Autoimmune polyendocrine syndrome type 1: an Italian survey on 158 patients.

BACKGROUND: Autoimmune Polyglandular Syndrome type 1 (APS-1) is a rare recessive inherited disease, caused by AutoImmune Regulator (AIRE) gene mutations and characterized by three major manifestations: chronic mucocutaneous candidiasis (CMC), chronic hypoparathyroidism (CH) and Addison's disease (AD). METHODS: Autoimmune conditions and associated autoantibodies (Abs) were analyzed in 158 Italian patients (103 females and 55 males; F/M 1.9/1) at the onset and during a follow-up of 23.7 ± 15.1 years. AIRE mutations were determined. RESULTS: The prevalence of APS-1 was 2.6 cases/million (range 0.5-17 in different regions). At the onset 93% of patients presented with one or more components of the classical triad and 7% with other components. At the end of follow-up, 86.1% had CH, 77.2% AD, 74.7% CMC, 49.5% premature menopause, 29.7% autoimmune intestinal dysfunction, 27.8% autoimmune thyroid diseases, 25.9% autoimmune gastritis/pernicious anemia, 25.3% ectodermal dystrophy, 24% alopecia, 21.5% autoimmune hepatitis, 17% vitiligo, 13.3% cholelithiasis, 5.7% connective diseases, 4.4% asplenia, 2.5% celiac disease and 13.9% cancer. Overall, 991 diseases (6.3 diseases/patient) were found. Interferon-ω Abs (IFNωAbs) were positive in 91.1% of patients. Overall mortality was 14.6%. The AIRE mutation R139X was found in 21.3% of tested alleles, R257X in 11.8%, W78R in 11.4%, C322fsX372 in 8.8%, T16M in 6.2%, R203X in 4%, and A21V in 2.9%. Less frequent mutations were present in 12.9%, very rare in 9.6% while no mutations in 11% of the cases. CONCLUSIONS: In Italy, APS-1 is a rare disorder presenting with the three major manifestations and associated with different AIRE gene mutations. IFNωAbs are markers of APS-1 and other organ-specific autoantibodies are markers of clinical, subclinical or potential autoimmune conditions.

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.

A genetic epidemiology study of congenital adrenal hyperplasia in Italy.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD-CAH) is an autosomal recessive disorder affecting steroidogenesis, due to mutations in CYP21A2 (6p21.3). 21OHD-CAH neonatal screening is based on 17-hydroxyprogesterone (17OHP) serum levels, showing high type I error rate and low sensitivity to mild CAH forms. Here, we used an epidemiological approach, which estimates the allelic frequency (q) of an autosomal recessive disorder using the proportion of homozygous patients, the mutational spectrum and the inbreeding coefficient in a sample of affected individuals. We applied this approach to 2 independent Italian cohorts of patients with both clinical and molecular diagnosis of 21OHD-CAH from mainland Italy (N = 240) and Sardinia (N = 53). We inferred q estimates of 2.87% and 1.83%, corresponding to a prevalence of 1/1214 and 1/2986, respectively. CYP21A2 mutational spectra were quite discrepant between the 2 cohorts, with V281L representing 74% of all the mutations detected in Sardinia vs 37% in mainland Italy. These findings provide an updated fine-grained picture of 21OHD-CAH genetic epidemiology in Italy and suggest the need for a screening approach suitable to the detection of the largest number of clinically significant forms of CAH.

Secondary solid cancer following hematopoietic cell transplantation in patients with thalassemia major.

Hematopoietic cell transplant (HCT) recipients have a substantial risk of developing secondary solid cancers (SSCs). The aim of this retrospective study was to compare the incidence of SSC in a monocentric cohort of thalassemia major (TM) patients (n=122) who received HCT versus an hematopoietic cell donor monocentric cohort (n=122) and versus a large multicenter cohort of age- and sex-matched TM patients (n=244) who received conventional therapy. With a median follow-up of 24 years, 8 transplanted patients were diagnosed with SSC at a median of 18 years after HCT and at a median age of 33 years. Three patients died of cancer progression and 5 are living after a follow-up ranging from 10 months to 16 years after SSC diagnosis. The 30-year cumulative incidence of developing SSC was 13.24%. The occurrence of solid cancers in the hematopoietic cell donor cohort was limited to only one case for a significantly lower cumulative incidence (3.23%, P=0.02) and to 3 cases in the cohort of nontransplant patients for a significantly lower cumulative incidence (1.32%, P=0.005). This study shows that the magnitude of increased risk of SST is fourfold to sixfold for patients treated with HCT as compared with hematopoietic cell donors and nontransplant patients.

