Alessia Gozzini

Multiple endocrine neoplasia type 1

Multiple Endocrine Neoplasia type 1 (MEN1) is a rare autosomal dominant hereditary cancer syndrome presented mostly by tumours of the parathyroids, endocrine pancreas and anterior pituitary, and characterised by a very high penetrance and an equal sex distribution. It occurs in approximately one in 30,000 individuals. Two different forms, sporadic and familial, have been described. The sporadic form presents with two of the three principal MEN1-related endocrine tumours (parathyroid adenomas, entero-pancreatic tumours and pituitary tumours) within a single patient, while the familial form consists of a MEN1 case with at least one first degree relative showing one of the endocrine characterising tumours. Other endocrine and non-endocrine lesions, such as adrenal cortical tumours, carcinoids of the bronchi, gastrointestinal tract and thymus, lipomas, angiofibromas, collagenomas have been described. The responsible gene, MEN1, maps on chromosome 11q13 and encodes a 610 aminoacid nuclear protein, menin, with no sequence homology to other known human proteins. MEN1 syndrome is caused by inactivating mutations of the MEN1 tumour suppressor gene. This gene is probably involved in the regulation of several cell functions such as DNA replication and repair and transcriptional machinery. The combination of clinical and genetic investigations, together with the improving of molecular genetics knowledge of the syndrome, helps in the clinical management of patients. Treatment consists of surgery and/or drug therapy, often in association with radiotherapy or chemotherapy. Currently, DNA testing allows the early identification of germline mutations in asymptomatic gene carriers, to whom routine surveillance (regular biochemical and/or radiological screenings to detect the development of MEN1-associated tumours and lesions) is recommended.

Bioinformatics for Next Generation Sequencing Data

The emergence of next-generation sequencing (NGS) platforms imposes increasing demands on statistical methods and bioinformatic tools for the analysis and the management of the huge amounts of data generated by these technologies. Even at the early stages of their commercial availability, a large number of softwares already exist for analyzing NGS data. These tools can be fit into many general categories including alignment of sequence reads to a reference, base-calling and/or polymorphism detection, de novo assembly from paired or unpaired reads, structural variant detection and genome browsing. This manuscript aims to guide readers in the choice of the available computational tools that can be used to face the several steps of the data analysis workflow.

Estrogen Receptor Expression in Cutaneous Melanoma A Real-Time Reverse Transcriptase-Polymerase Chain Reaction and Immunohistochemical Study

OBJECTIVE To evaluate estrogen receptor (ER) expression in human melanoma tissues and in the adjacent healthy skin with the aim of explaining whether the ERalpha:ERbeta expression ratio has a role in neoplastic progression. DESIGN Prospective study. SETTING Department of Dermatology, University of Florence, Florence, Italy. Patients Fourteen patients, 12 with cutaneous melanoma (6 women and 6 men) and 2 with melanocytic nevi (1 woman and 1 man). MAIN OUTCOME MEASURES Using quantitative reverse transcriptase-polymerase chain reaction and immunohistochemical analysis, we analyzed ERalpha and ERbeta messenger RNA (mRNA) and ERbeta protein expression in cutaneous melanoma and in the healthy skin surrounding the lesions. RESULTS All melanocytic lesions expressed detectable levels of ERalpha and ERbeta mRNA as well as ERbeta protein. Dividing melanoma cases into 2 groups according to Breslow thickness, we found lower ERalpha and ERbeta mRNA levels and lower ERbeta protein levels in thicker, more invasive tumors. CONCLUSIONS These observations suggest a role for ERs in the metastatic process of melanoma cells, pointing at the possibility of using ERbeta expression as a prognostic indicator of melanoma. The possibility of distinguishing proliferative melanomas, which are associated with dismal prognosis, from the so-called dormant melanomas opens up novel avenues in tailoring individual treatments, as already happens for other tumors.

Myostatin Polymorphisms and Age-Related Sarcopenia in the Italian Population

13. Posnett DN, Stein RS, Graber SE, et al. Cimetidine-induced neutropenia: A possible dose-related phenomenon. Arch Intern Med 1979;139:584–586. 14. Watts RG, Emanuel PD, Zuckerman KS, et al. Valproic acid-induced cytopenias: Evidence for a dose-related suppression of hematopoiesis. J Pediatr 1990;117:495–499. 15. Pisciotta AV. Immune and toxic mechanisms in drug-induced agranulocytosis. Semin Hematol 1973;10:279–310.

