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author:("pilus, GIULIO")
1.  LEOPARD syndrome: clinical dilemmas in differential diagnosis of RASopathies 
BMC Medical Genetics  2014;15:44.
Background
Diagnosis within RASopathies still represents a challenge. Nevertheless, many efforts have been made by clinicians to identify specific clinical features which might help in differentiating one disorder from another. Here, we describe a child initially diagnosed with Neurofibromatosis-Noonan syndrome. The follow-up of the proband, the clinical evaluation of his father together with a gene-by-gene testing approach led us to the proper diagnosis.
Case presentation
We report a 8-year-old male with multiple café-au-lait macules, several lentigines and dysmorphic features that suggest Noonan syndrome initially diagnosed with Neurofibromatosis-Noonan syndrome. However, after a few years of clinical and ophthalmological follow-up, the absence of typical features of Neurofibromatosis type 1 and the lack of NF1 mutation led us to reconsider the original diagnosis. A new examination of the patient and his similarly affected father, who was initially referred as healthy, led us to suspect LEOPARD syndrome, The diagnosis was then confirmed by the occurrence in both patients of a heterozygous mutation c.1403 C > T, p.(Thr468Met), of PTPN11. Subsequently, the proband was also found to have type-1 Arnold-Chiari malformation in association with syringomyelia.
Conclusion
Our experience suggests that differential clinical diagnosis among RASopathies remains ambiguous and raises doubts on the current diagnostic clinical criteria. In some cases, genetic tests represent the only conclusive proof for a correct diagnosis and, consequently, for establishing individual prognosis and providing adequate follow-up. Thus, molecular testing represents an essential tool in differential diagnosis of RASophaties. This view is further strengthened by the increasing accessibility of new sequencing techniques.
Finally, to our knowledge, the described case represents the third report of the occurrence of Arnold Chiari malformation and the second description of syringomyelia with LEOPARD syndrome.
doi:10.1186/1471-2350-15-44
PMCID: PMC4005403  PMID: 24767283
LEOPARD syndrome; Neurofibromatosis type 1; RASopathy; PTPN11
2.  Giant thrombosed intracavernous carotid artery aneurysm presenting as Tolosa–Hunt syndrome in a patient harboring a new pathogenic neurofibromatosis type 1 mutation: a case report and review of the literature 
Neurofibromatosis type 1 (NF1) is a relatively common single-gene disorder, and is caused by heterozygous mutations in the NF1 gene that result in a loss of activity or in a nonfunctional neurofibromin protein. Despite the common association of NF1 with neurocutaneous features, its pathology can extend to numerous tissues not derived from the neural crest. Among the rare cerebrovascular abnormalities in NF1, more than 85% of cases are of purely occlusive or stenotic nature, with intracranial aneurysm being uncommon. Predominantly, the aneurysms are located in the internal carotid arteries (ICAs), being very rare bilateral aneurysms. This report describes a very unusual case of fusiform aneurysms of both ICAs in a Caucasian NF1 patient, with a new pathogenic intragenic heterozygous deletion of the NF1 gene, presenting at age 22 years with Tolosa–Hunt syndrome, because of partial thrombosis of the left giant intracavernous aneurysm. Medical treatment with anticoagulant therapy allowed a good outcome for the patient. In conclusion, early identification of cerebral arteriopathy in NF1 and close follow-up of its progression by neuroimaging may lead to early medical or surgical intervention and prevention of significant neurologic complications.
doi:10.2147/NDT.S49784
PMCID: PMC3901779  PMID: 24476631
neurofibromatosis type 1; NF1 gene; multiplex ligation-dependent probe amplification (MLPA); intracranial aneurysms; Tolosa–Hunt syndrome
3.  Next-Generation Sequencing Identifies Transportin 3 as the Causative Gene for LGMD1F 
PLoS ONE  2013;8(5):e63536.