Amniocentesis and chorionic villus sampling in HIV-infected pregnant women: a multicentre case series.

OBJECTIVES: To assess in pregnant women with HIV the rates of amniocentesis and chorionic villus sampling (CVS), and the outcomes associated with such procedures. DESIGN: Observational study. Data from the Italian National Program on Surveillance on Antiretroviral Treatment in Pregnancy were used. SETTING: University and hospital clinics. POPULATION: Pregnant women with HIV. METHODS: Temporal trends were analysed by analysis of variance and by the Chi-square test for trend. Quantitative variables were compared by Student's t-test and categorical data by the Chi-square test, with odds ratios and 95% confidence intervals calculated. MAIN OUTCOME MEASURES: Rate of invasive testing, intrauterine death, HIV transmission. RESULTS: Between 2001 and 2015, among 2065 pregnancies in women with HIV, 113 (5.5%) had invasive tests performed. The procedures were conducted under antiretroviral treatment in 99 cases (87.6%), with a significant increase over time in the proportion of tests performed under highly active antiretroviral therapy (HAART) (100% in 2011-2015). Three intrauterine deaths were observed (2.6%), and 14 pregnancies were terminated because of fetal anomalies. Among 96 live newborns, eight had no information available on HIV status. Among the remaining 88 cases with either amniocentesis (n = 75), CVS (n = 12), or both (n = 1), two HIV transmissions occurred (2.3%). No HIV transmission occurred among the women who were on HAART at the time of invasive testing, and none after 2005. CONCLUSIONS: The findings reinforce the assumption that invasive prenatal testing does not increase the risk of HIV vertical transmission among pregnant women under suppressive antiretroviral treatment. TWEETABLE ABSTRACT: No HIV transmission occurred among women who underwent amniocentesis or CVS under effective anti-HIV regimens.

Anti-cytokine autoantibodies preceding onset of autoimmune polyendocrine syndrome type I features in early childhood.

PURPOSE: Almost all patients with autoimmune polyendocrine syndrome (APS)-I have high titer neutralizing autoantibodies to type I interferons (IFN), especially IFN-ω and IFN-α2, whatever their clinical features and onset-ages. About 90 % also have antibodies to interleukin (IL)-17A, IL-17F and/or IL-22; they correlate with the chronic mucocutaneous candidiasis (CMC) that affects ~90 % of patients. Our aim was to explore how early the manifestations and endocrine and cytokine autoantibodies appear in young APS-I patients. That may hold clues to very early events in the autoimmunization process in these patients. METHODS: Clinical investigations and autoantibody measurements in 13 APS-I patients sampled before age 7 years, and 3 pre-symptomatic siblings with AIRE-mutations in both alleles. RESULTS: Antibody titers were already high against IFN-α2 and IFN-ω at age 6 months in one sibling-8 months before onset of APS-I-and also against IL-22 at 7 months in another (still unaffected at age 5 years). In 12 of the 13 APS-I patients, antibody levels were high against IFN-ω and/or IL-22 when first tested, but only modestly positive against IFN-ω in one patient who had only hypo-parathyroidism. Endocrine organ-specific antibodies were present at age 6 months in one sibling, and as early as 36 and 48 months in two of the six informative subjects. CONCLUSION: This is the first study to collate the onset of clinical features, cytokine and endocrine autoantibodies in APS-I infants and siblings. The highly restricted early autoantibody responses and clinical features they show are not easily explained by mere loss of broad-specific self-tolerance inducing mechanisms, but hint at some more sharply focused early event(s) in autoimmunization.

GLP-1 receptor activated insulin secretion from pancreatic ß-cells: mechanism and glucose dependence.

The major goal in the treatment of type 2 diabetes mellitus is to control the hyperglycaemia characteristic of the disease. However, treatment with common therapies such as insulin or insulinotrophic sulphonylureas (SU), while effective in reducing hyperglycaemia, may impose a greater risk of hypoglycaemia, as neither therapy is self-regulated by ambient blood glucose concentrations. Hypoglycaemia has been associated with adverse physical and psychological outcomes and may contribute to negative cardiovascular events; hence minimization of hypoglycaemia risk is clinically advantageous. Stimulation of insulin secretion from pancreatic ß-cells by glucagon-like peptide 1 receptor (GLP-1R) agonists is known to be glucose-dependent. GLP-1R agonists potentiate glucose-stimulated insulin secretion and have little or no activity on insulin secretion in the absence of elevated blood glucose concentrations. This 'glucose-regulated' activity of GLP-1R agonists makes them useful and potentially safer therapeutics for overall glucose control compared to non-regulated therapies; hyperglycaemia can be reduced with minimal hypoglycaemia. While the inherent mechanism of action of GLP-1R agonists mediates their glucose dependence, studies in rats suggest that SUs may uncouple this dependence. This hypothesis is supported by clinical studies showing that the majority of events of hypoglycaemia in patients treated with GLP-1R agonists occur in patients treated with a concomitant SU. This review aims to discuss the current understanding of the mechanisms by which GLP-1R signalling promotes insulin secretion from pancreatic ß-cells via a glucose-dependent process.

