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Homozygous or compound heterozygous mutations in the GJC2 gene, encoding the gap junction protein connexin47 (Cx47), cause the autosomal recessive hypomyelinating Pelizaeus-Merzbacher-like disease (PMLD1, MIM# 608804). Although clinical and neuroradiological findings resemble those of the classic Pelizaeus-Merzbacher disease, PMLD patients usually show a greater level of cognitive and motor functions. Unpredictably a homozygous missense GJC2 mutation (p.Glu260Lys) was found in a patient presenting with a very severe clinical picture characterised by congenital nystagmus and severe neurological impairment. Also magnetic resonance imaging was unusually severe, showing an abnormal supra- and infratentorial white matter involvement extending to the spinal cord. The novel p.Glu260Lys (c.778G>A) mutation, occurring in a highly conserved motif (SRPTEK) of the Cx47 extracellular loop-2 domain, was predicted, by modelling analysis, to break a 'salt bridge network', crucial for a proper connexin-connexin interaction to form a connexon, thus hampering the correct formation of the connexon pore. The same structural analysis, extended to the previously reported missense mutations, predicted that most changes were expected to have less severe impact on protein functions, correlating with the mild PMLD1 form of the patients. Our study expands the spectrum of PMLD1 and provides evidence that the extremely severe clinical and neuroradiological PMLD1 form of our patient likely correlates with the predicted impairment of gap junction channel assembly resulting from the detrimental effect of the new p.Glu260Lys mutant allele on Cx47 protein.

We report the first newborn screening pilot study in an Italian region for four lysosomal disorders including Pompe disease, Gaucher disease, Fabry disease and mucopolysaccharidosis type 1. The screening has been performed using enzymatic assay on Dry Blood Spot on filter paper. A total of 3403 newborns were screened. One newborn showed a reduction of β-glucosidase activity in leucocytes. Molecular analysis revealed a status of compound heterozygous for the panethnic mutation N370S and for the sequence variation E388K, not yet correlated to Gaucher disease onset. The functional consequences of the E388K replacement on β-glucosidase activity were evaluated by in vitro expression, showing that the mutant protein retained 48% of wild type activity. Structural modeling predicted that the E388K replacement, localized to a surface of the enzyme, would change the local charges distribution which, in the native protein, displays an overwhelming presence of negative charges. However, the newborn, and a 4 year old sister showing the same genomic alterations, are currently asymptomatic. This pilot newborn screening for lysosomal diseases appears to be feasible and affordable to be extended to large populations. Moreover other lysosomal diseases for which a therapy is available or will be available, could be included in the screening., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Mutational analysis of the GNPTAB gene was performed in 46 apparently unrelated patients with mucolipidosis IIalpha/beta or IIIalpha/beta, characterized by the mistargeting of multiple lysosomal enzymes as a consequence of a UDP-GlcNAc-1-phosphotransferase defect. The GNPTAB mutational spectrum comprised 25 distinct mutant alleles, 22 of which were novel, including 3 nonsense mutations (p.Q314X, p.R375X, p.Q507X), 5 missense mutations (p.I403T, p.C442Y, p.C461G, p.Q926P, p.L1001P), 6 microduplications (c.749dupA, c.857dupA, c.1191_1194dupGCTG, c.1206dupT, c.1331dupG, c.2220_2221dupGA) and 8 microdeletions (c.755_759delCCTCT, c.1399delG, c.1959_1962delTAGT, c.1965delC, c.2550_2554delGAAAA, c.3443_3446delTTTG, c.3487_3490delACAG, c.3523_3529delATGTTCC). All micro-duplications/deletions were predicted to result in the premature termination of translation. A novel exonic SNP (c.303G>A; E101E) was identified which is predicted to create an SFRS1 (SF2/ASF) binding site that may be of potential functional/clinical relevance. This study of mutations in the GNPTAB gene, the largest yet reported, extends our knowledge of the mutational heterogeneity evident in MLIIalpha/beta/MLIIIalpha/beta.

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.