Limb-girdle muscular dystrophies (LGMD) are genetically and clinically heterogeneous conditions. We investigated a large family with autosomal dominant transmission pattern, previously classified as LGMD1F and mapped to chromosome 7q32. Affected members are characterized by muscle weakness affecting earlier the pelvic girdle and the ileopsoas muscles. We sequenced the whole exome of four family members and identified a shared heterozygous frame-shift variant in the Transportin 3 (TNPO3) gene, encoding a member of the importin-β super-family. The TNPO3 gene is mapped within the LGMD1F critical interval and its 923-amino acid human gene product is also expressed in skeletal muscle. In addition, we identified an isolated case of LGMD with a new missense mutation in the same gene. We localized the mutant TNPO3 around the nucleus, but not inside. The involvement of gene related to the nuclear transport suggests a novel disease mechanism leading to muscular dystrophy.
doi:10.1371/journal.pone.0063536
PMCID: PMC3646821  PMID: 23667635
4.  Use of a Lower Dosage Liver-Detargeted AAV Vector to Prevent Hamster Muscular Dystrophy 
Human Gene Therapy  2013;24(4):424-430.
Abstract
The BIO14.6 hamster carries a mutation in the delta sarcoglycan gene causing muscular dystrophy and cardiomyopathy. The disease can be prevented by systemic delivery of delta sarcoglycan cDNA using adeno-associated viruses (AAVs). However, all AAVs also target the liver, raising concerns about their therapeutic efficacy in human applications. We compared the AAV2/8 with the chimeric AAV2/2i8, in which the 585-QQNTAP-590 motif of the AAV8 serotype was added to the heparan sulfate receptor footprint of the AAV2 strain. Both vectors carrying the human delta sarcoglycan cDNA were delivered into 24 14-day-old BIO14.6 hamsters. We followed transgene expression in muscle and liver for 7 months. We detected a sustained ectopic expression of delta sarcoglycan in the liver when using AAV2/8 but not AAV2/2i8. Genomic copies of AAV2/2i8 were not detectable in the liver, while at least 100-fold more copies of AAV2/8 were counted. In contrast, the hamster skeletal muscle expressed more delta sarcoglycan using AAV2/2i8 and were still healthy after 7 months at the lower dosage. We conclude that this chimeric vector is a robust option for safer and longer-term diseased muscle targeting.
Rotundo and colleagues generate a chimeric adeno-associated virus (AAV) 2/8 vector called AAV2/2i8, in which the 585-QQNTAP-590 motif of the AAV8 serotype is added to the heparan sulfate receptor footprint of the AAV2 strain. They show that muscle delivery of AAV2/2i8 works in the BIO14.6 hamster at lower dosages than AAV2/8 and that this occurs without any liver targeting, even within the context of intraperitoneal injections.
doi:10.1089/hum.2012.121
PMCID: PMC3631017  PMID: 23427808
5.  Enhancer Chip: Detecting Human Copy Number Variations in Regulatory Elements 
PLoS ONE  2012;7(12):e52264.
Critical functional properties are embedded in the non-coding portion of the human genome. Recent successful studies have shown that variations in distant-acting gene enhancer sequences can contribute to disease. In fact, various disorders, such as thalassaemias, preaxial polydactyly or susceptibility to Hirschsprung’s disease, may be the result of rearrangements of enhancer elements. We have analyzed the distribution of enhancer loci in the genome and compared their localization to that of previously described copy-number variations (CNVs). These data suggest a negative selection of copy number variable enhancers. To identify CNVs covering enhancer elements, we have developed a simple and cost-effective test. Here we describe the gene selection, design strategy and experimental validation of a customized oligonucleotide Array-Based Comparative Genomic Hybridization (aCGH), designated Enhancer Chip. It has been designed to investigate CNVs, allowing the analysis of all the genome with a 300 Kb resolution and specific disease regions (telomeres, centromeres and selected disease loci) at a tenfold higher resolution. Moreover, this is the first aCGH able to test over 1,250 enhancers, in order to investigate their potential pathogenic role. Validation experiments have demonstrated that Enhancer Chip efficiently detects duplications and deletions covering enhancer loci, demonstrating that it is a powerful instrument to detect and characterize copy number variable enhancers.
doi:10.1371/journal.pone.0052264
PMCID: PMC3527541  PMID: 23284961
6.  Next generation sequencing (NGS) strategies for the genetic testing of myopathies 
Acta Myologica  2012;31(3):196-200.
Next generation sequencing (NGS) technologies offer the possibility to map entire genomes at affordable costs. This brings the genetic testing procedure to a higher level of complexity. The positive aspect is the ease to cope with the complex diagnosis of genetically heterogeneous disorders and to identify novel disease genes. Worries arise from the management of too many DNA variations with unpredictable meaning and incidental findings that can cause ethical and clinical dilemmas. The technology of enrichment makes possible to focus the sequencing to the exome or to a more specific DNA target. This is being used to provide insights into the genetics underlying Mendelian traits involved in myopathies and to set up cost-effective diagnostic tests. This huge potential of the NGS applications makes likely that these will soon become the first approach in genetic diagnostic laboratories.