TSGA10 - A target for autoantibodies in autoimmune polyendocrine syndrome type 1 and systemic lupus erythematosus.

Autoimmune polyendocrine syndrome type 1 (APS1) is a rare monogenic autoimmune disorder caused by mutations in the autoimmune regulator (AIRE) gene. High-titre autoantibodies are a characteristic feature of APS1 and are often associated with particular disease manifestations. Pituitary deficits are reported in approximately 7% of APS1 patients, with immunoreactivity to pituitary tissue frequently described. Using APS1 patient serum to immunoscreen a pituitary cDNA expression library, testis specific, 10 (TSGA10) was isolated. Immunoreactivity against TSGA10 was detected in 5/99 (5.05%) patients with APS1, but also in 5/135 (3.70%) systemic lupus erythematosus (SLE) patients and 1/188 (0.53%) healthy controls. TSGA10 autoantibodies were not detected in the serum from patients with any other autoimmune disease. Autoantibodies against TSGA10 were detectable from a young age in 4/5 positive APS1 patients with autoantibody titres remaining relatively constant over time. Furthermore, real-time PCR confirmed TSGA10 mRNA to be most abundantly expressed in the testis and also showed moderate and low expression levels throughout the entire body. TSGA10 should be considered as an autoantigen in a subset of APS1 patients and also in a minority of SLE patients. No recognizable clinical phenotype could be found to correlate with positive autoantibody reactivity.

A novel missense mutation (C84R) in a patient with type II vitamin d-dependent rickets.

A 7-year-old boy with severe rickets that by clinical analysis was diagnosed as affected by type II vitamin D-dependent rickets, was evaluated for mutations in the vitamin D receptor gene (VDR). The molecular analysis showed a homozygous state for a novel missense mutation (C84R) in a highly conserved nucleotide in the second Zn finger of the DNA binding domain.

Evaluation of a web-based network for reproducible T2* MRI assessment of iron overload in thalassemia.

PURPOSE: To build and evaluate a national network able to improve the care of thalassemia, a genetic disorder in haemoglobin synthesis often associated with iron accumulation in a variety of organs, due to the continuous blood transfusions. METHODS: The MIOT (Myocardial Iron Overload in Thalassemia) network is constituted by thalassemia and magnetic resonance imaging (MRI) centers. Thalassemia centers are responsible for patient recruitment and collection of anamnestic and clinical data. MRI centers have been equipped with a standardized acquisition technique and an affordable workstation for image analysis. They are able to perform feasible and reproducible heart and liver iron overload assessments for a consistent number of thalassemia patients in a robust manner. All centers are linked by a web-based network, configured to collect and share patient data. RESULTS: On 30th March 2008, 695 thalassemia patients were involved in the network. The completion percentage of the patient records in the database was 85+/-6.5%. Six hundred and thirteen patients (88%) successfully underwent MRI examination. Each MRI center had a specific absorption capacity that remained constant over time, but the network was capable of sustaining an increasing number of patients due to continuous enrollment of new centers. The patient's comfort, assessed as the mean distance from the patient home locations to the MRI centers, significantly increased during the network's evolution. CONCLUSION: The MIOT network seems to be a robust and scalable system in which T2* MRI-based cardiac and liver iron overload assessment is available, accessible and reachable for a significant and increasing number of thalassemia patients in Italy (about 420 per year), reducing the mean distance from the patient locations to the MRI sites from 951km to 387km. A solid, wide and homogeneous database will constitute an important scientific resource, shortening the time scale for diagnostic, prognostic and therapeutical evidence-based research on the management of thalassemia disease.

Pregnancy outcomes and antiretroviral treatment in a national cohort of pregnant women with HIV: overall rates and differences according to nationality.

