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Figure S3. Sequence analysis of the aberrant mRNA transcript generated as a consequence of the c.899–167 A > G lesion identified in the GALNS gene of Pt1. A. GALNS mRNA sequence and schematic representation of the wild-type splicing event. B. Aberrant mRNA splicing resulting from the intronic c.899–167 A > G transition. (PPT 425 kb)

Negli ultimi anni grandi progressi sono stati effettuati per il trattamento di alcuni errori congeniti del metabolismo, al fine di migliorare la qualità di vita, la crescita e lo sviluppo dei pazienti affetti. Da sempre il trattamento delle LSD rappresenta una grande sfida per i medici coinvolti nel management dei pazienti affetti da tali patologie. In passato l’unica terapia possibile per le LSD era una terapia di supporto, mirata a combattere le complicanze associate alle LSD. Al contrario non era disponibile alcun approccio mirato alla correzione del difetto di base della malattia. Negli ultimi 20 anni, le maggiori conoscenze sulle basi molecolari e biochimiche di varie malattie da accumulo lisosomiale, e la disponibilità di nuove tecnologie hanno reso possibile l’introduzione e la sperimentazione, con risultati anche molto incoraggianti, di diverse strategie terapeutiche innovative quali la terapia enzimatica sostitutiva (Enzyme Replacement Therapy, ERT). Oltre alle malattie d’accumulo lisosmali, nuove strategie terapeutiche sono state sperimentate anche in altri errori congeniti del metabolismo, quali le aminoacidopatie, in particolare nelle iperfenilalaninemie. Storicamente, il trattamento della HPA è stato unicamente non-farmacologico e i pazienti affetti dovevano essere trattati con una dieta a ridotto contenuto di Phe entro le prime settimane di vita. È attualmente noto che il trattamento deve essere necessariamente continuato per tutta la vita. Sfortunatamente, la compliance alla terapia dietetica risulta essere sub-ottimale, e molti pazienti, in particolare gli adolescenti, non seguono o sospendono lo schema dietetico prescritto. Lo studio qui riportato è il risultato di quattro anni di ricerca effettuato nell’ambito del programma del Dottorato, sviluppato presso il Dipartimento di Pediatria dell’Università “Federico II” di Napoli da Novembre 2006 ad Ottobre 2010. Tale studio ha permesso la realizzazione di due progetti, incentrati su nuovi approcci terapeutici in due errori congeniti del metabolismo: la Mucopolisaccaridosi tipo I (MPS I) e le iperfenilalaninemia (HPA).

Type 1 Gaucher's disease (GD1) is traditionally regarded as "non-neurological". Spatiotemporal and kinematic analysis of gait was carried on thirteen GD1 patients and thirteen healthy controls. We identified a previously unknown subclinical reduction of amplitude of movements in GD1. Articular excursion of ankle, knee and hip was reduced during the swing phase of gait (p < 0.0001). Furthermore, the excursion of the knee appeared also significantly more asymmetric in GD1 patients (p = 0.02). Correction for age, BMI and basal walking speed did not modify the significance. Accordingly to the recent observations that GD1 predisposes to Parkinson's disease, the impaired and asymmetric gait kinematics that we observed might be interpreted as a form of extrapyramidal involvement.

Mucolipidosis type III (MLIII) is an autosomal recessive disorder affecting lysosomal hydrolase trafficking. In a study of 10 patients from seven families with a clinical phenotype and enzymatic diagnosis of MLIII, six novel GNPTG gene mutations were identified. These included missense (p.T286M) and nonsense (p.W111X) mutations and a transition in the obligate AG-dinucleotide of the intron 8 acceptor splice site (c.610–2A>G). Three microdeletions were also identified, two of which (c.611delG and c.640_667del28) were located within the coding region whereas one (c.609+28_610-16del) was located entirely within intron 8. RT-PCR analysis of the c.610–2A>G transition demonstrated that the change altered splicing, leading to the production of two distinct aberrantly spliced forms, viz. the skipping of exon 9 (p.G204_K247del) or the retention of introns 8 and 9 (p.G204VfsX28). RT-PCR analysis, performed on a patient homozygous for the intronic deletion (c.609+28_610-16del), failed to detect any GNPTG RNA transcripts. To determine whether c.609+28_610-16del allele-derived transcripts were subject to nonsense-mediated mRNA decay (NMD), patient fibroblasts were incubated with the protein synthesis inhibitor anisomycin. An RT-PCR fragment retaining 43 bp of intron 8 was consistently detected suggesting that the 33-bp genomic deletion had elicited NMD. Quantitative real-time PCR and GNPTG western blot analysis confirmed that the homozygous microdeletion p.G204VfsX17 had elicited NMD resulting in failure to synthesize GNPTG protein. Analysis of the sequences surrounding the microdeletion breakpoints revealed either intrinsic repetitivity of the deleted region or short direct repeats adjacent to the breakpoint junctions. This is consistent with these repeats having mediated the microdeletions via replication slippage and supports the view that the mutational spectrum of the GNPTG gene is strongly influenced by the properties of the local DNA sequence environment.