Laboratory of Clinical Molecular Biology

San Raffaele Cimena, Italy

Laboratory of Clinical Molecular Biology

San Raffaele Cimena, Italy
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Ferrari M.,Vita-Salute San Raffaele University | Presi S.,Laboratory of Clinical Molecular Biology | Ventura L.,MBBM Foundation | Vergani B.,University of Milan Bicocca | And 4 more authors.
Pediatric Pulmonology | Year: 2014

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.

PubMed | Laboratory of Clinical Molecular Biology, Unit of Genomics for Diagnosis of Human Pathologies, Vita-Salute San Raffaele University and Center for Translational Genomics and Bioinformatics
Type: Journal Article | Journal: Clinica chimica acta; international journal of clinical chemistry | Year: 2015

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in infancy, affecting preterm children with low birth weight. The disease has a multifactorial aetiology with a significant genetic component; until now published association studies have identified several candidate genes but only few of these data has been replicated. In this pilot study, we approached exome sequencing aimed at identifying non-common variants, which are expected to have a stronger phenotypic effect.We performed this study on 26 Italian severely affected BPD preterm unrelated newborns, homogeneously selected from a large prospective cohort. We used an Illumina HiSeq 2000 for sequencing. Data analysis was focussed on genes previously associated to BPD susceptibility and to new candidates in related pathways, highlighted by a prioritization analysis performed using ToppGene Suite.By exome sequencing, we identified 3369 novel variants, with a median of 400 variations per sample. The top candidate genes highlighted were NOS2, MMP1, CRP, LBP and the toll-like receptor (TLR) family. All of them have been confirmed with Sanger sequencing.Potential candidate genes have been discovered in this preliminary study; the pathogenic role of identified variants will need to be confirmed with functional and segregation studies and possibly with further methods, able to evaluate the collective influence of rare variants. Moreover, additional candidates will be tested and genetic analysis will be extended to all affected children.

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 | Year: 2012

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.

Ferrari M.,San Raffaele Scientific Institute | Ferrari M.,Laboratory of Clinical Molecular Biology | Ferrari M.,Vita-Salute San Raffaele University | Carrera P.,San Raffaele Scientific Institute | And 3 more authors.
Clinica Chimica Acta | Year: 2015

The presence of fetal DNA in maternal plasma represents a source of genetic material which can be obtained non-invasively. To date, the translation of noninvasive prenatal diagnosis from research into clinical practice has been rather fragmented, and despite the advances in improving the analytical sensitivity of methods, distinguishing between fetal and maternal sequences remains very challenging. Thus, the field of noninvasive prenatal diagnosis of genetic diseases has yet to attain a routine application in clinical diagnostics. On the contrary, fetal sex determination in pregnancies at high risk of sex-linked disorders, tests for fetal RHD genotyping and non-invasive assessment of chromosomal aneuploidies are now available worldwide. © 2014 Elsevier B.V.

Marangoni S.,University of Milan Bicocca | Di Resta C.,San Raffaele Scientific Institute | Rocchetti M.,University of Milan Bicocca | Barile L.,University of Milan Bicocca | And 10 more authors.
Cardiovascular Research | Year: 2011

Aims The Na channel mutation (p.S216L), previously associated with type 3 long-QT syndrome (LQT3) phenotype, and a common polymorphism (p.H558R) were detected in a patient with an intermittent Brugada syndrome (BS) ECG pattern. The study was aimed to assess the p.S216L electrical phenotype, its modulation by p.H558R, and to identify abnormalities compatible with a mixed BS-LQT3 phenotype. Methods and resultsThe mutation was expressed alone (S216L channels), or in combination with the polymorphism (S216LH558R channels), in a mammalian cell line (TSA201). Functional analysis included standard voltage clamp and dynamic clamp with endo-and epicardial action potential waveforms. Expression of S216L channels was associated with a 60 reduction in maximum Na current (INa) density, attributable to protein misfolding (rescued by mexiletine pretreatment) and moderate slowing of inactivation. INa density partially recovered in S216LH558R channels, but INa inactivation and its recovery were further delayed. The persistent component of INa (INaL) was unchanged. Under dynamic clamp conditions, INa decreased in S216L channels and displayed a 'resurgent component during late repolarization. In S216LH558R channels, INa density partially recovered and did not display a resurgent component. INa changes during dynamic clamp were interpreted by numerical modelling. ConclusionThe BS pattern of p.S216L might result from a decrease in INa density, which masked gating abnormalities that might otherwise result in a LQT phenotype. The p.H558R polymorphism decreased p.S216L expressivity, partly by lessening p.S216L effects and partly through the induction of further gating abnormalities suitable to blunt p.S216L effects during repolarization. © 2011 The Author.

