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

Clinical efficacy of Enzyme Replacement Therapy in paediatric Hunter patients, an independent study of 3.5 years

BACKGROUND: Hunter Syndrome is an X-linked lysosomal storage disorder due to the deficit of iduronate 2-sulfatase, an enzyme catalysing the degradation of the glycosaminoglycans (GAG) dermatan- and heparan-sulfate. Treatment of the disease is mainly performed by Enzyme Replacement Therapy (ERT) with idursulfase, in use since 2006. Clinical efficacy of ERT has been monitored mainly by the Hunter Outcome Survey (HOS) while very few independent studies have been so far conducted. The present study is a 3.5-years independent follow-up of 27 Hunter patients, starting ERT between 1.6 and 27 years of age, with the primary aim to evaluate efficacy of the therapy started at an early age (<12 years). METHODS: In this study, we evaluated: urinary GAG content, hepato/splenomegaly, heart valvulopathies, otorinolaryngological symptoms, joint range of motion, growth, distance covered in the 6-minute walk test, neurological involvement. For data analysis, the 27 patients were divided into three groups according to the age at start of ERT: ≤5 years, >5 and ≤ 12 years and > 12 years. Patients were analysed both as 3 separate groups and also as one group; in addition, the 20 patients who started ERT up to 12 years of age were analysed as one group. Finally, patients presenting a “severe” phenotype were compared with “attenuated” ones. RESULTS: Data analysis revealed a statistically significant reduction of the urinary GAG in patients ≤5 years and ≤ 12 years and of the hepatomegaly in the group aged >5 and ≤ 12 years. Although other clinical signs improved in some of the patients monitored, statistical analysis of their variation did not reveal any significant changes following enzyme administration. The evaluation of ERT efficacy in relation to the severity of the disease evidenced slightly higher improvements as for hepatomegaly, splenomegaly, otological disorders and adenotonsillar hypertrophy in severe vs attenuated patients. CONCLUSIONS: Although the present protocol of idursulfase administration may result efficacious in delaying the MPS II somatic disease progression at some extent, in this study we observed that several signs and symptoms did not improve during the therapy. Therefore, a strict monitoring of the efficacy obtained in the patients under ERT is becoming mandatory for clinical, ethical and economic reasons. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13023-014-0129-1) contains supplementary material, which is available to authorized users

Analisi molecolare dei geni implicati nelle mucopolisaccaridosi: valutazione della casistica di uno studio multicentrico.

Introduzione Nell'ambito di un Progetto PRIN, sono state rivalutate le diagnosi molecolari dei pazienti arruolati nello studio, per un totale di 60 soggetti, affetti da diverse forme di MPS. Metodi I dati genetici dei singoli pazienti, resi disponibili dalle diverse Unità Cliniche, sono stati ri-analizzati. Le varianti sono state ri-annottate secondo l'annotazione HGVS corrente. Risultati La diagnosi molecolare è risultata disponibile per 50 dei 60 pazienti. Per i 10 pazienti non caratterizzati era disponibile l'analisi enzimatica, sulla quale si era basata la formulazione della diagnosi di malattia. Sono state identificate 86 varianti causanti patologia (55 diverse). Il 70% erano missenso, 11.6% non senso e una variante senso; il 9.3% erano ampie delezioni/riarrangiamenti, il 3.5% piccole delezioni/inserzioni, il 4.6% varianti di splicing. Nove varianti non erano riportate in letteratura. Una spiccata disomogeneità nell'annotazione delle varianti è risultata evidente; ciò è imputabile al fatto che l'analisi genetica di molti pazienti risale a periodi storici diversi, in cui erano in vigore regole di annotazione differenti. Tale aspetto potrebbe causare difficoltà nell'interpretazione delle varianti, con inevitabili ricadute sulla consulenza genetica. Discussione Lo studio evidenzia la necessità di completare la diagnosi molecolare nei pazienti MPS già diagnosticati in passato con procedure di tipo biochimico. Emerge poi la necessità di aggiornare periodicamente l'annotazione delle varianti e di depositarle presso database dedicati (LOVD , ClinVAr, ecc.) in modo da renderle disponibili alla comunità scientifica. E' necessario, inoltre, stabilire un percorso diagnostico-molecolare condiviso, che preveda anche la ricerca delle varianti nei genitori per facilitare la diagnosi e consentire di identificare anche le varianti de novo, che richiedono un diverso percorso di consulenza

