Central Hypogonadotropic Hypogonadism: Genetic Complexity of a Complex Disease

Central hypogonadotropic hypogonadism (CHH) is an emerging pathological condition frequently associated with overweight, metabolic syndrome, diabetes, and midline defects. The genetic mechanisms involve mutations in at least twenty-four genes regulating GnRH neuronal migration, secretion, and activity. So far, the mechanisms underlying CHH, both in prepubertal and in adulthood onset forms, remain unknown in most of the cases. Indeed, all detected gene variants may explain a small proportion of the affected patients (43%), indicating that other genes or epigenetic mechanisms are involved in the onset of CHH. The aim of this review is to summarize the current knowledge on genetic background of CHH, organizing the large amount of data present in the literature in a clear and concise manner, to produce a useful guide available for researchers and clinicians

Role of Multidrug-Resistance Protein 2 in coproporphyrin transport: results from experimental studies in bile fistula rat models

Coproporphyrin (CP) is one of the main by-products of heme biosynthesis and its abnormal accumulation is associated with different forms of porphyria. Indirect data obtained from animal and human models have suggested a possible role for Multidrug Resistance-associated Protein 2 (MRP2) and other MRPs in hepatocyte excretion of CP. Using normal, MRP2-deficient and a cholestatic rat model, we have assessed the role of MRPs in CP disposition. MRP levels were assayed using immunofluorescence. Biliary and urinary excretion patterns of CP and conjugate bilirubin were measured during equimolar infusions of CP isomers with and without phenoldibromopthalein sulfonate (BSP), a well-known MRP2 substrate. Our results suggest a role for the MRP system as a possible regulator of CP traffic and accumulation in normal and pathological conditions. Alteration in this systems (as observed in cholestatic disease) may play an important role in triggering clinical expression of porphyria in individuals with underlying mutations leading to porphyrin accumulation and may help explain the phenotypic heterogeneity in patients affected by different forms of porphyrias

MTHFR C677T polymorhism and hyperhomocysteinemia in patients with liver cirrhosis complicated by portal vein thrombosis and hepatocellular carcinoma

BACKGROUND: Mild hyperhomocysteinemia (HHcy) is an independent risk factor for deep vein thrombosis; the liver plays a key role in homocysteine (Hcy) metabolism, being the sole tissue provided with the whole enzymatic set of transsulfuration and remethylation pathways; the liver has also a key-role in storage and metabolism of vitamins needed as cofactor in sulphur aminocid metabolism. Among different determinants of HHcy, the methylen-tetrahydrofolate reductase (MTHFR) C677T polymorphism has an high prevalence in general population (the homozygous state (TT) have been estimated in about 18% of italians). MTHFR gene anomalies have been also involved in cancer development, through a mechanism involving the impairment of folate metabolism and hence inducing alterations in DNA repair and methylation. Liver cirrhosis (LC) is by far the most important risk factor of portal vein thrombosis (PVT) especially if complicated by hepatocellular carcinoma (HCC). AIM: Considering the complex interaction between HHcy, MTHFR status and liver function impairment , aim of our study was to investigate a possible relation between HHcy, MTHFR status, HCC and PVT in patients with liver cirrhosisMATERIALS and METHODS: To these purposes, we have studied 86 patients (35 f, mean age 65±10 years) affected by LC, 53 without portal thrombosis (LC group) and 33 with portal thrombosis (doppler ultrasonography, angio-CT or angiography dcumented) (PVT group) . Patients in the two groups were age- ,Child Pugh score- , etiology- and sex–matched. All patients were assessed for plasma homocysteine (HPLC) , MTHFR C677T and vitamin (reb blood cell folate, serum B12 and B6) status. RESULTS: The table resumes the results regarding HCC presence and homocysteine parameters in the two studied groups.PVT (n=33)LC (n=53)pOR (95% CI)HCC presence (y/n)21/12 (63.6 %)12/41 (22.6%).0073.26 (1.31÷8.12)Mean plasma Hcy16.4 ± 6.112.1 ± 6.1.0151.07 (1.01÷1.15)HHcy prevalence (y/n)§21/12 (63.6%)15/38 (28.3%).0023.08 (1.61÷7.41)MTHFR status (CC/CT/TT)8/12/1335/14/4.001* .013***TT prevalence; **CT plus TT status vs. CC prevalence; §= >15 nmol/mlNo significant differences in vitamin status (red blood cell folate, and serum B12 and B6) were observed between groups. Patients with HCC (n=33) showed significant higher levels of plasma Hcy (17.7±5.9 vs. 13.5±5.8, p.039) and significant higher prevalence of HHcy (19/33, 57.5% vs.17/53, 32.1%, p=.025, OR 1.79, 95% CI 1.12÷2.99) than patients without HCC(n=53). Patients with HCC had also a significant higher prevalence of MTHFR TT status (10/33, 30.3% vs. 7/53, 13.2 %, p=.044, OR 2.67, 95% CI 1.07÷6.67). Interestingly, all patients with MTHFR TT status had HHcy and all patients MTHFR TT and HCC had PVT.CONCLUSION: Mild HHcy associated to liver cirrhosis may play a possible role for PVT development. Even if confirmations by larger and prospective studies are needed, assessment of homocysteine (simple and cheap) may be suggested in patients with liver cirrhosis (especially in case of advanced liver disease or in the presence of HCC).Our data concerning the association between HCC and MTHFR TT status is intriguing, due the postulated role for this polymorphism in cancerogenesis: it may represent a further link between HCC and PV

