X-linked kallman's syndrome: a molecular genetic and developmental analysis

Kallmann's syndrome (KS) is defined as the association of hypogonadotrophic hypogonadism (IHH), caused by hypothalamic gonadotrophin releasing hormone (GnRH) deficiency, and anosmia, due to malformation of the olfactory bulbs and tracts. Patients most commonly present with delayed puberty and may also present in childhood with cryptorchidism. Renal agenesis seen in 40% and synkinesis observed in up to 90[percent] of patients with X-linked KS (XKS) are likely to originate in the aberrant expression of KAL (Xp22.3) during early foetal development. Failure of the olfactory axons in the accessory olfactory nerves to project through the cribriform plate and establish synaptic contact with the developing olfactory bulb is thought to be central to the pathogenesis of these clinical features. GnRH neurons originating in the primitive nasal area consequently fail to migrate into the forebrain because of the absence of the "scaffolding" provided by these olfactory nerves. This study has examined areas of KAL protein (680 amino acid protein containing a signal peptide but no membrane insertion or anchorage sequence, suggesting that this is a secreted protein) expression in the developing foetus and adult using anti-protein peptide antibodies generated in mice. A short sequence of the putative KAL sequence was used to synthesise a multiple antigenic peptide, which was then used as the antigen. Due to the non specificity of these antibodies the areas and stages of KAL gene expression were further investigated using in situ hybridisation and reverse transcriptase polymerase chain reaction (RT-PCR) on first trimester foetal tissue. This revealed KAL transcript in the olfactory bulbs, neuroretina and developing kidney. Patients with XKS and sporadic KS were investigated for mutations in KAL using established molecular biology techniques including PCR, direct DNA sequencing and single stranded conformational polymorphism (SSCP). Previously characterised mutations were confirmed and two new mutations identified. Using restriction fragment length polymorphism (RFLP) analysis, a methodology was established to successfully screen XKS earners in a selection of patient pedigrees

Prognostic Implications of NOTCH1 and FBXW7 Mutations in Adults With T-Cell Acute Lymphoblastic Leukemia Treated on the MRC UKALLXII/ECOG E2993 Protocol

PURPOSE: Notch pathway activation by mutations in either NOTCH1 and/or FBXW7 is one of the most common molecular events in T-cell acute lymphoblastic leukemia (T-ALL) and, in pediatric disease, predicts for favorable outcome. Their prognostic significance in adult T-ALL is unclear. We sought to evaluate the outcome according to mutation status of patients with adult T-ALL treated on the United Kingdom Acute Lymphoblastic Leukaemia XII (UKALLXII)/Eastern Cooperative Oncology Group (ECOG) E2993 protocol. METHODS: NOTCH1 and FBXW7 were screened by a combination of denaturing high-performance liquid chromatography and sequencing in 88 adult patients with T-ALL treated on the UKALLXII/ECOG E2993 protocol and compared with clinical characteristics and outcome. RESULTS: NOTCH1 and FBXW7 mutations were common (60% and 18%, respectively) and were not associated with age or WBC count. NOTCH1 heterodimerization domain mutations were associated with FBXW7 mutations (P = .02), and NOTCH1 proline, glutamic acid, serine, threonine (PEST) rich domain and FBXW7 mutations were mutually exclusive. There were an equal number of high- and standard-risk patients in the NOTCH1 and FBXW7 mutated (MUT) groups. Patients wild type (WT) for both markers trended toward poorer event-free survival (EFS; MUT v WT, 51% v 27%, P = .10; hazard ratio, 0.6). Analysis by each marker individually was not significantly predictive of outcome (NOTCH1 MUT v WT, EFS 49% v 34%, P = .20; FBXW7 MUT v WT, EFS 53% v 41%, P.72). CONCLUSION: NOTCH1 and FBXW7 mutant-positive patients do not fare sufficiently well to warrant an individualized treatment approach in future studies

p53-mediated apoptosis of CLL cells: evidence for a transcription-independent mechanism

The p53 protein plays a key role in securing the apoptotic response of chronic lymphocytic leukemia (CLL) cells to genotoxic agents. Transcriptional induction of proapoptotic proteins including Puma are thought to mediate p53-dependent apoptosis. In contrast, recent studies have identified a novel nontranscriptional mechanism, involving direct binding of p53 to antiapoptotic proteins including Bcl-2 at the mitochondrial surface. Here we show that the major fraction of p53 induced in CLL cells by chlorambucil, fludarabine, or nutlin 3a was stably associated with mitochondria, where it binds to Bcl-2. The Puma protein, which was constitutively expressed in a p53-independent manner, was modestly up-regulated following p53 induction. Pifithrin alpha, an inhibitor of p53-mediated transcription, blocked the up-regulation of Puma and also of p21(CIP1). Surprisingly, pifithrin alpha dramatically augmented apoptosis induction by p53-elevating agents and also accelerated the proapoptotic conformation change of the Bax protein. These data suggest that direct interaction of p53 with mitochondrial antiapoptotic proteins including Bcl-2 is the major route for apoptosis induction in CLL cells and that p53's transcriptional targets include proteins that impede this nontranscriptional pathway. Therefore, strategies that block up-regulation of p53-mediated transcription may be of value in enhancing apoptosis induction of CLL cells by p53-elevating drugs