We used data from the main surveillance study of HIV and pregnancy in Italy to evaluate possible differences in pregnancy care and outcomes according to nationality. Among 960 women followed in 2001-06, 33.5% were of foreign nationality, mostly from African countries. Foreign women had lower rates of preconception counselling and planning of pregnancy. They had more frequently HIV diagnosed during pregnancy, with a later start of antiretroviral treatment and lower treatment rates at all trimesters but not when the entire pregnancy, including delivery, was considered. No differences were observed between the two groups in ultrasonography assessments, hospitalisations, AIDS events, intrauterine or neonatal deaths, and mode and complications of delivery. Foreign women had a slightly lower occurrence of preterm delivery and infants with low birthweight. The results indicate good standards of care and low rates of adverse outcomes in pregnant women with HIV in Italy, irrespective of nationality. Specific interventions, however, are needed to increase the rates of counselling and HIV testing before pregnancy in foreign women.

Two novel mutations of the AIRE protein affecting its homodimerization properties.

We report two novel mutations, c.230T>C (p.F77S) and c.64_69del (p.V22_D23del) within the HSR domain of the AIRE protein in two patients of Italian descent affected by APECED. Both mutations were found in the compound heterozygous state respectively with c.994+5G>T and c.232T>A (p.W78R). With the two-hybrid assay in the yeast system we found that constructs containing the two mutations fail to interact with the wild-type protein. These findings indicate that both mutations negatively affected the homodimerization properties of the AIRE protein, thereby leading to a defective function.

Non-invasive electrophysiologic study with amagnetic transesophageal pacing during unshielded multichannel magnetocardiographic mapping.

UNLABELLED: Multichannel magnetocardiographic (MCG) mapping is a non-invasive method, which can provide reproducible three-dimensional (3D) localization of accessory pathways (AP) and ventricular arrhythmias, before ablation procedures. More recently MCG imaging of intra-atrial reentry circuits has also been reported. So far, reported cases of MCG localization and imaging of arrhythmias were investigated during spontaneous rhythm only, although more relevant information can be obtained during dynamic electrophysiologic study (EPS). For cardiac pacing one could use an amagnetic intracardiac catheter; but this, however, would add invasivity to a non-invasive method. The aim of this study was to validate a novel approach for dynamic non-invasive EPS based on MCG in combination with amagnetic transesophageal pacing (TEP). METHOD: A tetrapolar 7 French amagnetic catheter was developed, which provides effective TEP (with an average stimulation threshold of 10-15 mA) and simultaneous recording of two esophageal atrial electrograms. MCG data were acquired at rest, with a 36-channel MCG system (sensitivity of 20 fT/Hz(1/2)), for 90 to 300 seconds (sampling rate of 1 KHz; bandwidth of DC Hz to 100 Hz), as a function of the type of pacing procedure. 10 patients were investigated, during both continuous and programmed TEP. RESULTS AND CONCLUSIONS: MCG during TEP was feasible and reproducible. It provided: 1) more accurate localization of AP during pacing-induced maximal preexcitation; 2) inducibility of supraventricular AR and imaging of atrial reentry circuits, not spontaneously present; 3) stabilization of the heart rate to improve the accuracy of quantitative estimate of ventricular repolarization parameters.

Magnetocardiographic study of ventricular repolarization in hypertensive patients with and without left ventricular hypertrophy.

UNLABELLED: Previous studies in magnetically shielded rooms have shown that magnetocardiographic (MCG) mapping can be useful to detect early signs of left ventricular hypertrophy (LVH). The aim of this study was to evaluate ventricular repolarization parameters in patients with essential hypertension, associated or not with LVH, by means of unshielded multichannel MCG mapping. METHODS: 31 patients with pharmacologically treated essential hypertension (average BP systolic: 147.8+/-11.2, diastolic: 92.2+/-4.9) since 6.5+/-5.6 years, 13 without and 18 with evidence of LVH (4 by ECG, 11 by echocardiography, and 3 at both), were studied with a 36-channel MCG system (sensitivity of 20 fT/square root of Hz1/2) and with 12-lead ECG, in an unshielded hospital setting. To assess ventricular repolarization, HR-corrected, QTend, JTpeak, JTend, Tpeak-end intervals and QT dispersion (QTd) were measured from both MCG and ECG waveforms. The magnetic field gradient orientation (alpha angle) during the ST interval and at the Tpeak was also computed. 20 normal age-matched volunteers were used for comparison. RESULTS: As compared to normal volunteers, MCG JTend, QTend, Tpeak-end and QTd were significantly longer in hypertensive patients. The difference was not significant, if only patients with essential hypertension but no LVH were considered. The magnetic field alpha angle during the ST was significantly abnormal in patients with essential hypertension (p < 0.01). CONCLUSIONS: In patients with essential hypertension, MCG detects alterations of ventricular repolarization, not evidenced by 12-lead ECG.

Magnetocardiographic differences of ventricular repolarization parameters between Wistar rats and guinea pigs.