Valutazione della storia naturale dei pazienti affetti da mucopolisaccaridosi e dell'efficacia terapeutica mediante indicatori clinici e biochimici, individuazione di marcatori precoci di severità : studio osservazionale

In the present thesis, a clinical, neuropsychiatric and biochemical evaluation of patients affected by different types of Mucopolysaccharidosis (MPS) was performed, with the aim to analyze the natural history of these pathologies, and therefore their onset and their progression over time, but also to evaluate the effects, on the natural history, of the therapies available today and to identify early markers of severity. This project is part of a more extensive multi-center project on mucopolysaccharidoses, which also includes a preclinical evaluation performed on mouse models, not reported in this thesis. Furthermore, the project produced an IT platform, accessible remotely from all the Operating Units involved in the project, on which it will be possible to continue patients' data uploading over time, in order to increase as much as possible, the number of samples to be analyzed. This thesis reports the results of the analysis performed on the 58 MPS patients so far available, at 3 different stages of progressive evaluation. The analysis of the sample here evaluated confirmed that MPS patients are diagnosed with some delay, and this is especially true for those suffering from MPS III, diagnosed on average after 5 years of age; this may also be due to the particular clinical manifestations of this specific syndrome. Moreover, this data could be influenced by the heterogeneity of the analyzed sample, which includes both recent diagnoses and dated diagnostic pathways. The clinical suspicion on these diseases has certainly increased in recent years and this will undoubtedly help an early diagnosis. With respect to the signs and symptoms at the onset, 41% of the patients in the sample present at the same time more signs and symptoms, and for almost 30% of the MPS subjects the suspicion that leads to the diagnosis is of musculoskeletal origin. Among the neuronopathic MPS analyzed, 37.5% of MPS II and 38.5% of MPS III patients present at the onset neurological signs and symptoms. This last data is particularly interesting, since we commonly consider the peripheral involvement in MPS III as being limited. From the present analysis, this does not appear to be true at the onset, when in more than 60% of cases, MPS III patients presented with non-neurological signs and symptoms, being this an important aspect to consider during diagnosis. From a neurological point of view, it was evident how, independently from the different forms of MPS, both neuronopathic and not, on average half of the patients present macrocephaly at diagnosis, with peaks of 85% in the case of MPS I. In neuronopathic forms, there are different signs and symptoms that occur at diagnosis and/or during disease progression; in this thesis, in particular language disorders and intellectual disability were analyzed. As already known, the analysis carried out in this thesis confirms that the intellectual disability does not receive any benefits from the administration of the enzyme replacement therapy, in particular for patients suffering from the severe form of MPS II, in which a significant increase in the degree of neuro-cognitive severity is progressively registered. As expected, the psychiatric features of the different MPS, such as sleep disturbances and behavioral disorders, also do not receive benefits from ERT administration. On the other hand, some peripheral variables have shown benefits from replacement therapy, although in most cases only a tendency to an improvement has been shown, while a statistically significant improvement has been registered for hepatosplenomegaly and urinary glycosaminoglycan (GAG) levels. Interestingly, the analysis carried out highlighted some significant correlations between quantitative urinary GAG analysis and the neuropsychiatric involvement, and between the urinary levels of heparan-sulfate, identified by qualitative test, and the same clinical aspects. In these diseases, the identification of biomarkers helping to understand, even in the very early stage of the disease, what could be its progression, would be of considerable importance for the management of the patient and his family, and for the evaluation of a correct therapeutic choice. It is hoped that soon a critical analysis of many clinical and biochemical data obtained from a large cohort of patients will allow to identify other biomarkers, useful for an early diagnosis and possibly a prognosis on the severity of its progression.In