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 Paget's 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 patient's 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.     

Circulating gammadelta T cells in young/adult and old patients with cutaneous primary melanoma

BACKGROUND: In a previous study we demonstrated the existence of numerical and functional alterations of gammadelta T cells in healthy elderly. Recently, we analysed the involvement of gammadelta T lymphocytes in malignant melanoma, describing a lower frequency of circulating gammadelta T cells, an altered pattern of cytokine production, and an impaired in vitro expansion of these cells in primary cutaneous melanoma patients. METHODS: In this study we investigated the existence of numerical and functional alterations of circulating gammadelta T cells in young/adult and old melanoma patients, comparing the data obtained with age-matched healthy subjects. RESULTS: We demonstrated that the number of circulating gammadelta+ T cells was significantly and similarly reduced in young/adult and old melanoma patients and in old healthy subjects in comparison with young healthy donors. The decrease was due to a reduction of Vdelta2 T cells whereas the number of Vdelta1 T cells was not affected. A higher percentage of gammadelta+ T cells producing TNF-alpha was found in old healthy donors, whereas a reduced number of TNF-alpha-producing gammadelta+ T cells was present in melanoma patients independently by age. No significant difference was observed in IFN-gamma production. After a 10-day in vitro culture, both the percentage and the expansion index of gammadelta T cells, and in particular of Vdelta2 subset, were significantly and similarly reduced both in young/adult and old melanoma patients, and in healthy aged people, in comparison with young/adult healthy subjects. CONCLUSIONS: Our study demonstrates that the numerical and functional impairment of gammadelta T cells found in melanoma patients is not correlated with age and that it has characteristics very similar to the alterations of gammadelta T cells found in old healthy subjects. We suggest that a similar impairment of gammadelta T cell population may be related to the increased susceptibility to tumors present in the elderly as well as in the pathogenesis of malignant melanoma.     

Structure of the chromo barrel domain from the MOF acetyltransferase

We report here the structure of the putative chromo domain from MOF, a member of the MYST family of histone acetyltransferases that acetylates histone H4 at Lys-16 and is part of the dosage compensation complex in Drosophila. We found that the structure of this domain is a beta-barrel that is distinct from the alpha + beta fold of the canonical chromo domain. Despite the differences, there are similarities that support an evolutionary relationship between the two domains, and we propose the name "chromo barrel." The chromo barrel domains may be divided into two groups, MSL3-like and MOF-like, on the basis of whether a group of conserved aromatic residues is present or not. The structure suggests that, although the MOF-like domains may have a role in RNA binding, the MSL3-like domains could instead bind methylated residues. The MOF chromo barrel shares a common fold with other chromatin-associated modules, including the MBT-like repeat, Tudor, and PWWP domains. This structural similarity suggests a probable evolutionary pathway from these other modules to the canonical chromo domains (or vice versa) with the chromo barrel domain representing an intermediate structure.     

Caspase-3 inhibits the growth of breast cancer cells independent of protease activity

In this study, the MCF-7 breast cancer cells that lack caspase-3 were transfected with a wild type (WT) or mutant caspase-3 cDNA. Expression of the WT, but not of the mutant, caspase-3 was associated with increased caspase activity and susceptibility to staurosporine (STS)-induced apoptosis. Both derivatives displayed inhibition of cell growth compared with vector control cells. Growth inhibition was associated with increased expression of the cyclin dependent kinase (CDK) inhibitor p27Kip1 in the WT, but not in the mutant caspase-3 expressing cells. Cyclin D1 expression level was not affected by caspase-3 expression. Phosphorylation of the Akt protein was decreased in both WT and mutant caspase transfected cells, although Akt expression level remained unchanged. These results suggest that caspase-3 might have biological functions independent of its protease activity and that its loss might contribute to tumor development by increasing the growth potential of cancer cells.     

Identification of distinct N-terminal truncated forms of prion protein in different Creutzfeldt-Jakob disease subtypes

In prion diseases, the cellular prion protein (PrP(C)) is converted to an insoluble and protease-resistant abnormal isoform termed PrP(Sc). In different prion strains, PrP(Sc) shows distinct sites of endogenous or exogenous proteolysis generating a core fragment named PrP27-30. Sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human prion disease, clinically presents with a variety of neurological signs. As yet, the clinical variability observed in sCJD has not been fully explained by molecular studies relating two major types of PrP27-30 with unglycosylated peptides of 21 (type 1) and 19 kDa (type 2) and the amino acid methionine or valine at position 129. Recently, smaller C-terminal fragments migrating at 12 and 13 kDa have been detected in different sCJD phenotypes, but their significance remains unclear. By using two-dimensional immunoblot with anti-PrP antibodies, we identified two novel groups of protease-resistant PrP fragments in sCJD brain tissues. All sCJD cases with type 1 PrP27-30, in addition to MM subjects with type 2 PrP27-30, were characterized by the presence of unglycosylated PrP fragments of 16-17 kDa. Conversely, brain homogenates from patients VV and MV with type 2 PrP27-30 contained fully glycosylated PrP fragments, which after deglycosylation migrated at 17.5-18 kDa. Interestingly, PrP species of 17.5-18 kDa matched deglycosylated forms of the C1 PrP(C) fragment and were associated with tissue PrP deposition as plaque-like aggregates or amyloid plaques. These data show the presence of multiple PrP(Sc) conformations in sCJD and, in addition, shed new light on the correlation between sCJD phenotypes and disease-associated PrP molecules.     