Estrogen receptor alpha and beta polymorphisms: is there an association with bone mineral density, plasma lipids, and response to postmenopausal hormone therapy?

Objective and design: A cross-sectional segregation analysis of polymorphisms in the estrogen receptor (ER) genes (Pvull and Xbal in ERα, and Alul in ERAβ with bone mineral density in the lumbar spine and forearm and with lipid profile was performed in 1098 postmenopausal women. Additionally, in a subpopulation of 280 women, who completed 1 year of treatment with estrogen plus progestin, the association between genotypes and the response to treatment in both plasma lipids and bone was investigated. In another untreated subpopulation of 443 women, genotype influence on the prevalence of vertebral fractures and on annual rate of bone loss during a mean follow-up period of 11 years was estimated. Results: Baseline plasma lipids, bone mineral density, annual rate of bone loss and prevalence of spinal fractures were not significantly associated with polymorphisms in the ERβ gene. The ERA polymorphism was significantly associated with bone loss from the distal forearm (P = 0.04) but not with bone loss from the spine. After 1 year of treatment with hormone therapy there was also a significant association between the ERβ polymorphism and the response in total cholesterol (P = 0.02); while the ERα gene polymorphisms did not significantly influence the response to hormone therapy. Conclusions: In a large white population of postmenopausal women, ERα gene polymorphisms were not associated with bone mineral density or lipid profile at baseline or after hormone therapy. Conversely, the ERβ genotype appeared to segregate with bone loss from the forearm and to modulate the decrease in total cholesterol during hormone therapy.

A Novel Recessive Mutation of Fibroblast Growth Factor-23 in Tumoral Calcinosis

BACKGROUND Tumoral calcinosis is a rare disease characterized by hyperphosphatemia due to hypophosphaturia and by ectopic calcifications. Phosphatonins are important hormones that regulate phosphorus homeostasis. Tumoral calcinosis is a rare congenital disorder in which the differential diagnosis from other syndromes associated with extraskeletal calcifications may be difficult. Mutations in the UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-3 (GALNT3) and fibroblast growth factor-23 (FGF23) genes have been described. Mutational analysis is important for the early recognition of the disorder, for prevention of its complications, and for family screening strategies. We examined two unrelated white patients affected by tumoral calcinosis. METHODS The first patient was a woman with a history of an ectopic calcification in the left shoulder. The second patient was a man with a history of an ectopic calcification in the right buttock. Routine biochemistry and FGF-23 assays were performed on serum samples. Genomic DNA was extracted from peripheral blood. The FGF23 and GALNT3 genes were analyzed by direct sequencing. RESULTS A new homozygous H41Q codon 41, C-->A transversion at position 123 (c.123C>A) in exon 1 of the FGF23 gene was evidenced in both patients. No mutation of the GALNT3 gene was detected in these patients. As determined by an ELISA assay, intact FGF-23 circulating protein was low in both patients. CONCLUSIONS This is the fourth mutation of the FGF23 gene described in subjects with tumoral calcinosis.

FokI Polymorphism of the Vitamin D Receptor Gene Correlates with Parameters of Bone Mass and Turnover in a Female Population of the Italian Island of Lampedusa

One of the most promising genetic approaches to dissecting a multifactorial disease is represented by genetically isolated population studies. We studied a genetic marker in a cohort of women living on the Mediterranean island of Lampedusa, a geographically isolated population. Lampedusa, located between the African coast and Sicily, consists of a young genetic isolate (<20 generations) with an exponential growth in the last generations. We analyzed the association between the FokI vitamin D receptor (VDR) gene polymorphism, previously proposed as a predictor of bone mass, with parameters of bone mass and turnover in a cohort of pre- and postmenopausal women living on Lampedusa. In 424 women (277 postmenopausal and 147 premenopausal), allelic frequencies were 49% for the F allele and 51% for the f allele. Using analysis of covariance, we found that subjects with ff genotype exhibited a significantly (P < 0.001) lower lumbar spine bone mass, by dual-energy X-ray absorptiometry, and lower values of bone ultrasonographic parameters (speed of sound and broadband ultrasound attenuation) relative to those with Ff and FF genotypes. Conversely, osteocalcin and serum cross-laps were significantly higher in ff and Ff compared to FF genotype. Our data suggest that FokI VDR polymorphism may contribute to the determination of bone mass and turnover in both pre- and postmenopausal women in this geographically isolated population.