questa tesi di dottorato è stata effettuata una valutazione clinica, neuropsichiatrica e biochimica di pazienti affetti da diversi tipi di Mucopolisaccaridosi (MPS), con lo scopo di analizzare la storia naturale di queste patologie, e perciò la loro insorgenza e la loro progressione nel tempo, ma anche di valutare gli effetti, sulla storia naturale, delle terapie ad oggi disponibili e di individuare marcatori precoci di severità. Questo progetto di tesi è parte di un progetto multicentrico più esteso sulle mucopolisaccaridosi, che prevede anche una valutazione preclinica effettuata sui modelli murini, non riportata in questa tesi. Inoltre, il progetto ha prodotto una piattaforma informatica, accessibile da remoto da tutte le Unità Operative afferenti al progetto stesso, sulla quale sarà  possibile continuare a caricare i dati nei pazienti nel corso del tempo, in modo da aumentare quanto più possibile la numerosità del campione da analizzare. Questa tesi riporta i risultati dell'analisi effettuata sui 58 pazienti MPS finora disponibili, a 3 diversi tempi di valutazione progressiva. L'analisi del campione qui valutato ha confermato come i pazienti MPS siano diagnosticati con un certo ritardo, e ciò vale soprattutto per gli affetti da MPS III, diagnosticati mediamente dopo i 5 anni di età; questo forse anche a causa delle particolari manifestazioni cliniche di questa specifica sindrome. Inoltre, tale dato potrebbe essere influenzato dall'eterogeneità  del campione analizzato, che comprende sia diagnosi recenti che percorsi diagnostici datati. Il sospetto clinico su queste patologie è sicuramente aumentato negli ultimi anni e ciò indubbiamente aiuterà una diagnosi precoce. Per quanto riguarda i segni e i sintomi all'esordio, il 41% dei pazienti del campione presenta contemporaneamente più segni e sintomi, e per quasi il 30% dei soggetti MPS il sospetto che porta a formulare la diagnosi è di origine muscolo-scheletrica. Tra le MPS neuronopatiche analizzate, presentano all'esordio segni e sintomi di origine neurologica il 37.5% delle MPS II e il 38.5% delle MPS III. Quest'ultimo dato risulta particolarmente interessante, dato che comunemente si considera che il coinvolgimento periferico nelle MPS III sia relativo. Dai dati qui analizzati, ciò non sembra vero all'esordio, quando in oltre il 60% dei casi i pazienti possono presentare segni e sintomi non neurologici, dato questo importante da considerare in fase di diagnosi. Da un punto di vista neurologico, èrisultato evidente come, indipendentemente dalle diverse forme di MPS, sia neuronopatiche che non, mediamente la metà dei pazienti presenti macrocefalia alla diagnosi, con punte del 85% nel caso della MPS I. Nelle forme neuronopatiche, diversi sono i segni e i sintomi che si presentano alla diagnosi e/o in corso di progressione; in questa tesi sono stati in particolare analizzati i disturbi del linguaggio e la disabilità intellettiva. Come era già noto, l'analisi effettuata in questo lavoro di tesi conferma come la disabilità intellettiva non riceva alcun giovamento dalla somministrazione della terapia enzimatica sostitutiva, in particolare per quanto riguarda i pazienti affetti dalla forma severa di MPS II, nei quali si evidenzia un aumento significativo nel tempo del grado di severità neuro-cognitiva. Come atteso, anche le caratteristiche psichiatriche delle diverse MPS, come i disturbi del sonno e del comportamento, non ricevono beneficio dall'ERT. Alcune variabili periferiche hanno evidenziato, invece, di trarre giovamento dalla terapia sostitutiva; tuttavia, nella maggior parte dei casi si apprezza solo una tendenza a un miglioramento, mentre un miglioramento statisticamente significativo si è evidenziato per l'epatosplenomegalia e i livelli di glicosaminoglicani (GAG) urinari. In modo interessante, l'analisi qui effettuata ha messo in evidenza alcune correlazioni significative tra la concentrazione dei GAG quantitativi e il coinvolgimento neuropsichiatrico e tra i livelli urinari di eparan-solfato, identificati tramite saggio qualitativo, e le medesime problematiche. In queste patologie, l'identificazione di biomarcatori che aiutassero a comprendere, ancora in fase molto precoce di malattia, quale potrebbe essere la sua progressione, sarebbe di notevole importanza per la gestione del paziente e dei suoi famigliari, e anche per la valutazione di una corretta scelta terapeutica. E' auspicabile che in un prossimo futuro un'analisi critica di molti dati clinici e biochimici ricavati da un'ampia popolazione di pazienti consenta di identificare altri biomarcatori, utili ad una diagnosi precoce e possibilmente ad una prognosi sulla severità della sua progressione

