Characterization of an Italian founder mutation in the RING-finger domain of BRCA1

The identification of founder mutations in cancer predisposing genes is important to improve risk assessment in geographically defined populations, since it may provide specific targets resulting in cost-effective genetic testing. Here, we report the characterization of the BRCA1 c.190T>C (p.Cys64Arg) mutation, mapped to the RING-finger domain coding region, that we detected in 43 hereditary breast/ovarian cancer (HBOC) families, for the large part originating from the province of Bergamo (Northern Italy). Haplotype analysis was performed in 21 families, and led to the identification of a shared haplotype extending over three BRCA1-associated marker loci (0.4 cM). Using the DMLE+2.2 software program and regional population demographic data, we were able to estimate the age of the mutation to vary between 3,100 and 3,350 years old. Functional characterization of the mutation was carried out at both transcript and protein level. Reverse transcriptase-PCR analysis on lymphoblastoid cells revealed expression of full length mRNA from the mutant allele. A green fluorescent protein (GFP)-fragment reassembly assay showed that the p.Cys64Arg substitution prevents the binding of the BRCA1 protein to the interacting protein BARD1, in a similar way as proven deleterious mutations in the RING-domain. Overall, 55 of 83 (66%) female mutation carriers had a diagnosis of breast and/or ovarian cancer. Our observations indicate that the BRCA1 c.190T>C is a pathogenic founder mutation present in the Italian population. Further analyses will evaluate whether screening for this mutation can be suggested as an effective strategy for the rapid identification of at-risk individuals in the Bergamo area

Molecular genetics of male infertility: Stem cell factor/c-kit system

PROBLEM: Infertility, affects about 5% of human males and genetic factors are recognized in approximately 30% of them. The mouse represents a good model to study autosomal genes that might play a role in spermatogenesis. In mice, mutations in the c-kit gene and in the gene encoding stem cell factor (SCF) cause pleiotropic defects among which sterility. A possible involvement of the SCF/c-kit system in human spermatogenesis was investigated. METHODS OF STUDY: A group of 65 idiopathic azoospermic patients was screened for the presence of mutations in the human c-kit gene codon encoding tyrosine 721 (Y721), analogous to Y719 in the murine c-kit gene (a residue known to be essential for a normal spermatogonial proliferation). Furthermore we have used a mouse model for studying the molecular mechanisms that regulate the transcription of the endogenous SCF gene. RESULTS: No mutations have been detected on codon encoding Y721 of the human e-kit gene, in our group of infertile patients. CONCLUSIONS: A larger group of azoospermic patients, including preferentially patients affected by Sertoli-cell-only syndrome, should be screened in order to exclude a role of c-kit mutations in Y721 in spermatogenesis defects. In this study we also show that the murine SCF promoter is transcriptionally active and stimulated by follicle stimulating hormone (FSH), 3'-5' cyclic adenosine monophosphate (cAMP) analogs, and IBMX in primary mouse Sertoli cells, and that the cAMP effect is cell-specific, as the SCF promoter is not stimulated in other SCF-expressing cell types tested

Soluble and insoluble signals and the induction of bone formation: molecular therapeutics recapitulating development

The osteogenic molecular signals of the transforming growth factor-β (TGF-β) superfamily, the bone morphogenetic/osteogenic proteins (BMPs/OPs) and uniquely in primates the TGF-β isoforms per se, pleiotropic members of the TGF-β supergene family, induce de novo endochondral bone formation as a recapitulation of embryonic development. Naturally derived BMPs/OPs and gamma-irradiated human recombinant osteogenic protein-1 (hOP-1) delivered by allogeneic and xenogeneic insoluble collagenous matrices initiate de novo bone induction in heterotopic and orthotopic sites of the primate Papio ursinus, culminating in complete calvarial regeneration by day 90 and maintaining the regenerated structures by day 365. The induction of bone by hOP-1 in P. ursinus develops as a mosaic structure with distinct spatial and temporal patterns of gene expression of members of the TGF-β superfamily that singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis. The temporal and spatial expressions of TGF-β1 mRNA indicate a specific temporal transcriptional window during which expression of TGF-β1 is mandatory for successful and optimal osteogenesis. Highly purified naturally derived bovine BMPs/OPs and hOP-1 delivered by human collagenous bone matrices and porous hydroxyapatite, respectively, induce bone formation in mandibular defects of human patients. By using healthy body sites as bioreactors it is possible to recapitulate embryonic developments by inducing selected biomaterials combined with recombinant proteins to transform into custom-made prefabricated bone grafts for human reconstruction. The osteogenic proteins of the TGF-β superfamily, BMPs/OPs and TGF-βs, the last endowed with the striking prerogative of inducing endochondral bone formation in primates only, are helping to engineer skeletal reconstruction in molecular terms

Prognostic impact of c-Kit mutations in core binding factor leukemias: an Italian retrospective study

Distinct forms of tyrosine kinase domain (TKD), juxtamembrane domain, exon 8, and internal tandem duplication (ITD) mutations of c-KIT, were observed in about 46% of core binding factor leukemia (CBFL) patients. To evaluate their prognostic significance, 67 adult patients with CBFL were analyzed to ascertain the c-KIT mutation status. In acute myeloid leukemia (AML) with t(8;21), the presence of c-KIT TKD mutation at codon 816 (TKD(816)) was associated with a high white blood cell count at diagnosis (median, 29.60 x 10(9)/L) and a higher incidence (33%) of extramedullary leukemia (EML) during the course of the disease. Data also showed that the TKD(816) mutated patients (n = 12) had a significantly higher incidence of relapse and a lower overall survival (OS) at 24 months, compared with the 17 c-KIT unmutated (c-KIT(-)) patients (90% vs 35.3%, P = .002; 25% vs 76.5%, P = .006, respectively). No difference in relapse incidence (P = .126) and OS (P = .474) was observed between the c-KIT mutated other than TKD(816) (n = 7) and the c-KIT(-) patients. These findings indicate that c-KIT TKD(816) mutation has a negative impact on the outcome of AML with t(8;21)

Molecular analysis of PDGFRA and PDGFRB genes by rapid single-strand conformation polymorphism (SSCP) in patients with core-binding factor leukaemias with KIT or FLT3 mutation

Mutations involving KIT and FLT3 genes, encoding tyrosine kinase (TK) membrane receptors, are detected in core-binding factor leukaemia (CBFL) patients. PDFGRA and PDGFRB encode class III TK receptors and are involved both in physiological processes and in the pathogenesis of haematological and solid tumours. The aim of this study was to investigate if PDGFR mutations are involved in CBFL. PATIENTS AND METHODS: In order to detect PDGFR mutations in CBFL, 35 patients without KIT or FLT3 mutations patients were screened by rapid and sensitive single-strand conformation polymorphism (SSCP) analysis. Sequence analysis was performed in polymerase chain reaction (PCR) products showing altered mobility in SSCP analysis in order to determine the nucleotide changes. RESULTS: Three types of single-nucleotide polymorphism (SNP) were detected in the PDGFRA gene (exon 12, exon 13 and exon 18) while no mutation of PDGFRB was detected in the tested CBFLs. CONCLUSION: These data showed that no pathogenic mutations in PDGFRA and PDGFRB were detected in the context of CBFL without KIT and FLT3 mutations. Thus, PDGFR genes do not seem to be involved in CBFL and future studies are needed to establish the genetic causes of the disease in these particular patients