PMCID: PMC3631804  PMID: 23620651
Next generation sequencing; NGS; neuromuscular disorders
7.  Improvement of survival in Duchenne Muscular Dystrophy: retrospective analysis of 835 patients 
Acta Myologica  2012;31(2):121-125.
Duchenne Muscular Dystrophy (DMD) is the most common muscle disease in children. Historically, DMD results in loss of ambulation between ages 7 and 13 years and death in the teens or 20s. In order to determine whether survival has improved over the decades and whether the impact of nocturnal ventilation combined with a better management of cardiac involvement has been able to modify the pattern of survival, we reviewed the notes of 835 DMD patients followed at the Naples Centre of Cardiomyology and Medical Genetics from 1961 to 2006. Patients were divided, by decade of birth, into 3 groups: 1) DMD born between 1961 and 1970; 2) DMD born between 1971 and 1980; 3) DMD born between 1981 and 1990; each group was in turn subdivided into 15 two-year classes, from 14 to 40 years of age. Age and causes of death, type of cardiac treatment and use of a mechanical ventilator were carefully analyzed.
The percentage of survivors in the different decades was statistically compared by chi-square test and Kaplan-Meier survival curves analyses. A significant decade on decade improvement in survival rate was observed at both the age of 20, where it passed from 23.3% of patients in group 1 to 54% of patients in group 2 and to 59,8% in patients in group 3 (p < 0.001) and at the age of 25 where the survival rate passed from 13.5% of patients in group 1 to 31.6% of patients in group 2 and to 49.2% in patients in group 3 (p < 0.001).
The causes of death were both cardiac and respiratory, with a prevalence of the respiratory ones till 1980s. The overall mean age for cardiac deaths was 19.6 years (range 13.4-27.5), with an increasing age in the last 15 years. The overall mean age for respiratory deaths was 17.7 years (range 11.6-27.5) in patients without a ventilator support while increased to 27.9 years (range 23-38.6) in patients who could benefit of mechanical ventilation.
This report documents that DMD should be now considered an adulthood disease as well, and as a consequence more public health interventions are needed to support these patients and their families as they pass from childhood into adult age.
PMCID: PMC3476854  PMID: 23097603
Duchenne; survival; cardiomyopathy
8.  Worsening of Cardiomyopathy Using Deflazacort in an Animal Model Rescued by Gene Therapy 
PLoS ONE  2011;6(9):e24729.
We have previously demonstrated that gene therapy can rescue the phenotype and extend lifespan in the delta-sarcoglycan deficient cardiomyopathic hamster. In patients with similar genetic defects, steroids have been largely used to slow down disease progression. Aim of our study was to evaluate the combined effects of steroid treatment and gene therapy on cardiac function. We injected the human delta-sarcoglycan cDNA by adeno-associated virus (AAV) 2/8 by a single intraperitoneal injection into BIO14.6 Syrian hamsters at ten days of age to rescue the phenotype. We then treated the hamsters with deflazacort. Treatment was administered to half of the hamsters that had received the AAV and the other hamsters without AAV, as well as to normal hamsters. Both horizontal and vertical activities were greatly enhanced by deflazacort in all groups. As in previous experiments, the AAV treatment alone was able to preserve the ejection fraction (70±7% EF). However, the EF value declined (52±14%) with a combination of AAV and deflazacort. This was similar with all the other groups of affected animals. We confirm that gene therapy improves cardiac function in the BIO14.6 hamsters. Our results suggest that deflazacort is ineffective and may also have a negative impact on the cardiomyopathy rescue, possibly by boosting motor activity. This is unexpected and may have significance in terms of the lifestyle recommendations for patients.
doi:10.1371/journal.pone.0024729
PMCID: PMC3170375  PMID: 21931833
9.  Combined deficiency of alpha and epsilon sarcoglycan disrupts the cardiac dystrophin complex 
Human Molecular Genetics  2011;20(23):4644-4654.
