Showing papers by "Mirella Filocamo published in 2014"

Functional analysis of 11 novel GBA alleles.

Abstract: Gaucher disease is the most frequent lysosomal storage disorder due to the deficiency of the acid β-glucosidase, encoded by the GBA gene. In this study, we report the structural and functional characterization of 11 novel GBA alleles. Seven single missense alleles, P159S, N188I, E235K, P245T, W312S, S366R and W381C, and two alleles carrying in cis mutations, (N188S; G265R) and (E326K; D380N), were studied for enzyme activity in transiently transfected cells. All mutants were inactive except the P159S, which retained 15% of wild-type activity. To further characterize the alleles carrying two in cis mutations, we expressed constructs bearing singly each mutation. The presence of G265R or D380N mutations completely abolished enzyme activity, while N188S and E326K mutants retained 25 and 54% of wild-type activity, respectively. Two mutations, affecting the acceptor splice site of introns 5 (c.589-1G>A) and 9 (c.1389-1G>A), led to the synthesis of aberrant mRNA. Unpredictably, family studies showed that two alleles resulted from germline or ‘de novo’ mutations. These results strengthen the importance of performing a complete and accurate molecular analysis of the GBA gene in order to avoid misleading conclusions and provide a comprehensive functional analysis of new GBA mutations.

Cell Line and DNA Biobank From Patients Affected by Genetic Diseases

Abstract: The Bioresource, presently storing 10,279 biospecimens, was initially established in 1976 as a private laboratory-collection to maintain rare mutant cell lines from genetic-metabolic diseases. Shortly afterwards, however, data from the sample collection was organised in a database and the sample collection was released to the scientific community. The Biobank has received Telethon grants since 1993, as individual facility, and from 2008 as part of the Telethon Network of Genetic Biobanks ( www.biobanknetwork.org ). In 2010, the Biobank has obtained official recognition from Regione Liguria. The Biobank has always provided essential services by establishing, analysing, maintaining, and distributing biospecimens from patients affected by rare genetic diseases. Up to now, the contribution of the Biobank to the scientific community has been expressed in acknowledgement notes in 145 scientific manuscripts.

SMAD4 mutations causing Myhre syndrome result in disorganization of extracellular matrix improved by losartan

Abstract: Myhre syndrome (MS, MIM 139210) is a connective tissue disorder that presents with short stature, short hands and feet, facial dysmorphic features, muscle hypertrophy, thickened skin, and deafness. Recurrent missense mutations in SMAD4 encoding for a transducer mediating transforming growth factor β (TGF-β) signaling are responsible for MS. We found that MS fibroblasts showed increased SMAD4 protein levels, impaired matrix deposition, and altered expression of genes encoding matrix metalloproteinases and related inhibitors. Increased TGF-β signaling and progression of aortic root dilation in Marfan syndrome can be prevented by the antihypertensive drug losartan, a TGF-β antagonists and angiotensin-II type 1 receptor blocker. Herein, we showed that losartan normalizes metalloproteinase and related inhibitor transcript levels and corrects the extracellular matrix deposition defect in fibroblasts from MS patients. The results of this study may pave the way toward therapeutic applications of losartan in MS.

Mucopolysaccharidosis type II in a female patient with a reciprocal X;9 translocation and skewed X chromosome inactivation.

Abstract: Mucopolysaccharidosis type II (MPS II or Hunter syndrome) is a rare X-linked disorder caused by deficient activity of the lysosomal enzyme, iduronate-2-sulfatase (IDS). Phenotypic expression of MPS II in female patients rarely occurs and may be the result of (i) structural abnormalities of the X chromosome, (ii) homozygosity for disease-causing mutations, or (iii) skewed X-chromosome inactivation, in which the normal IDS allele is preferentially inactivated and the abnormal IDS allele is active. We report here on a female patient with clinical MPS II manifestations, deficiency of IDS enzyme activity and a de novo balanced reciprocal X;9 translocation. As our patient has a skewed XCI pattern, but neither genomic IDS mutations nor abnormal IDS transcripts were detected, we speculate about the possible role of the chromosomal rearrangement in reducing the IDS translation efficiency.

A novel homozygous splicing mutation in PSAP gene causes metachromatic leukodystrophy in two Moroccan brothers

Abstract: Prosaposin (PSAP) gene mutations, affecting saposin B (Sap-B) domain, cause a rare metachromatic leukodystrophy (MLD) variant in which arylsulfatase A (ARSA) activity is normal To date, only 10 different PSAP mutations have been associated with a total of 18 unrelated MLD patients worldwide In this study, we report for the first time a family with Moroccan origins in which the proband, presenting with a late-infantile onset of neurological involvement and a brain MRI with the typical tigroid MLD pattern, showed normal values of ARSA activity in the presence of an abnormal pattern of urinary sulfatides In view of these findings, PSAP gene was analyzed, identifying the newly genomic homozygous c909 + 1G > A mutation occurring within the invariant GT dinucleotide of the intron 8 donor splice site Reverse transcriptase-polymerase chain reaction (RT-PCR), showing the direct junction of exon 7 to exon 9, confirmed the skipping of the entire exon 8 (pGln260_Lys303) which normally contains two cysteine residues (Cys271 and Cys265) involved in disulfide bridges Our report provides further evidence that phenotypes of patients with Sap-B deficiency vary widely depending on age of onset, type, and severity of symptoms Awareness of this rare MLD variant is crucial to prevent delayed diagnosis or misdiagnosis and to promptly provide an accurate genetic counseling, including prenatal diagnosis, to families

Further genotype–phenotype correlation emerging from two families with PLP1 exon 4 skipping

Abstract: Proteolipid protein 1 (PLP1) gene-related disorders due to mutations in the PLP1 include a wide spectrum of X-linked disorders ranging from severe connatal Pelizaeus–Merzbacher disease (PMD) to spastic paraplegia 2 (SPG2). Duplications, deletions or point mutations in coding and noncoding regions of the PLP1 gene may occur. We report the clinical, neuroradiologic and molecular findings in six patients from two unrelated families. The affected males showed severe mental retardation, spastic tetraparesis, inability of walking and pes cavus at onset in early infancy. Brain magnetic resonance imaging (MRI) showed hypomyelination and brain atrophy. Nystagmus was never observed. The affected females showed adult-onset progressive spastic paraparesis leading to wheel-chair dependency and subtle white matter changes on brain MRI. Molecular studies in the two families identified two different intronic mutations, the novel c.622+2T>C and the known c.622+1G>A, leading to the skipping of PLP1-exon 4. The clinical presentation of the affected males did not consistently fit in any of the PLP1-related disorder subtypes (i.e., connatal or classic PMD, SPG2 and ‘PLP1 null syndrome’), and in addition, the carrier females were symptomatic despite the severe clinical picture of their respective probands. This study provides new insight into the genotype–phenotype correlations of patients with PLP1 splice-site mutations.