Simona Mellone

Amedeo Avogadro University of Eastern Piedmont, Alessandria, Piedmont, Italy

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Publications (12)47.09 Total impact

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    ABSTRACT: Mutations affecting exon3 splicing are the main cause of autosomal dominant isolated GH deficiency (IGHDII) by increasing the level of exon 3-skipped mRNA encoding the functionally inactive dominant-negative 17.5-kDa isoform. The exons and introns of the gene encoding GH (GH1) were screened for the presence of mutations in 103 sporadic IGHD cases. Four different variations within exon 3 were identified in three patients. One carried c.261C>T (p.Pro87Pro) and c.272A>T (p.Glu91Val), the second c.255G>A (p.Pro85Pro) and c.261 C>T and the third c.246G>C (p.Glu82Asp). All the variants were likely generated by gene conversion from an homologous gene in the GH1 cluster. In silico analysis predicted that positions c.255 and c.272 were included within two putative novel Exon Splicing Enhancers (ESEs). Their effect on splicing was confirmed in vitro. Constructs bearing these two variants induced consistently higher levels both of transcript and protein corresponding to the 17.5 kDa isoform. When c.255 and c.272 were combined in cis with the c.261 variant, as in our patients, their effect was weaker. In conclusion we identified two variations, c.255G>A and c.272A>T, located in two novel putative ESEs and affecting GH1 splicing in vitro by increasing the production of alternatively spliced isoforms. The amount of aberrant isoforms is further regulated by the presence in cis of the c.261 variant. Thus our results evidenced novel putative splicing regulatory elements within exon 3 confirming the crucial role of this exon in mRNA processing.
    Endocrinology 03/2014; · 4.72 Impact Factor
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    ABSTRACT: HNF1B gene mutations might be an underdiagnosed cause of nephropathy in adult patients mainly because of their pleomorphic clinical presentations. As most studies are based on paediatric populations, it is difficult to assess the likelihood of finding HNF1B mutations in adult patients and consequently define clinical settings in which genetic analysis is indicated. The aim of this study was the search for mutations in the HNF1B gene in a cohort of unrelated adult patients with nephropathy of unknown aetiology. Patients were tested for the HNF1B gene if they had chronic kidney disease of unknown origin and renal structure abnormalities (RSA) or a positive family history of nephropathy. The HNF1B coding sequence and intron-exon boundaries were analyzed by direct sequencing. The search for gene deletions was performed by Multiple Ligation Probe Analysis (MLPA). Heterozygous mutations were identified in 6 out of 67 screened patients (9.0%) and included two whole gene deletions, one nonsense (p.Gln136Stop), two missense (p.Gly76Cys and p.Ala314Thr) mutations and a frameshift microdeletion (c.384_390 delCATGCAG), the latter two (c.384_390 del and p.Ala314Thr) not ever being reported to date. Mean age of the mutated patients at screening was 48.5 years with a M/F ratio of 2/4. The clinical manifestations of affected patients were extremely pleomorphic, including several urological and extra-renal manifestations. Mutations of HNF1B could explain chronic kidney disease in up to 9% of adult patients with a nephropathy of unknown aetiology and RSA: therefore an HNF1B mutation analysis should be considered in this group of patients.
    Nephrology 01/2014; · 1.69 Impact Factor
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    ABSTRACT: TREX1 (DNase III) is an exonuclease involved in response to oxidative stress and apoptosis. Heterozygous mutations in TREX1 were previously observed in patients with systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS). We performed a mutational analysis of the TREX1 gene on three autoimmune diseases: SLE (210 patients) and SS (58 patients), to confirm a TREX1 involvement in the Italian population, and systemic sclerosis (SSc, 150 patients) because it shares similarities with SLE (presence of antinuclear antibodies and connective tissue damage). We observed 7 variations; two of these are novel nonsynonymous variants (p.Glu198Lys and p.Met232Val). They were detected in one SS and in one SSc patient, respectively, and in none of the 200 healthy controls typed in this study and of the 1712 published controls. In silico analysis predicts a possibly damaging role on protein function for both variants. The other 5 variations are synonymous and only one of them is novel (p.Pro48Pro). This study contributes to the demonstration that TREX1 is involved in autoimmune diseases and proposes that the spectrum of involved autoimmune diseases can be broader and includes SSc. We do not confirm a role of TREX1 variants in SLE.
