Clinically, GSDII encompasses a continuous spectrum of phenotypes, from a rapidly progressive infantile form leading to death within the first year of life to a slowly progressive late-onset form of the disease that affects mobility and respiratory function. Classic infantile GSDII manifests soon after birth and is characterized by absent or nearly absent enzyme activity, severe muscle weakness, cardiomegaly/cardiomyopathy and respiratory insufficiency, that typically lead to death within

the first year of life (1–4). Some infantile patients have less severe cardiac involvement without left cardiac output obstruction, Inhibitors,research,lifescience,medical survive longer and die because of pulmonary infections with secondary ventilatory insufficiency (5, 6). Late onset GSDII comprises all milder subtypes: partial enzyme deficiency manifests in children and adults as slowly progressive skeletal muscle weakness without cardiac involvement. Respiratory muscle weakness, particularly Inhibitors,research,lifescience,medical of the diaphragm, is the leading cause of death in the

late-onset cases. (1, 2, 4, 7–9). The GAA gene (MIM# 606800) located in the human chromosome 17q25.2-25.3 produces an inactive 110 kD precursor which is transported to the lysosomal compartment and processed into the 95 kD intermediate and the fully active forms of 76 and 70 kD (1, 10–13). More than 200 mutations in the GAA gene have been described up to date (http://www2.eur.nl/fgg/ch1/pompe). Inhibitors,research,lifescience,medical In an extensive collaborative study we analysed the complete mutational profile in 45 Italian patients affected by the late onset GSDII. We were able

to characterize 27 mutant alleles leading to the identification of 12 novel mutations. Missense mutations Inhibitors,research,lifescience,medical were functionally characterized in vitro by enzyme activity and protein processing and splicing mutations were studied by RT-PCR or in silico analysis. This work offers a complete picture of the late onset GSDII molecular genetics in Italy which contributes in the understanding of the natural history and in the evaluation of emerging ERT efficacy. Material and Methods Patients We studied 45 Italian patients with late onset GSDII (19 females and 26 males). The diagnosis Inhibitors,research,lifescience,medical was based on clinical data and Perifosine nmr confirmed by reduced GAA activity in lymphocytes or muscle. The age at diagnosis varied from 2 to 68 years. Almost all the patients underwent a program of physiotherapy, high protein diet and respiratory management in their reference centres. Most of the patients have had mild muscular symptoms since childhood. First complaints Florfenicol were mostly related to mobility problems, weakness and fatigue. GAA mutation analysis Genomic DNA was extracted with the QIAamp DNA blood Mini Kit (Qiagen GmbH, Hilden, Germany). GAA gene was amplified as described (14). PCR products were screened by denaturing High Performance Liquid Chromatography (dHPLC, Varian, Palo Alto, CA, USA) and in the presence of heteroduplex, sequenced on the ABI PRISM 3700 DNA Analyzer.