Valutazione della storia naturale dei pazienti affetti da mucopolisaccaridosi e dell'efficacia terapeutica mediante indicatori clinici e biochimici, individuazione di marcatori precoci di severità : studio osservazionale

In the present thesis, a clinical, neuropsychiatric and biochemical evaluation of patients affected by different types of Mucopolysaccharidosis (MPS) was performed, with the aim to analyze the natural history of these pathologies, and therefore their onset and their progression over time, but also to evaluate the effects, on the natural history, of the therapies available today and to identify early markers of severity. This project is part of a more extensive multi-center project on mucopolysaccharidoses, which also includes a preclinical evaluation performed on mouse models, not reported in this thesis. Furthermore, the project produced an IT platform, accessible remotely from all the Operating Units involved in the project, on which it will be possible to continue patients' data uploading over time, in order to increase as much as possible, the number of samples to be analyzed. This thesis reports the results of the analysis performed on the 58 MPS patients so far available, at 3 different stages of progressive evaluation. The analysis of the sample here evaluated confirmed that MPS patients are diagnosed with some delay, and this is especially true for those suffering from MPS III, diagnosed on average after 5 years of age; this may also be due to the particular clinical manifestations of this specific syndrome. Moreover, this data could be influenced by the heterogeneity of the analyzed sample, which includes both recent diagnoses and dated diagnostic pathways. The clinical suspicion on these diseases has certainly increased in recent years and this will undoubtedly help an early diagnosis. With respect to the signs and symptoms at the onset, 41% of the patients in the sample present at the same time more signs and symptoms, and for almost 30% of the MPS subjects the suspicion that leads to the diagnosis is of musculoskeletal origin. Among the neuronopathic MPS analyzed, 37.5% of MPS II and 38.5% of MPS III patients present at the onset neurological signs and symptoms. This last data is particularly interesting, since we commonly consider the peripheral involvement in MPS III as being limited. From the present analysis, this does not appear to be true at the onset, when in more than 60% of cases, MPS III patients presented with non-neurological signs and symptoms, being this an important aspect to consider during diagnosis. From a neurological point of view, it was evident how, independently from the different forms of MPS, both neuronopathic and not, on average half of the patients present macrocephaly at diagnosis, with peaks of 85% in the case of MPS I. In neuronopathic forms, there are different signs and symptoms that occur at diagnosis and/or during disease progression; in this thesis, in particular language disorders and intellectual disability were analyzed. As already known, the analysis carried out in this thesis confirms that the intellectual disability does not receive any benefits from the administration of the enzyme replacement therapy, in particular for patients suffering from the severe form of MPS II, in which a significant increase in the degree of neuro-cognitive severity is progressively registered. As expected, the psychiatric features of the different MPS, such as sleep disturbances and behavioral disorders, also do not receive benefits from ERT administration. On the other hand, some peripheral variables have shown benefits from replacement therapy, although in most cases only a tendency to an improvement has been shown, while a statistically significant improvement has been registered for hepatosplenomegaly and urinary glycosaminoglycan (GAG) levels. Interestingly, the analysis carried out highlighted some significant correlations between quantitative urinary GAG analysis and the neuropsychiatric involvement, and between the urinary levels of heparan-sulfate, identified by qualitative test, and the same clinical aspects. In these diseases, the identification of biomarkers helping to understand, even in the very early stage of the disease, what could be its progression, would be of considerable importance for the management of the patient and his family, and for the evaluation of a correct therapeutic choice. It is hoped that soon a critical analysis of many clinical and biochemical data obtained from a large cohort of patients will allow to identify other biomarkers, useful for an early diagnosis and possibly a prognosis on the severity of its progression