    BioMed research international. 01/2013; 2013:471703.
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    ABSTRACT: Context: Mutations within the PROP1 gene represent one of the main causes of familial combined pituitary hormone deficiency (CPHD). However, most of the cases are sporadic with an unknown genetic cause. Objective: The aim of this study was the search for low penetrance variations within and around a conserved regulatory element in the intron 1 of PROP1, contributing to a multifactorial form of the disease in sporadic patients. Methods and Patients: A fragment of 570 bp encompassing the conserved region was sequenced in 107 CPHD patients and 294 controls, and an association study was performed with the four identified variants, namely c.109+435G>A (rs73346254), c.109+463C>T (rs4498267), c.109+768C>G (rs4431364), and c.109+915_917ins/delTAG (rs148607624). The functional role of the associated polymorphisms was evaluated by luciferase reporter gene expression analyses and EMSA. Results: A statistically significant increased frequency was observed in the patients for rs73346254A (P = 5 × 10(-4)) and rs148607624delTAG (P = 0.01) alleles. Among all the possible allele combinations, only the haplotype bearing both risk alleles showed a significantly higher frequency in the patients vs. controls (P = 4.7 × 10(-4)) and conferred a carrier risk of 4.19 (P = 1.2 × 10(-4)). This haplotype determined a significant decrease of the luciferase activity in comparison with a basal promoter and the other allelic combinations in GH4C and MCF7 cells (P = 4.6 × 10(-6); P = 5.5 × 10(-4), respectively). The EMSA showed a differential affinity for nuclear proteins for the alternative alleles of the two associated variations. Conclusions: Variations with a functional significance conferring susceptibility to CPHD have been identified in the PROP1 gene, indicating a multifactorial origin of this disorder in sporadic cases.
    The Journal of clinical endocrinology and metabolism 06/2012; 97(9):E1791-7. · 6.50 Impact Factor
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    ABSTRACT: To test the involvement of osteopontin gene (OPN) in systemic sclerosis (SSc) susceptibility, two OPN single nucleotide polymorphisms previously reported to be associated with systemic lupus erythematosus, namely -156G/GG (proximal promoter) and +1239A/C (3' untranslated region (UTR)), were tested in 357 Italian patients and 864 matched control subjects. OPN serum levels were determined by enzyme-linked immunosorbent assay in 32 patients and 116 controls. Compared with the controls, in SSc patients there was a significantly increased frequency of the alleles -156G (p = 0.0086), and +1239C (p = 0.00064), paralleling the association reported for systemic lupus erythematosus. According to logistic regression analysis, this association is primarily due to the effect of +1239 single nucleotide polymorphism. OPN serum levels were significantly higher in SSc patients than in controls (p = 0.00025). These data suggest that OPN genetic variations have a role in SSc susceptibility, reporting for the first time an involvement of this molecule in SSc pathogenesis and emphasizing that SSc shares pathogenetic mechanisms with other autoimmune diseases.
    Human immunology 07/2011; 72(10):930-4. · 2.55 Impact Factor
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    ABSTRACT: Mutations in HESX1 represent a rare cause of GH deficiency (GHD) associated with a broad spectrum of other anomalies. We searched for causative mutations in a cohort of 244 Italian patients affected by combined and isolated GHD (IGHD). The HESX1 gene-coding region and exon-intron boundaries were screened by denaturing HPLC scanning. A novel mutation adjacent to the invariant donor splice site of intron 2 (c.357+3G>A) was identified at the heterozygous state in an IGHD patient. The in vitro and in vivo mRNA analysis of the wild-type HESX1 allele revealed the presence of the whole cDNA and two isoforms lacking exon 2 and exons 2-3 respectively. The mutant HESX1 allele yielded only two splicing products, the whole cDNA and the cDNA missing exons 2-3, whereas the mRNA lacking exon 2 was absent. An in vitro assay demonstrated that the exon 2-deleted mRNA, predicting a prematurely truncated protein, is subjected to nonsense-mediated mRNA decay (NMD). The c.357+3G>A mutation prevents the generation of one of the alternative isoforms normally produced by the wild-type allele, predicting a truncated HESX1 protein. The mutation is likely to cause IGHD in the heterozygous patient by interfering with the downregulation of HESX1 expression mediated by alternative splicing and NMD. Our results open new insight into the mechanism of HESX1 regulation suggesting that the coupling of alternative splicing and NMD might play a fundamental role in directing the HESX1 expression, and that the alteration of this process might lead to severe consequences.
