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San Raffaele Cimena, Italy

Crimella C.,E Medea Scientific Institute | Baschirotto C.,E Medea Scientific Institute | Arnoldi A.,E Medea Scientific Institute | Tonelli A.,E Medea Scientific Institute | And 12 more authors.
Clinical Genetics

Spastic paraplegia type 10 (SPG10) is an autosomal dominant form of hereditary spastic paraplegia (HSP) due to mutations in KIF5A, a gene encoding the neuronal kinesin heavy chain implicated in anterograde axonal transport. KIF5A mutations were found in both pure and complicated forms of the disease; a single KIF5A mutation was also detected in a CMT2 patient belonging to an SPG10 mutant family. To confirm the involvement of the KIF5A gene in both CMT2 and SPG10 phenotypes and to define the frequency of KIF5A mutations in an Italian HSP patient population, we performed a genetic screening of this gene in a series of 139 HSP and 36 CMT2 affected subjects. We identified five missense changes, four in five HSP patients and one in a CMT2 subject. All mutations, including the one segregating in the CMT2 patient, are localized in the kinesin motor domain except for one, falling within the stalk domain and predicted to generate protein structure destabilization. The results obtained indicate a KIF5A mutation frequency of 8.8% in the Italian HSP population and identify a region of the kinesin protein, the stalk domain, as a novel target for mutation. In addition, the mutation found in the CMT2 patient strengthens the hypothesis that CMT2 and SPG10 are the extreme phenotypes resulting from mutations in the same gene. © 2011 John Wiley & Sons A/S. Source

Battistini S.,University of Siena | Ricci C.,University of Siena | Giannini F.,University of Siena | Calzavara S.,Laboratory of Clinical Molecular Biology | And 7 more authors.
Amyotrophic Lateral Sclerosis

More than 140 different mutations have been reported in the Cu/Zn superoxide dismutase-1 (SOD1) gene in patients with amyotrophic lateral sclerosis (ALS), some occurring as founder mutations. Occasionally, specific mutations are associated with a particular phenotype. We evaluated a possible genotype-phenotype correlation and looked for a founder effect in nine patients from six unrelated families with ALS, all carrying the G41S mutation, originating from north-west Tuscany in central Italy. Mutational analysis of the SOD1 gene was carried out by direct sequencing. A haplotype study was carried out using eight polymorphic markers flanking the SOD1 gene. The clinical pattern of the nine familial ALS (FALS) patients was characterized by spinal onset with early upper and lower motor neuron involvement, appearance of bulbar signs within one year, and death a few months later. Mean age at onset was 49.3 years and mean duration of disease was 0.9 years. Genotyping revealed a common haplotype for the G41S allele. We provide the first evidence that the G41S mutation in Italy originates from a common founder. In addition, our findings strengthen the data reported previously and indicate that the G41S mutation is consistently associated with a uniform and dramatic, fast-progressing phenotype. Source

Ferrari M.,Vita-Salute San Raffaele University | Presi S.,Laboratory of Clinical Molecular Biology | Ventura L.,Neonatology and Neonatal Intensive Care | Vergani B.,University of Milan Bicocca | And 4 more authors.
Pediatric Pulmonology

A study was undertaken to analyze the clinical presentation, pulmonary function, and pathological features in two female siblings with neonatal pulmonary surfactant metabolism dysfunction, type 3 (MIM 610921). The clinical records of the siblings were examined; the genes encoding surfactant protein B (SFTPB), surfactant protein C (SFTPC), and ATP-binding cassette transporter 3 protein (ABCA3) were analyzed with direct sequencing and Southern blotting. The infants were homozygous for a 5,983 bp deletion in ABCA3 including exons 2-5 as well as the start AUG codon and a putative Golgi exit signal motif. Dense abnormalities of lamellar bodies at electron microscopy and absence of ABCA3 at immunohistochemical staining were in agreement with the presence of two null alleles. In addition, an increased lipid synthesis suggested a compensatory mechanism. The clinical course in the two sisters was influenced by different environmental factors like the time needed for molecular confirmation, the ventilatory assistance adopted, the occurrence of infections. A less aggressive clinical approach did not improve the course of the disease; the prognosis was always poor. Development of a fast molecular test, able to detect also structural variants, is needed. © 2014 Wiley Periodicals, Inc. Source