The Complexity of Pain Management in Children Affected by Mucopolysaccharidoses

Mucopolysaccharidoses (MPSs) are a group of rare, genetic lysosomal storage disorders. They are caused by deficiencies of the lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). Pain is a common feature in mucopolysaccharidoses. However, the pathophysiology of pain in this group of diseases is still unclear and genesis of pain is multifactorial. Currently, poor data about pain management in these patients are available. Here, we present our clinical experience in complex pain management in three children with MPS

The Role of Visual Electrophysiology in Mucopolysaccharidoses

none7Visual electrophysiological techniques represent excellent means for assessing retinal, optic pathways and visual cortex function. Electroretinograms, visual evoked potentials, and clinical records of 17 patients with mucopolysaccharidosis registered in the neurophysiological database of our institution were reviewed retrospectively. Ten patients were on enzyme replacement therapy, 2 underwent bone marrow transplantation, one also keratoplasty. Changes in the electroretinogram pointed to the diagnosis of retinal dystrophy type rod-cone in 8 patients. In patients in whom severe corneal clouding precluded fundus oculi inspection and at an early stage before typical fundus appearance diagnosis was possible only using the electroretinogram. Visual evoked potentials were useful to confirm the loss of visual function in patients difficult to test clinically. The authors suggest the use of electroretinogram and visual evoked potentials primarily as research tools to describe the natural history and ophthalmologic outcome in mucopolysaccharidoses, although they may have clinical utility in very selected cases.mixedSuppiej A; Rampazzo A; Cappellari A; Traverso A; Tormene A P; Pinello L; Scarpa MSuppiej, A; Rampazzo, A; Cappellari, A; Traverso, A; Tormene, A P; Pinello, L; Scarpa,

Clinical efficacy of Enzyme Replacement Therapy in paediatric Hunter patients, an independent study of 3.5\ua0years

Hunter Syndrome is an X-linked lysosomal storage disorder due to the deficit of iduronate 2-sulfatase, an enzyme catalysing the degradation of the glycosaminoglycans (GAG) dermatan- and heparan-sulfate. Treatment of the disease is mainly performed by Enzyme Replacement Therapy (ERT) with idursulfase, in use since 2006. Clinical efficacy of ERT has been monitored mainly by the Hunter Outcome Survey (HOS) while very few independent studies have been so far conducted. The present study is a 3.5-years independent follow-up of 27 Hunter patients, starting ERT between 1.6 and 27 years of age, with the primary aim to evaluate efficacy of the therapy started at an early age (<12 years). METHODS: In this study, we evaluated: urinary GAG content, hepato/splenomegaly, heart valvulopathies, otorinolaryngological symptoms, joint range of motion, growth, distance covered in the 6-minute walk test, neurological involvement. For data analysis, the 27 patients were divided into three groups according to the age at start of ERT: 645 years, >5 and\u2009 64\u200912 years and\u2009>\u200912 years. Patients were analysed both as 3 separate groups and also as one group; in addition, the 20 patients who started ERT up to 12 years of age were analysed as one group. Finally, patients presenting a "severe" phenotype were compared with "attenuated" ones. RESULTS: Data analysis revealed a statistically significant reduction of the urinary GAG in patients 645 years and\u2009 64\u200912 years and of the hepatomegaly in the group aged >5 and\u2009 64\u200912 years. Although other clinical signs improved in some of the patients monitored, statistical analysis of their variation did not reveal any significant changes following enzyme administration. The evaluation of ERT efficacy in relation to the severity of the disease evidenced slightly higher improvements as for hepatomegaly, splenomegaly, otological disorders and adenotonsillar hypertrophy in severe vs attenuated patients. CONCLUSIONS: Although the present protocol of idursulfase administration may result efficacious in delaying the MPS II somatic disease progression at some extent, in this study we observed that several signs and symptoms did not improve during the therapy. Therefore, a strict monitoring of the efficacy obtained in the patients under ERT is becoming mandatory for clinical, ethical and economic reasons