Two novel truncating variants of the AAAS gene causative of the triple A syndrome.

PURPOSE: The triple A syndrome (AAAS) is an inherited condition associated with mutations in the AAAS gene, which encodes a protein of 546 amino acids known as ALADIN (alacrima achalasia adrenal insufficiency neurologic disorder) whose function is not well understood. This protein belongs to the WD-repeat family of regulatory proteins and is located in the nuclear pore complexes. Only a few cohorts of AAAS patients have been reported and fully characterized. Thus, the objective of the present study was to report on a mini cohort of Italian AAAS patients and to get insights on their predisposing genetic defects. METHODS: Genetic analysis of AAAS gene in triple A syndrome patient and molecular and functional characterization of the novel identified allelic variants. RESULTS: Here we describe three newly diagnosed cases of AAAS, in whom genetic analysis allowed us to identify two novel allelic variants in the AAAS gene: the frameshift substitution c.765 dupT (p.Gly256Trp fsX67) in exon 8 and the splice site mutation in intron 11(c.997-2 A > G, IVS11-2A > G). Both variants result in a truncated non-functional protein, as we demonstrate by transcript analysis and expression studies. CONCLUSIONS: Our findings establish a pathogenic role for both new variants. Moreover, our data highlight the essential role of the C-terminal domain of the protein for its correct targeting and function and underline the importance of sequencing splice sites surrounding the intron-exon junctions to ensure accurate molecular diagnosis and correct genetic counseling in AAAS patients.

Molecular markers for the classification of cytologically indeterminate thyroid nodules.

BACKGROUND: The diagnosis of indeterminate lesions of the thyroid is a challenge in cytopathology practice. Indeed, up to 30% of cases lack the morphological features needed to provide definitive classification. Molecular tests have been developed to assist in the diagnosis of these indeterminate cases. The first studies dealing with the preoperative molecular evaluation of FNA samples focused on the analysis of BRAFV600E or on the combined evaluation of two or three genetic alterations. The sensitivity of molecular testing was then improved through the introduction of gene panels, which became available for clinical use in the late 2000s. Two different categories of molecular tests have been developed, the 'rule-out' methods, which aim to reduce the avoidable treatment of benign nodules, and the 'rule-in' tests that have the purpose to optimize surgical management. The genetic evaluation of indeterminate thyroid nodules is predicted to improve patient care, particularly if molecular tests are used appropriately and with the awareness of their advantages and weaknesses. The main disadvantage of these tests is the cost, which makes them rarely used in Europe. To overcome this limitation, customized panels have been set up, which are able to detect the most frequent genetic alterations of thyroid cancer. CONCLUSIONS: In the present review, the most recent available versions of commercial molecular tests and of custom, non-commercial panels are described. Their characteristics and accuracy in the differential diagnosis of indeterminate nodules, namely Bethesda classes III (Atypical follicular lesion of undetermined significance, AUS/FLUS) and IV (Suspicious for follicular neoplasm, FN/SFN) are fully analyzed and discussed.

Hepatocyte growth factor-induced expression of ornithine decarboxylase, c-met, and c-myc is differently affected by protein kinase inhibitors in human hepatoma cells HepG2.

Binding of hepatocyte growth factor (HGF) to its receptor Met induces autophosphorylation and activation of the tyrosine kinase activity. In HGF-treated HepG2 cells, we studied: (i) the expression patterns of early (c-myc, c-jun, and c-fos) and delayed-early (ornithine decarboxylase and c-met) response genes and (ii) the possible involvement of protein kinase transducers in the control of the expression of c-met and of other genes eventually induced downstream. c-met and c-myc mRNAs peaked 1-2 h after HGF, while c-jun and c-fos mRNAs slightly increased at 1 h. Ornithine decarboxylase activity was induced earlier (4 h) than the mRNA (8-10 h). The transducers involved in HGF-triggered gene inductions were investigated using different protein kinase inhibitors: genistein for the receptor tyrosine kinase, herbimycin A for the nonreceptor tyrosine kinase (pp60(c-src)), wortmannin for phosphatidylinositol 3-kinase (PI3K) and H7 for protein kinase C (PKC). The similarity of responses to PKC inhibition led to suppose that c-myc and ornithine decarboxylase mRNAs were induced sequentially along the same transduction pathway triggered by HGF. Ornithine decarboxylase activity seemed to be largely regulated by phosphorylation(s). The mRNA expression of c-jun was likely to undergo a negative regulation through a mechanism involving PI3K, while that of c-met seemed to be almost independent from various protein kinases (PI3K, pp60(c-src), and PKC).

Regulation of spermidine/spermine N1-acetyltransferase expression by cytokines and polyamines in human hepatocarcinoma cells (HepG2).