Regulation of A2B adenosine receptor functioning by tumour necrosis factor a in human astroglial cells

Low-affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real-time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-alpha) treatment (24- h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-alpha markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A2B ARs desensitization. In the presence of TNF-alpha, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF-alpha. These results suggest that, in ADF cells, TNF-alpha selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.     

Identification of an envelope protein from the FRD family of human endogenous retroviruses (HERV-FRD) conferring infectivity and functional conservation among simians

A member of the HERV-W family of human endogenous retroviruses (HERV) had previously been demonstrated to encode a functional envelope which can form pseudotypes with human immunodeficiency virus type 1 virions and confer infectivity on the resulting retrovirus particles. Here we show that a second envelope protein sorted out by a systematic search for fusogenic proteins that we made among all the HERV coding envelope genes and belonging to the HERV-FRD family can also make pseudotypes and confer infectivity. We further show that the orthologous envelope genes that were isolated from simians-from New World monkeys to humans-are also functional in the infectivity assay, with one singular exception for the gibbon HERV-FRD gene, which is found to be fusogenic in a cell-cell fusion assay, as observed for the other simian envelopes, but which is not infectious. Sequence comparison of the FRD envelopes revealed a limited number of mutations among simians, and one point mutation-located in the TM subunit-was shown to be responsible for the loss of infectivity of the gibbon envelope. The functional characterization of the identified envelopes is strongly indicative of an ancestral retrovirus infection and endogenization, with some of the envelope functions subsequently retained in evolution.     

Co-localization of susceptibility loci for psoriasis (PSORS4) and atopic dermatitis (ATOD2) on human chromosome 1q21

Psoriasis (PS) is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation and altered differentiation. Atopic dermatitis (ATOD) is a chronic inflammatory, pruritic and eczematous disease frequently associated with respiratory atopy. These diseases are associated with distinct immunologic abnormalities and represent typical examples of complex diseases triggered by both genetic and environmental factors, as demonstrated by independent twin studies. Genome wide linkage studies have mapped susceptibility loci on several chromosomes (PSORS1-9; ATOD1-5). Four of them overlap on chromosomes 1q21, 3q21, 17q25 and 20p although ATOD is quite distinct from PS and these two diseases rarely occur together in the same patient. An association fine-mapping study has been performed to refine PSORS4 and ATOD2 susceptibility loci on chromosome 1q21 analyzing two independently collected cohorts of 128 PS and 120 ATOD trios. Genotype and haplotype analysis of PSORS4 and ATOD2 led us to detect significant p value for haplotypes defined by MIDDLE and ENDAL16 markers in both PS (p = 0.0000036) and ATOD (p = 0.0276), suggesting a strict co-localization within an interval of 42 kb. This genomic interval contains a single gene, LOR, encoding for loricrin. Polymorphic markers mapping in regulatory and coding regions did not show evidence of association in neither of the two diseases. However, expression profiles of LOR in skin biopsies have shown reduced levels in PS and increased levels in ATOD, suggesting the existence of a specific misregulation in LOR mRNA production.     

Multiple endocrine neoplasia type 1

Solitary median maxillary central incisor syndrome (SMMCI) is a complex disorder consisting of multiple, mainly midline defects of development resulting from unknown factor(s) operating in utero about the 35th–38th day(s) from conception. It is estimated to occur in 1:50,000 live births. Aetiology is uncertain. Missense mutation in the SHH gene (I111F) at 7q36 may be associated with SMMCI. The SMMCI tooth differs from the normal central incisor, in that the crown form is symmetric; it develops and erupts precisely in the midline of the maxillary dental arch in both primary and permanent dentitions. Congenital nasal malformation (choanal atresia, midnasal stenosis or congenital pyriform aperture stenosis) is positively associated with SMMCI. The presence of an SMMCI tooth can predict associated anomalies and in particular the serious anomaly holoprosencephaly. Common congenital anomalies associated with SMMCI are: severe to mild intellectual disability, congenital heart disease, cleft lip and/or palate and less frequently, microcephaly, hypopituitarism, hypotelorism, convergent strabismus, oesophageal and duodenal atresia, cervical hemivertebrae, cervical dermoid, hypothyroidism, scoliosis, absent kidney, micropenis and ambiguous genitalia. Short stature is present in half the children. Diagnosis should be made by eight months of age, but can be made at birth and even prenatally at 18–22 weeks from the routine mid-trimester ultrasound scan. Management depends upon the individual anomalies present. Choanal stenosis requires emergency surgical treatment. Short stature may require growth hormone therapy. SMMCI tooth itself is mainly an aesthetic problem, which is ideally managed by combined orthodontic, prosthodontic and oral surgical treatment; alternatively, it can be left untreated.