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 | Year: 2010

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.

Salvianti F.,University of Florence | Inversetti A.,San Raffaele Hospital | Smid M.,San Raffaele Hospital | Valsecchi L.,San Raffaele Hospital | And 8 more authors.
Placenta | Year: 2015

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.

Damin F.,CNR Institute of Chemistry of Molecular Recognition | Galbiati S.,San Raffaele Scientific Institute | Ferrari M.,San Raffaele Scientific Institute | Ferrari M.,Laboratory of Clinical Molecular Biology | And 2 more authors.
Biosensors and Bioelectronics | Year: 2016

In a previous study we developed a highly sensitive DNA microarray for the detection of common KRAS oncogenic mutations, which has been proven to be highly specific in assigning the correct genotype without any enrichment strategy even in the presence of minority mutated alleles. However, in this approach, the need of a spotter for the deposition of the purified PCR products on the substrates and the purification step of the conventional PCR are serious drawbacks. To overcome these limitations we have introduced the solid-phase polymerase chain reaction (SP-PCR) to form the array of PCR products starting from the oligonucleotide primers. This work was possible thanks to the great thermal stability of the copoly (DMA-NAS-MAPS) coating which withstands PCR thermal cycling temperatures. As an example of the application of this platform we performed the analysis of six common mutations in the codon 12 of KRAS gene (G12A, G12C, G12D, G12R, G12S, and G12V). In conclusion solid-phase PCR, combined with dual-color hybridization, allows mutation analysis in a shorter time span and is more suitable for automation. © 2015 Elsevier B.V.

PubMed | Vita-Salute San Raffaele University, University of Modena and Reggio Emilia, Leiden University, Urology and Transplant and 6 more.
Type: | Journal: Scientific reports | Year: 2016

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common hereditary kidney disease. We analysed PKD1 and PKD2, in a large cohort of 440 unrelated Italian patients with ADPKD and 203 relatives by direct sequencing and MLPA. Molecular and detailed phenotypic data have been collected and submitted to the PKD1/PKD2 LOVD database. This is the first large retrospective study in Italian patients, describing 701 variants, 249 (35.5%) already associated with ADPKD and 452 (64.5%) novel. According to the criteria adopted, the overall detection rate was 80% (352/440). Novel variants with uncertain significance were found in 14% of patients. Among patients with pathogenic variants, in 301 (85.5%) the disease is associated with PKD1, 196 (55.7%) truncating, 81 (23%) non truncating, 24 (6.8%) IF indels, and in 51 (14.5%) with PKD2. Our results outline the high allelic heterogeneity of variants, complicated by the presence of variants of uncertain significance as well as of multiple variants in the same subject. Classification of novel variants may be particularly cumbersome having an important impact on the genetic counselling. Our study confirms the importance to improve the assessment of variant pathogenicity for ADPKD; to this point databasing of both clinical and molecular data is crucial.

Avemaria F.,University of Milan | Avemaria F.,Laboratory of Clinical Molecular Biology | Carrera P.,Laboratory of Clinical Molecular Biology | Lapolla A.,University of Padua | And 7 more authors.
Clinical Chemistry and Laboratory Medicine | Year: 2015

Diabetes mellitus is a global pandemic and continues to increase in numbers and significance. Several pathogenic processes are involved in the development of such disease and these mechanisms could be influenced by genetic, epigenetic and environmental factors. Non-enzymatic glycation reactions of proteins have been strongly related to pathogenesis of chronic diabetic complications. The identification of fructosamine 3-kinase (FN3K), an enzyme involved in protein deglycation, a new form of protein repair, is of great interest. FN3K phosphorylates fructosamines on the third carbon of their sugar moiety, making them unstable and causing them to detach from proteins, suggesting a protective role of this enzyme. Moreover, the variability in FN3K activity has been associated with some polymorphisms in the FN3K gene. Here we argue about genetic studies and evidence of FN3K involvement in diabetes, together with results of our analysis of the FN3K gene on a Caucasian cohort of diabetic patients. Present knowledge suggests that FN3K could act in concert with other molecular mechanisms and may impact on gene expression and activity of other enzymes involved in deglycation process. © 2015 by De Gruyter.

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