Segregation of a M404V mutation of the p62/sequestosome 1 (p62/SQSTM1) gene with polyostotic Paget's disease of bone in an Italian family

Mutations of the p62/Sequestosome 1 gene (p62/SQSTM1) account for both sporadic and familial forms of Pagets disease of bone (PDB). We originally described a methionine→valine substitution at codon 404 (M404V) of exon 8, in the ubiquitin protein-binding domain of p62/SQSTM1 gene in an Italian PDB patient. The collection of data from the patients pedigree provided evidence for a familial form of PDB. Extension of the genetic analysis to other relatives in this family demonstrated segregation of the M404V mutation with the polyostotic PDB phenotype and provided the identification of six asymptomatic gene carriers. DNA for mutational analysis of the exon 8 coding sequence was obtained from 22 subjects, 4 PDB patients and 18 clinically unaffected members. Of the five clinically ascertained affected members of the family, four possessed the M404V mutation and exhibited the polyostotic form of PDB, except one patient with a single X-ray-assessed skeletal localization and one with a polyostotic disease who had died several years before the DNA analysis. By both reconstitution and mutational analysis of the pedigree, six unaffected subjects were shown to bear the M404V mutation, representing potential asymptomatic gene carriers whose circulating levels of alkaline phosphatase were recently assessed as still within the normal range. Taken together, these results support a genotype–phenotype correlation between the M404V mutation in the p62/SQSTM1 gene and a polyostotic form of PDB in this family. The high penetrance of the PDB trait in this family together with the study of the asymptomatic gene carriers will allow us to confirm the proposed genotype–phenotype correlation and to evaluate the potential use of mutational analysis of the p62/SQSTM1 gene in the early detection of relatives at risk for PDB.

Methodological models for in vitro amplification and maintenance of human articular chondrocytes from elderly patients.

Articular cartilage defects, an exceedingly common problem closely correlated with advancing age, is characterized by lack of spontaneous resolution because of the limited regenerative capacity of adult articular chondrocytes. Medical and surgical therapies yield unsatisfactory short-lasting results. Recently, cultured autologous chondrocytes have been proposed as a source to promote repair of deep cartilage defects. Despite encouraging preliminary results, this approach is not yet routinely applicable in clinical practice, but for young patients. One critical points is the isolation and ex vivo expansion of large enough number of differentiated articular chondrocytes. In general, human articular chondrocytes grown in monolayer cultures tend to undergo dedifferentiation. This reversible process produces morphological changes by which cells acquire fibroblast-like features, loosing typical functional characteristics, such as the ability to synthesize type II collagen. The aim of this study was to isolate human articular chondrocytes from elderly patients and to carefully characterize their morphological, proliferative, and differentiative features. Cells were morphologically analyzed by optic and transmission electron microscopy (TEM). Production of periodic acid-schiff (PAS)-positive cellular products and of type II collagen mRNA was monitored at different cellular passages. Typical chondrocytic characteristics were also studied in a suspension culture system with cells encapsulated in alginate-polylysine-alginate (APA) membranes. Results showed that human articular chondrocytes can be expanded in monolayers for several passages, and then microencapsulated, retaining their morphological and functional characteristics. The results obtained could contribute to optimize expansion and redifferentiation sequences for applying cartilage tissue engineering in the elderly patients.

In vitro effects of oestrogens, antioestrogens and SERMs on pancreatic solid pseudopapillary neoplasm-derived primary cell culture

Background: Solid-pseudopapillary neoplasms of the pancreas (SPNs) are uncommon tumours usually frequent in young women. Although the pathogenesis of SPNs is uncertain a potential influence of the sex hormone milieu on the biology of these tumours has been suggested. The controversial expression of oestrogen receptors (ERs) in SPNs, provide a rationale for studying the effects of oestrogenic molecules on SPN development. Methods: The expression of a large series of hormonal ligands and receptors was evaluated in tissue specimens and in a primary cell culture (SPNC), obtained from a SPN in young female patient. The effects of 17β-oestradiol (17βE2), ICI 182,780 and tamoxifen (Tam) on cell replication and growth were examined. Results: We have established SPNC primary line. Immunocytochemical analysis was positive for vimentin, cyclin D1 and β-catenin and negative for cytokeratin, CD10 and neuroendocrine markers, in line with the immunostaining features of the tumoral tissue. Expression of ERα, ERβ and progesterone mRNAs was demonstrated in SPNC and tumor tissue. A proliferative and antiproliferative action of 17βE2 and Tam respectively were proved in SPNC. Conclusions: In conclusion, we provide the first direct evidence that oestrogenic molecules can influence proliferation of SPNC, offering future strategies in the control of this neoplasia via selective ER modulators.