    European Journal of Endocrinology 02/2011; 164(5):705-13. · 3.14 Impact Factor
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    ABSTRACT: Motor neurons in amyotrophic lateral sclerosis (ALS) are characterized by the presence of inclusion bodies composed of intermediate filament (IF) proteins. Peripherin protein is as components of these inclusions and rare mutations in peripherin gene (PRPH) were identified in sporadic ALS cases. The aim of this study was to further define the spectrum of PRPH mutations in a cohort of 122 Italian ALS patients. We screened the coding sequence, the exon/intron boundaries, and the 5'-3' un-translated regions (UTRs) in 122 ALS patients. Eighteen sequence variations were detected. Seven variants were not identified in a panel of at least 245 matched controls, including 2 missense variations, namely p.R133P and p.D141Y, each identified in one heterozygous patient. p.R133P was newly identified whereas p.D141Y was previously described in one homozygous sporadic ALS patient. These 2 variants were predicted to have a deleterious effect on protein structure or function. This work contributes to determine the role of PRPH gene variants in ALS. Further studies are necessary to define the mechanisms through which the mutant peripherin could cause ALS phenotype.
    Neurobiology of aging 04/2010; 32(3):552.e1-6. · 5.94 Impact Factor
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    ABSTRACT: Mutations in the GHRH receptor (GHRHR) have been detected in the familial type-IB isolated GH deficiency (IGHD-IB) inherited as an autosomal recessive disorder and characterized by a low but detectable serum GH level and good response to substitutive GH therapy. The aim of our study was the identification of mutations in sporadic patients with a IGHD-IB phenotype. The GHRHR gene was systematically screened by DHPLC in 134 IGHD patients with no family history of the disorder or declared parental consanguinity. We identified a novel variation, Val10Gly, within the signal peptide at the heterozygous state in three patients and in one of 1084 controls (P = 0.004), suggesting that it might contribute to IGHD. The functional analysis showed that the signal peptide is not cleaved from the mutant GHRHR, which in turn is not translocated to the cellular surface, demonstrating that 10Gly drastically affects the receptor correct processing. Because 10Gly was also present in normal-stature relatives of the patients as well as in a control, it is likely that it exerts its effects in the context of other genetic and environmental susceptibility factors. At difference from previous papers reporting GHRHR mutations in familial cases with a clear recessive mode of inheritance, our study was conducted on a large sample of sporadic patients and allowed to discover a novel mechanism of the disease caused by a recurrent dominant mutation in the GHRHR signal peptide associated with incomplete penetrance.
    The Journal of clinical endocrinology and metabolism 08/2009; 94(10):3939-47. · 6.50 Impact Factor
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    ABSTRACT: Mutations in the gene encoding the pituitary transcription factor POU1F1 (Pit-1, pituitary transcription factor-1) have been described in combined pituitary hormone deficiency (CPHD). The aim of this study was the characterisation of the molecular defect causing CPHD in a patient born to consanguineous parents. The case of a 12.5-yr-old girl presenting with severe growth failure at diagnosis (-3 SD score at 3 months) and deficiency of GH, PRL, and TSH was investigated for the presence of POU1F1 gene mutations by denaturing high performance liquid chromatography analysis. A novel mutation adjacent to the IVS2 splicing acceptor site (IVS2-3insA) was identified in the patient at the homozygous state. Analysis of patient's lymphocyte mRNA and an in vitro splicing assay revealed the presence of 2 aberrant splicing products: a) deletion of the first 71 nucleotides of exon 3, altering the open reading frame and generating a premature stop codon, b) total exon 3 skipping resulting in an in frame deleted mRNA encoding a putative protein lacking part of the transactivation domain and of the POUspecific homeodomain. Notably, the patient's relatives heterozygous for the mutation had PRL levels under the normal range with no evident clinical symptoms. The IVS2- 3insAmutation, responsible for CPHD at the homozygous state, causes the presence of 2 aberrant splicing products encoding non-functional products. In the heterozygotes one normal allele might not guarantee a complete pituitary function.