Urinary coproporhyrin isomers during therapy with IFn-RIBA in patients with chronic hepatitis due to HCV infection

Background : MRP2 transporter has a proven role in biliary export of many different products of hepatocytic metabolism. Particularly, human and animal models have demonstrated MRP2 role in biliary excretion of coproporhyrins: its preference for isomer I (CPI) export with respect to isomer III (CPIII) is responsable for the biliary CPI/CPIII ratio about 2-3:1 and the plasma and urinary CPI/CPIII ratio about 1:2-3. A significant reduction of MRP2 expression has been described in many different acquired cholestatic liver diseases and in patients with chronic hepatitis (CH) due to HCV infection. Aim of our study was to evaluate quantitative and qualitative (CPI/CPIII ratio) urinary CP excretion in patients with CH due to HCV before (T0), at 3 months (T3) and at the end of antiviral therapy (ET) (peg-IFN + Ribavirin)Materials and Methods: 60 patients (18 females, aged 46±11 years, genotype: 1 (31); 2(10); 3(15); 4(4); 51 naive) with istologically proven HCV-inducted CH were consecutively enrolled; 5 (8.4%) did not conclude the study. 10 healthy subjects sex- and age-matched were also considered for comparing the basal values. None of considered subjects had overt laboratory [i.e. serum alkaline phosphatase (AP) over the normal range] or clinical signs of cholestasis. All patients underwent a urinary evaluation (HPLC) of CPI excretion at T0, T3 and ET. SVR (n=34) was defined as serum HCV-RNA undetectable after 6 months of follow-up. Results: Basal serum FA levels resulted significantly related both to basal urinary total CPI (r=.405, p=.001), and basal CPI/CPIII ratio (r=.692, p=.000). Table I show Total urinary CPI e urinary CPI/CPIII ratio in controls and in HCV patients with respect to serum PA percentile.Table IPA ≥ 75°percentile(≥ 232 u/L) (n=16)(group A)PA 60% at T3 showed a 88% PPV for SVR, reaching 95% if considering only patients in group A.Our data indirectly confirm the effect of liver chronic infection due to HCV on MRP2 expression, whose alteration may be considered as the main responsible for the observed urinary CPI modifications (greater excretion with CPI/CPIII ratio inversion). Even if more confirmative data are needed, monitoring of urinary CPI excretion (especially CPI/CPIII ratio) may be a useful, cheap and simple tool in early prediction of the response to antiviral therapy in subjects affected by HCV CH, especially in the presence of mild laboratory signs of cholestasis

The FSHR polymorphism p.N680S mediates different response kinetics to FSH in vitro

Introduction: FSH acts on its receptor (FSHR) resulting in signal transduction activation, gene expression and steroidogenesis. The FSHR common SNP p.N680S is a marker of gonadal response in vivo. However, in vitro dose–response experiments failed to demonstrate the molecular basis thereof so far. In this study, we systematically investigated whether p.N680S mediates different kinetics of FSH response in vitro. Design: We evaluated the activation kinetics of cAMP, phERK1/2, phCREB by ELISA and western blotting in FSHR homozygous, primary, human granulosa lutein cells (hGLC-680N, -680S) stimulated by 50 nM r-FSH for up to 2 h (short-term stimulation). Following short-term stimulation the expression of target genes was evaluated by real-time PCR after 12 h, and progesterone production kinetics over 24 h. Specific inhibitors/agonists (U0126, PMA) were used in the presence and in the absence of FSH. Results: Intracellular cAMP increased within 5–10 min in hGLC-680N, reaching the plateau in about 45 min. cAMP increase was delayed in hGLC-680S, reaching the plateau in 120 min, revealing different activation kinetics (Mann–Whitney U test; P<0.05; n=4). r-FSH-dependent cAMP stimulation kinetics resulted in different ERK1/2 and CREB phosphorylation, reaching maximal levels in 5–30 min in hGLC-680N, whereas, in hGLC-680S, these were weaker and steady over 2 h (Mann–Whitney U test; P<0.05; n=3). hGLC-680N stimulation resulted in higher expression levels of AREG and StAR (Mann–Whitney U test; P<0.05; n=4) and in subsequently different progesterone production kinetics, achieving overall higher levels in hGLC-680N vs -680S (Mann–Whitney U test; P<0.05; n=3). Interestingly, the different kinetics of progesterone production between hGLC-680N and -680S were interchanged by selective phospho-ERK1/2 blockade/activation through specific inhibitor/agonist, revealing a short-term cross-talk mediated by ERK1/2. Conclusions: This study demonstrates for the first time in vitro, how FSHR p.N680S mediates different response to FSH, resulting in different kinetics of cAMP, phERK1/2 and phCREB activation, and progesterone production