Cardiomyopathy is a puzzling complication in addition to skeletal muscle pathology for patients with mutations in β-, γ- or δ-sarcoglycan (SG) genes. Patients with mutations in α-SG rarely have associated cardiomyopathy, or their cardiac pathology is very mild. We hypothesize that a fifth SG, ɛ-SG, may compensate for α-SG deficiency in the heart. To investigate the function of ɛ-SG in striated muscle, we generated an Sgce-null mouse and a Sgca-;Sgce-null mouse, which lacks both α- and ɛ-SGs. While Sgce-null mice showed a wild-type phenotype, with no signs of muscular dystrophy or heart disease, the Sgca-;Sgce-null mouse developed a progressive muscular dystrophy and a more anticipated and severe cardiomyopathy. It shows a complete loss of residual SGs and a strong reduction in both dystrophin and dystroglycan. Our data indicate that ɛ-SG is important in preventing cardiomyopathy in α-SG deficiency.
doi:10.1093/hmg/ddr398
PMCID: PMC3209833  PMID: 21890494
10.  Muscular dystrophy with marked Dysferlin deficiency is consistently caused by primary dysferlin gene mutations 
Dysferlin is a 237-kDa transmembrane protein involved in calcium-mediated sarcolemma resealing. Dysferlin gene mutations cause limb-girdle muscular dystrophy (LGMD) 2B, Miyoshi myopathy (MM) and distal myopathy of the anterior tibialis. Considering that a secondary Dysferlin reduction has also been described in other myopathies, our original goal was to identify cases with a Dysferlin deficiency without dysferlin gene mutations. The dysferlin gene is huge, composed of 55 exons that span 233 140 bp of genomic DNA. We performed a thorough mutation analysis in 65 LGMD/MM patients with ≤20% Dysferlin. The screening was exhaustive, as we sequenced both genomic DNA and cDNA. When required, we used other methods, including real-time PCR, long PCR and array CGH. In all patients, we were able to recognize the primary involvement of the dysferlin gene. We identified 38 novel mutation types. Some of these, such as a dysferlin gene duplication, could have been missed by conventional screening strategies. Nonsense-mediated mRNA decay was evident in six cases, in three of which both alleles were only detectable in the genomic DNA but not in the mRNA. Among a wide spectrum of novel gene defects, we found the first example of a ‘nonstop' mutation causing a dysferlinopathy. This study presents the first direct and conclusive evidence that an amount of Dysferlin ≤20% is pathogenic and always caused by primary dysferlin gene mutations. This demonstrates the high specificity of a marked reduction of Dysferlin on western blot and the value of a comprehensive molecular approach for LGMD2B/MM diagnosis.
doi:10.1038/ejhg.2011.70
PMCID: PMC3179367  PMID: 21522182
dysferlin; limb-girdle muscular dystrophy; Miyoshi myopathy; nonsense-mediated mRNA decay; comparative genomic hybridization
11.  Interaction of Vault Particles with Estrogen Receptor in the MCF-7 Breast Cancer Cell  
The Journal of Cell Biology  1998;141(6):1301-1310.
A 104-kD protein was coimmunoprecipitated with the estrogen receptor from the flowtrough of a phosphocellulose chromatography of MCF-7 cell nuclear extract. mAbs to this protein identified several cDNA clones coding for the human 104-kD major vault protein. Vaults are large ribonucleoprotein particles of unknown function present in all eukaryotic cells. They have a complex morphology, including several small molecules of RNA, but a single protein species, the major vault protein, accounts for >70% of their mass. Their shape is reminiscent of the nucleopore central plug, but no proteins of known function have been described to interact with them. Western blot analysis of vaults purified on sucrose gradient showed the presence of estrogen receptor co-migrating with the vault peak. The AER317 antibody to estrogen receptor coimmunoprecipitated the major vault protein and the vault RNA also in the 20,000 g supernatant fraction. Reconstitution experiments of estrogen receptor fragments with the major vault protein mapped the site of the interaction between amino acids 241 and 280 of human estrogen receptor, where the nuclear localization signal sequences are located. Estradiol treatment of cells increased the amount of major vault protein present in the nuclear extract and coimmunoprecipitated with estrogen receptor, whereas the anti-estrogen ICI182,780 had no effect. The hormone-dependent interaction of vaults with estrogen receptor was reproducible in vitro and was prevented by sodium molybdate. Antibodies to progesterone and glucocorticoid receptors were able to coimmunoprecipitate the major vault protein. The association of nuclear receptors with vaults could be related to their intracellular traffic.
PMCID: PMC2132791  PMID: 9628887

Results 1-11 (11)