    Journal of endocrinological investigation 06/2009; 32(8):653-8. · 1.65 Impact Factor
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    ABSTRACT: Causal mutations have been detected only in a minority of isolated GH deficiency (IGHD) patients. Idiopathic IGHD might be the result of the interaction between several low-penetrance genetic factors and the environment. The aim of this study was to test the contribution to IGHD of genetic variations in the GH1 gene regulatory regions. A case-control association study was performed including 118 sporadic IGHD patients with a nonsevere phenotype (height -4/-1 sd score and partial GH deficiency) and two control groups, normal stature (n=200) and short-stature individuals with normal GH secretion (n=113). Seven single-nucleotide polymorphisms in the GH1 promoter, one in the IVS4 region, and two in the locus control region were analyzed. The -57T allele within the vitamin D-responsive element showed a positive significant association when comparing patients with normal (P=0.006) or short stature (P=0.0011) controls. The genotype -57TT showed an odds ratio of 2.93 (1.44-5.99) and 2.99 (1.42-6.31), respectively. The functional relevance of the -57 variation was demonstrated by the luciferase assay in the presence of vitamin D. The vitamin D-induced inhibition of luciferase activity was significantly (P=0.012) stronger for the promoter haplotype carrying the associated variation -57T [haplotype #1 (hp#1)] with respect to hp#2, bearing -57G. Replacement of the T with a G at -57 on hp#1 abolished the repression, demonstrating that the T at position -57 is necessary to determine the greater vitamin D-induced inhibitory effect of hp#1. EMSA experiments showed a different band-shift pattern of the T and G sequences. The common -57G-->T polymorphism contributes to IGHD susceptibility, indicating that it may have a multifactorial etiology.
    Journal of Clinical Endocrinology &amp Metabolism 04/2008; 93(3):1005-12. · 6.43 Impact Factor
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    ABSTRACT: The proximal promoter of the human growth hormone gene (GH1) is highly polymorphic. We tested if promoter haplotypes differing at possibly functional sites, namely -278T/G (in the NF1 binding site), -75A/G (in the proximal Pit-1 binding site) and -57G/T (in the VDR binding site), induced a different luciferase activity when transfected in a rat pituitary cell line. The presence of a G instead of an A at position -75 induced a more than two-fold reduced activity (p<0.0001). In accordance with this findings the electrophoretic mobility shift assay demonstrated a reduced affinity of the -75G for the pituitary transcription factor Pit-1. Despite the strong effect of this polymorphism in vitro, the -75G variation was not associated to an impairment of the GH secretion in vivo.
    Molecular and Cellular Endocrinology 04/2006; 249(1-2):51-7. · 4.04 Impact Factor
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    ABSTRACT: Mutations in the Prophet of Pit-1 (Prop-1), a paired-like homeodomain transcription factor involved in the early embryonic pituitary development, have been reported as a cause of combined hormone deficiency (CPHD) involving growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH), gonadotrophins and in some cases adrenocorticotrophic hormone (ACTH). We report two pre-pubertal siblings with short stature and deficiency of GH and TSH at presentation. Molecular analysis of the PROP1 gene revealed compound heterozygotes for two novel missense mutations of the PROP1 gene affecting the same amino acid (Arg71Cys and Arg71His) in the first alpha helix of the Prop-1 homeodomain.
    Clinical Genetics 09/2003; 64(2):142-7. · 3.94 Impact Factor