune potrebbe risultare di grande aiuto anche per la comprensione delle altre. Per la prima volta questo progetto ha valutato con tecnologia high throughput l’intero trascrittoma di cellule LSD mediante comparazione di fibroblasti cutanei ottenuti da pazienti Hunter e da controlli sani, consentendo uno studio approfondito della patogenesi della MPS II. Lo studio, condotto mediante sequenziamento di tutto l’mRNA cellulare, è stato effettuato utilizzando la tecnologia SOLiD. I risultati hanno evidenziato alterazioni a livello di: 1) processi cellulari di base, quali il ciclo cellulare, l’apopotosi, la comunicazione intercellulare; 2) processi metabolici quali il metabolismo dei proteoglicani, la sintesi dei lipidi, degli aminoacidi e dei nucleotidi; 3) la risposta agli stimoli quali lo stress ossidativo, l’insulina, le citochine; 4) l’alterazione dei processi dello sviluppo. Dal punto di vista del trattamento, nel caso della MPS II, valutata in questo progetto di studio, la terapia maggiormente applicata negli ultimi 5-6 anni è rappresentata dalla sostituzione enzimatica (Enzyme Replacement Therapy, ERT), che consiste nella somministrazione sistemica settimanale della forma attiva dell’enzima che è deficitario nei pazienti. Il monitoraggio clinico dei pazienti in trattamento, organizzato a partire dal 2005 dalla ditta che distribuisce il farmaco (Shire HGT) ha evidenziato, tra le altre cose, una importante soggettività nell’efficacia della terapia, come atteso per un trattamento effettuato per una patologia con diverse forme di severità, causata da un elevato numero di mutazioni diverse. Tuttavia l'ERT è di norma somministrato a tutti i pazienti con il medesimo protocollo. Da qui la necessità di effettuare uno studio approfondito sia clinico che molecolare allo scopo di individuare dei potenziali candidati a biomarcatori di efficacia che consentano un follow-up adeguato dei pazienti in ERT; ciò permetterebbe la messa a punto di un protocollo terapeutico personalizzato. A partire da queste considerazioni, in questo progetto è stata effettuata una valutazione dell’ERT in vitro, in fibroblasti primari Hunter trattati con l’enzima IDS terapeutico e raccolti 24 e 144 ore dall’inizio del trattamento. Il loro profilo trascrizionale è stato studiato allo scopo di caratterizzare la risposta cellulare precoce alla somministrazione dell’enzima. Tale analisi ha consentito di evidenziare una ventina di geni candidati a marcatori di efficacia terapeutica. Alcuni di questi sono stati poi valutati, mediante Real Time PCR, in alcuni campioni ematici provenienti da una popolazione di soggetti Hunter in ERT. Infine, è stato effettuato uno studio di correlazione tra l’andamento osservato dei marcatori molecolari e l’andamento di alcuni parametri clinici, provenienti dal follow-up clinico della popolazione Hunter studiata. Tale analisi ha evidenziato una buona correlazione per 8 appaiamenti gene candidato/parametro clinico valutato; in particolare, correlazioni con almeno un gene sono state trovate per la sordità, le crisi epilettiche, l’epatomegalia, la splenomegalia e altri parametri clinici. E’ auspicabile che l’estensione di tale valutazione ai rimanenti geni candidati metta in luce altri potenziali candidati biomarcatori di efficacia terapeutic