Spermidine/spermine N1-acetyltransferase (cSAT), a key enzyme in polyamine degradation, is induced by various hepatotoxins and liver tumor promoters. In this paper we demonstrate that physiological factors, such as cytokines, control cSAT expression in HepG2 human hepatocarcinoma cells. Hepatocyte growth factor (HGF) induced the cSAT mRNA precursor (3.5 kb) at 4 h. The mature form of mRNA (1.3 kb) increased 6-8-fold between 8 and 10 h, and remained elevated until 18 h. An increase in cSAT activity (2-fold) and high levels of N1-acetylspermidine were observed concomitantly. Interleukin-1 beta (IL-1 beta) enhanced cSAT expression (both mRNA and enzyme activity) similar to HGF, while tumor necrosis factor-alpha (TNF-alpha) was less effective. This system also provides a useful means for examining the involvement of negative and positive changes of polyamines in the induction of cSAT and c-jun, a gene that participates in the control of cSAT expression. alpha-Difluoromethylornithine (DFMO) pretreatment, by lowering putrescine and spermidine in HGF- or IL-1 beta-treated cells, prevented the induction of cSAT. This effect was reversed by exogenous putrescine or spermidine. IL-1 beta induced c-jun mRNA more than HGF. DFMO prevented almost completely the enhancement of c-jun mRNA expression by IL-1 beta, and this effect was reversed by exogenous putrescine or spermidine. Therefore, we suggest that cSAT and c-jun expression is specifically regulated by polyamine-mediated mechanisms in IL-1 beta treated HepG2 cells. Since cSAT is inducibile by cytokines that control tumor metabolism and growth as well as tumor-host interaction, we hypothesize an involvement of cSAT in hepatoma growth.

Differential activation of heat shock and nuclear factor kappaB transcription factors in postischemic reperfused rat liver.

The aim of this study was to investigate the behavior of the transcription factors, heat-shock factor (HSF) and nuclear factor kappaB (NF-kappaB), in postischemic reperfused liver, with particular attention paid to possible differences in the time-course and mechanism of activation, which may help in defining their role in the response of the liver to reperfusion. Ischemia was induced by clamping the hilar pedicle of the left lateral and median liver lobes; the clamp was removed after 1 hour. Some rats were treated intraperitoneally with IL-1 receptor antagonist (IL-1RA) 30 minutes before ischemia and at the time of reperfusion. Binding of NF-kappaB to the corresponding consensus sequence is activated after 30 minutes of reperfusion, and is still increased 1 hour after reperfusion. Activation is suppressed in rats treated with IL-1RA; NF-kappaB persists in the cytosol associated with the inhibitor, IkappaB, and can be artifactually activated in vitro. Super-gel shift experiments revealed that the two subunits, p50 and p65, are involved in the activation of binding. In contrast, binding of HSF to the corresponding consensus sequence, heat shock element (HSE), is already activated at the end of ischemia, shows a further increase after 30 minutes of reperfusion, but declines 1 hour after reperfusion; more importantly, it is not inhibited by pretreatment of the rat with IL-1RA. In conclusion, although both HSF and NF-kappaB are activated by ischemia-reperfusion, there are clear differences in time-course and mechanism of activation of the two transcription factors. Activation of HSF depends directly on some events occurring during ischemia; NF-kappaB is activated only after reperfusion and the concurrent oxidative stress, by an indirect mechanism that can be suppressed by IL-1RA. The possibility of dissociating the activation of these two transcription factors in postischemic reperfusion can have a prospective clinical relevance.

Effects of polyamine imbalance on the induction of stress genes in hepatocarcinoma cells exposed to heat shock.

The expression of hsp70-the inducible member of the corresponding heat shock gene family-of the oxidative stress marker gene heme oxygenase (HOx), and of the immediate early response genes c-fos and c-jun has been studied in FAO hepatocarcinoma cells depleted of polyamines and exposed to heat shock. Depletion of polyamines was obtained in short-term experiments (24-48 hours) by the use of alpha difluoromethylornithine (DFMO), a classical inhibitor of ornithine decarboxylase (ODC), or of the combination of the newly available inhibitors of ODC and S-adenosylmethionine decarboxylase, i.e., (2R,5R)-hept-6-yne-2,5-diamine (MAP) and 5'{[(Z)-4-aminobut-2-enyl]methylanino}-5-deoxyadeno-si ne (AbeAdo). Under our experimental conditions polyamine imbalance was realized without appreciable growth-related genes. Decreases of putrescine and spermidine 48 hours after DFMO prevented the induction of hsp70 messenger RNA (mRNA), whereas depletion spermidine and spermine obtained with MAP/AbeAdo decreased intensity and duration of post-heat shock accumulation of hsp70 mRNA. Inductions of HOx, c-jun and c-fos were also inhibited. Because MAP/AbeAdo caused also an intracelluar accumulation of putrescine, we tested the effect of exogenous putrescine, which was found to stabilize the mRNAs for hsp70 and c-jun. Hsp70 and HOx are thought to play a protective role, and the proteins of c-jun and c-fos constitute the transcription factor activator protein-1, which is involved in the transcription of many defensive products. Therefore, the integrity of polyamine pool seems to be a necessary permissive condition for an effective response of the cells to adverse environmental changes.