Galbiati S.,San Raffaele Scientific Institute | Monguzzi A.,San Raffaele Scientific Institute | Damin F.,CNR Institute of Chemistry of Molecular Recognition | Soriani N.,San Raffaele Scientific Institute | And 11 more authors.
Journal of Medical Genetics

Background Until now, non-invasive prenatal diagnosis of genetic diseases found only limited routine applications. In autosomal recessive diseases, it can be used to determine the carrier status of the fetus through the detection of a paternally inherited disease allele in cases where maternal and paternal mutated alleles differ. Methods Conditions for non-invasive identification of fetal paternally inherited mutations in maternal plasma were developed by two independent approaches: coamplification at lower denaturation temperature-PCR (COLD-PCR) and highly sensitive microarrays. Assays were designed for identifying 14 mutations, 7 causing ß-thalassaemia and 7 cystic fibrosis. Results In total, 87 non-invasive prenatal diagnoses were performed by COLD-PCR in 75 couples at risk for ß-thalassaemia and 12 for cystic fibrosis. First, to identify the more appropriate methodology for the analysis of minority mutated fetal alleles in maternal plasma, both fast and full COLD-PCR protocols were developed for the most common Italian ß-thalassaemia Cd39 and IVSI.110 mutations. In 5 out of 31 samples, no enrichment was obtained with the fast protocol, while full COLD-PCR provided the correct fetal genotypes. Thus, full COLD-PCR protocols were developed for all the remaining mutations and all analyses confirmed the fetal genotypes obtained by invasive prenatal diagnosis. Microarray analysis was performed on 40 samples from 28 couples at risk for ß- thalassaemia and 12 for cystic fibrosis. Results were in complete concordance with those obtained by both COLD-PCR and invasive procedures. Conclusions COLD-PCR and microarray approaches are not expensive, simple to handle, fast and can be easily set up in specialised clinical laboratories where prenatal diagnosis is routinely performed. © 2016 by the BMJ Publishing Group Ltd. Source

Salvianti F.,University of Florence | Inversetti A.,San Raffaele Hospital | Smid M.,San Raffaele Hospital | Valsecchi L.,San Raffaele Hospital | And 8 more authors.

Introduction This study aims to quantify total and fetal cell-free DNA (cfDNA) in maternal plasma at different gestational ages and to assess whether this could represent a reliable predictive marker of pre-eclampsia (PE) before clinical onset. Methods We performed a qPCR assay to compare the cfDNA concentration of hypermethylated and unmethylated RASSF1A promoter gene sequences in maternal plasma among 3 groups of pregnant women. These included 17 women with overt PE, 33 women at risk for the disease subsequently differentiated into 9 who developed PE and 24 who did not, and 73 controls. All women at risk were consecutively sampled throughout the whole gestation. Results Both total and fetal cfDNA had a good diagnostic performance in distinguishing patients with overt PE from healthy controls. When comparing women at risk who developed PE to women at risk who did not, the predictive capability was satisfactory at a gestational age ranging from 17 to 30 weeks. This allowed establishing within this time interval a cut-off value of 735 GE/ml for total cfDNA (87.5% sensitivity and 70.0% specificity), and a cut-off value of 7.49 GE/ml for fetal cfDNA (100% sensitivity and 50% specificity). cfDNA levels turned positive several weeks before the onset of the disease: from 2 to 18 weeks for total cfDNA and from 8 to 17 weeks for fetal cfDNA. Discussion The simultaneous use of total and fetal cfDNA would allow an accurate monitoring and prevention of PE development thus suggesting that RASSF1A could represent a potential biomarker of PE. © 2015 Elsevier Ltd. All rights reserved. Source

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