Metachromatic leukodystrophy (MLD), the demyelinating disorder resulting from impaired sulfatide catabolism, is caused by allelic mutations of the Arylsulfatase A (ARSA) locus except for extremely rare cases of Saposin-B (Sap-B) deficiency. We characterized twenty-one unrelated Italian patients among which seventeen were due to ARSA activity deficiency and 4 others resulted from Saposin-B defect. Overall, we found 20 different mutant ARSA alleles and 2 different Sap-B alleles. The eleven new ARSA alleles (c.53C>A; c.88G>C; c.372G>A; c.409_411delCCC; c.634G>C; [c.650G>A;c.1108C>T]; c.845A>G; c.906G>C; c.919G>T; c.1102-3C>G; c.1126T>A) were functionally characterized and the novel amino acid changes were also modelled into the three-dimensional structure. The present study is aimed at providing a broader picture of the molecular basis of MLD in the Italian population. It also emphasizes the importance of a comprehensive evaluation in MLD diagnosis including biochemical, enzymatic and molecular investigations., ((c) 2008 Wiley-Liss, Inc.)

Molecular characterization of twelve unrelated patients affected by the autosomal recessive osteosclerotic skeletal dysplasia, Pycnodysostosis (cathepsin k deficiency), revealed 11 different genotypes. The mutational profile consisted of 12 different mutations, including nine previously unreported ones, spread throughout the whole gene. One mutation occurred in regions coding predomain, two affected the prodomain and nine others occurred in the mature domain. The novel lesions consisted in six missense mutations c.20T>C (p.L7P), c.494A>G (p.Q165R), c.580G>A (p.G194S), c.746T>C (p.I249T), c.749A>G (p.D250G), c.955G>T (p.G319C), two frameshifts c.60_61dupGA (p.I21RfsX29), c.282dupA (p.S95VfsX9) and a splicing mutation c.890G>A (r.785_890del). The six new missense mutations were examined by western blots of COS-7 cells transfected with mutant CTSK genes. The L7P, occurring within the predicted hydrophobic domain of signal peptide, showed a significantly reduced expression level compared to the wild type control. These findings suggested that the mutation affected targeting and translocation of the nascent lysosomal protein across the endoplasmatic reticulum membrane. The novel amino acid changes were also modeled into the three-dimensional structure that predicted incorrect protein folding for all of them. Molecular characterization of the patients is of particular value for genetic counseling of patients and their families as diagnosis of Pycnodysostosis based on enzyme assay is unpractical and thus not offered routinely., (2007 Wiley-Liss, Inc.)

Hemophilia A (HA) is a disorder caused by mutations of the FVIII gene, which is located on the tip of the long arm of the X chromosome. In a cohort of 18 unrelated Italian patients affected with HA of varying severity, we performed mutational screening of the gene by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing of abnormal peaks. We identified five novel mutations and 9 previously reported DNA alterations. Two of the 9 previously reported alterations were each common to 3 unrelated patients. Six different mutations were characterized as missense alterations, while 8 were non-missense mutations. Among the new gene alterations, one created a stop codon, one consisted of an out-of frame deletion, and one was a splice-site mutation. The last two were missense alterations. In an attempt to better understand the causative effect of the mutations and the clinical variability of the patients, we investigated the consequences of each missense mutation and visualized the effect of the amino acid change on structural FVIII models., (Copyright 2005 Wiley-Liss, Inc.)

We describe 18 novel mutations, unreported in the Haemophilia A mutation Databases, that have been identified in a cohort of unrelated, Italian patients affected with haemophilia A (HA). Screening of the factor VIII gene (FVIII) was performed using denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. Eight mutations were characterized as non-missense alterations, and the remaining 10 were missense mutations. Heterozygosity for the identified mutations was observed in the female relatives of patients belonging to eight families with sporadic cases. In an attempt to understand better the causative effect of the mutations and the clinical variability of the patients, missense mutation consequences were investigated for: (1) the nature of the new amino acid; (2) the location of the substituted amino acid within crystallographic and theoretical models; and (3) the degree of conservation of the native residue in factor VIII (FVIII) protein and FVIII-related protein family aligned sequences. These research tools have provided evidence that the mutations we describe involve residues that were conserved, at least in FVIII proteins, in all the species we compared.