Post-transcriptional control of increased hepatic catalase gene expression in response to oxidative stress.

Catalase is an important member of the antioxidant network that protects the cell against reactive oxygen species (ROS). We studied catalase gene expression in the liver of rats exposed to oxidative stress induced by the ROS-generating drug nitrofurantoin (NF). Catalase enzymatic activity and content are enhanced by NF treatment. The corresponding increase in the steady state level of the messenger ribonucleic acid (mRNA) occurs without significant changes in transcription and seems therefore controlled post-transcriptionally. Indeed, RNA band-shift assays demonstrated a reduced binding of redox-sensitive cytoplasmic protein(s) to the 3' region of catalase mRNA in NF-treated rats, thus suggesting that the redox state of protein that binds to an antioxidant enzyme mRNA may play a role in the hepatic response to oxidative stress.

Heat shock activation of NFkB in rat liver is mediated by interleukin-1.

Exposure to high temperature (heat shock) activates the transcription factor NFkB in the liver of the living rat, but is not effective in hepatoblastoma cells in culture: on the contrary, activation of the heat shock transcription factor (HSF) occurs under both conditions. Pre-treatment of the rat with IL-1 receptor antagonist suppresses the activation of NFkB, which seems to be mediated by the release of this cytokine, but does not hamper the activation of HSF and the concurrent induction of hsp 70 mRNA. IL-1 activity actually shows a strong, albeit transient, increase in the blood of heat shocked rats.

Differential activation of heat-shock and oxidation-specific stress genes in chemically induced oxidative stress.

Post-ischaemic reperfusion increases the level of the major heat-shock (stress) protein hsp 70 and of its mRNA by transcriptional mechanisms, and activates the binding of the heat-shock factor HSF to the consensus sequence HSE. In common with CoCl2 treatment, post-ischaemic reperfusion increases the level of haem oxygenase mRNA, an indicator of oxidative stress, but CoCl2 does not seem to induce the expression of the hsp 70 gene [Tacchini, Schiaffonati, Pappalardo, Gatti and Bernelli-Zazzera (1993) Lab. Invest. 68, 465-471]. Starting from these observations, we have now studied the expression of two genes of the hsp 70 family and of other possibly related genes under conditions of oxidative stress. Three different chemicals, which cause oxidative stress by various mechanisms and induce haem oxygenase, enhance the expression of the cognate hsc 73 gene, but do not activate the inducible hsp 70 gene. Expression of the other genes that have been studied seems to vary in intensity and/or time course, in relation to the particular mechanism of action of any single agent. The pattern of induction of the early-immediate response genes c-fos and c-jun observed during oxidative stress differs from that found in post-ischaemic reperfused livers. Oxidative-stress-inducing agents do not promote the binding of HSF to its consensus sequence HSE, such as occurs in heat-shock and post-ischaemic reperfusion, and fail to activate AP-1 (activator protein 1). With the possible exception of Phorone, the oxidative stress chemically induced in rat liver activates NFkB (nuclear factor kB) and AP-2 (activator protein 2) transcription factors.

Induction of ferritin synthesis by oxidative stress. Transcriptional and post-transcriptional regulation by expansion of the "free" iron pool.

Ferritin, by regulating the "free" intracellular iron pool, controls iron-catalyzed generation of reactive oxygen species, but its role in oxidative damage is still unclear. We show that ferritin synthesis is significantly stimulated in the liver of rats subjected to oxidative stress by treatment with phorone, a glutathione-depleting drug. RNA-bandshift assays document reduced activity of iron regulatory factor, in particular of IRFB, the cytoplasmic protein that post-transcriptionally controls ferritin mRNA translation. Furthermore, Northern blot analysis shows increased accumulation of H and L subunit mRNAs, and nuclear run-on experiments provide evidence of transcriptional activation. Direct measurements of intracellular free iron levels by EPR indicate that the increased ferritin synthesis can be mediated by an expansion of the free iron pool. An early drop of ferritin content after phorone treatment indicates that part of the iron that fuels the free pool might derive from ferritin degradation. Present data seem to suggest that, under conditions of oxidative stress, liver ferritin can represent either a pro- or an anti-oxidant in a time-dependent manner. In fact, its early degradation contributes to expand the intracellular free iron pool that, later on, activates multiple molecular mechanisms to reconstitute ferritin content, thus limiting the pro-oxidant challenge of iron.