Insulin resistance in advanced liver cirrhosis : the role of liver dysfunction and the “dissociated” effect on glucose versus lipid and amino acid metabolism

Background: Chronic liver failure is characterized by the progression of multiple important metabolic derangements, simultaneously interesting all main metabolic pathways. In liver cirrhosis, an impaired glucose tolerance with insulin resistance is frequently observed (20% of these patients may develop overt diabetes mellitus during the disease progression). Aim: to evaluate (1) the role of liver dysfunction in inducing the alteration of insulin sensitivity in patients with advanced stage of liver cirrhosis and (2) the effect of this metabolic derangement on amino acid and lipid metabolism in patients with liver cirrhosis with respect to healthy controls and to patients with type II diabetes mellitus.Materials and methods: To this purpose 23 subjects with documented advanced liver cirrhosis (LC), 14 age and sex-matched healthy subjects (controls) and 10 patients with type II diabetes mellitus without liver disease (DM) were studied. A FSIVGTT test (Frequently Sample Intravenous Glucose Tolerance Test) with simultaneous evaluation of glucose, lipid and amino acid metabolism parameters was performed and the results for glucose, Insulin and C-peptide were analyzed by the MINMOD program, able to adapt data to three different mathematical models, generating predictions of kinetic of glucose disappearance and insulin and C-peptide production; FSIVGTT lipid metabolism parameters (NEFA levels) were measured by colorimetric kit and FSIVGTT amino acid metabolism parameters [Branched Chain Amino Acid, (BCAA) and Aromatic Amino Acid, (AAA) profile], by Amino Acid Analyzer. Results: The MINMOD integration of data concerning secretion and kinetics of C-peptide with those of insulin, confirm that advanced-stage liver cirrhosis is characterised by significant insulin-resistance with hyperinsulinemia and suggest the main role of reduction of insulin liver extraction (h) in insulin-resistance syndrome of liver cirrhosis. The effects of this liver-failure inducted insulin-resistance seem to have some peculiarities concerning lipid and amino acid metabolism, consisting in a relative conservation (greater effect than that observed in DM subjects) of insulin effect in peripheral tissues: in LC patients insulin seems to maintain a relative greater capacity to promote both BCAA and NEFA utilization by muscle and fat tissue (“dissociated effect”) with respect to patients with type II diabetes mellitus. Conclusion: The relative preservation of insulin sensibility by peripheral tissues represents an important and a positive fact, as rationalizes the use, for example, of BCAA solutions for nutritional purposes in presence of starvation or negative azoth balance, all conditions known to negatively influencing some neuropsychological complications of liver cirrhosis

FSHR polymorphism p.N680S mediates different responses to FSH in vitro

The single nucleotide polymorphism p.N680S of the follicle-stimulating hormone (FSH) receptor (FSHR) is a discrete marker of ovarian response but previous in vitro studies failed to demonstrate differences in the response to FSH between N and S carrier cells. Here we demonstrate that p.N680S mediates different kinetics of the response to FSH in vitro. Intracellular cAMP production is faster in p.N680S N than in S homozygous human granulosa cells (45 versus 90 min to achieve the plateau, respectively; Mann-Whitney's U-test; p < 0.005; n = 4). Reflecting the cAMP kinetics, phospho-ERK1/2 and -CREB activation, AREG and STARD1 gene expressions and progesterone production were qualitatively and quantitatively different in N versus S homozygous cells. Finally, the blockade of ERK pathway by U0126 abolishes the genotype-mediated different effects on gene expression and progesterone production (Mann-Whitney's U-test; p ≥ 0.005; n = 3)

Clinical, biochemical and genetic characteristics of Variegate Porphyria in Italy

Variegate Porphyria (VP) is an autosomal dominant disorder found worldwide but is rare in Italy. In this study we provide an overview of clinical, biochemical and genetic background of 33 Italian VP patients diagnosed in the last fifteen years. About 70% of patients had experienced clinical symptoms: 43.4% had photosensivity, 8.7% acute attacks and 47.8% both. Among the 33 patients, 14 different mutations were identified. Of these only 6 defects have been previously described in other countries and 8 are unique having been identified for the first time in Italy. Two of these, the c.851G&gt;T and the c.1013C&gt;G, were found in two and four unrelated families respectively. No mutation has been found in homozygosis and no significant correlation has been observed between specific clinical and biochemical manifestations and the type of mutation. In contrast, normal faecal protoporphyrin excretion was high predictive of silent phenotype. Normal urinary excretion of PBG and ALA, predicted absence of neurovisceral symptoms. This paper represents the first compilation of data on genotype-phenotype relation in Italian patients with VP