UNLABELLED: The morphology and range of duration of the action potential (AP) in normal Wistar rat's (WR) myocyte markedly differ from those of guinea pigs (GP), whose plateau (phase 2) duration is longer. Thus a clear-cut T wave can be easy defined in GP but not in WR. Aim of this study was to differentiate magnetocardiographic (MCG) ventricular repolarization (VR) parameters of healthy adult WR and GP. METHODS: 10 female animals (5 Guinea pigs and 5 Wistar rats) were studied with a 36-channel MCG system (sensitivity of 20 fT/square root of Hz1/2) and with one ECG lead, in an unshielded hospital room. To assess VR, HR-corrected, JTpeak, JTend, Tpeak-end and QTend, intervals were measured from both MCG waveforms. Timing was improved by MCG maps analysis. Magnetic field orientation (MFO), its dynamics (MFD) and stability (JTS) during the JT interval, were also automatically computed from MCG maps. RESULTS: All repolarization intervals were significantly shorter in WR than in GP, except the Tpeak-end, which was longer. MFO and MFD also differed. CONCLUSIONS: MCG estimate of VR parameters, in adult WR and GP, is precise enough to evidence breed-related differences, consistent with physiological heterogeneity of duration during phases 2 and 3 of the AP, and with an higher degree of transmural dispersion of repolarization in WR.

Effects of filtering on computer-aided analysis for detection of chronic ischemic heart disease with unshielded rest magnetocardiographic mapping.

UNLABELLED: Recent studies have reported better sensitivity of magnetocardiographic (MCG) mapping, as compared to ECG, in detecting ventricular repolarization (VR) abnormalities due to myocardial ischemia in patients (pts) with Ischemic Heart Disease (IHD). For quick data reduction, automatic analysis of MCG mapping is mostly used. The aim of our study was to evaluate if filtering modality could alter automatic analysis of MCG. METHOD: 39 subjects were studied: 20 normals and 19 IHD pts, with angiography-documented >70% coronary stenosis, positive stress/SPECT and ischemic 12-lead ECG in 12/19 (63%). Rest MCG was recorded with a 36-channel system (at 1 kHz; bandwidth DC-100 Hz). To assess VR, Hänninen's STalpha angle and three magnetic field dynamics parameters, [i.e. +/- poles: angle (A), distance (D) and ratio (R)] during the T-wave interval, were computed from the same MCG maps: 1) after digital 20 Hz low-pass filtering (LPF) and 2) after digital 50 Hz adaptive filtering (AF). The baseline was unchanged. Three quantitative MCG scores of the T-wave (EXT, ML, Q) were automatically calculated (with 20 Hz LPF only). RESULTS: Whereas the filtering modality didn't affect the predictivity of the STalpha angle, the predictive values of A, D, and R were different and partially contradicting. Automatic MCG scores had a predictive values ranging between 73% and 92%. CONCLUSIONS: The diagnostic power of unshielded MCG for detection of chronic IHD, with T-wave parameters (A, D and R) might be affected by LPF. The STalpha angle is not affected by LPF. Automatic EXT, ML and Q scores have better predictivity than ECG.

Clinical multichannel MCG in unshielded hospital environment.

UNLABELLED: From November 5th, 2001 to May 19th, 2004, 545 patients (177 with arrhythmias, 67 with WPW syndrome, 60 with Ischemic Heart Disease (IHD), 129 with different kinds of cardiomyopathy, 106 normals, 6 FMCG) have been consecutively investigated at the Catholic University of Rome, with unshielded Multichannel Magnetocardiographic Mapping (MMCG): 20 with the 9-channel system only and 525 with the 36-channel system (207 of them with both systems). 107 patients were investigated also after physical stress, carried out with a standard bicycle ergometer. In all patients MMCG was recorded at least three times, to check for reproducibility and/or for clinical follow-up, for a total of more than 1600 recordings. METHOD: MMCG was performed, with both the 9-channel and the 36-channel systems, at 1 kHz in the bandwidth DC-100 Hz. In the last 200 pts, 12-lead ECG was simultaneously recorded with amagnetic electrodes. On each patient file, post-processing and signal analysis for the quantitative assessment of ventricular repolarization and for 3D localization and electroanatomical imaging of cardiac arrhythmias, were carried out independently with two different approaches and software programs developed by CMI and by Neuromag (Finland). RESULTS: The results with the two methods have been compared. For 3D electroanatomical integration of MMCG localization results, 3D cardiac models have been used, constructed from patient MRI and/or from orthogonal fluoroscopic images taken at the moment of MCG recording. CONCLUSIONS: Qualitative reproducibility of MMCG was satisfactory. However the estimate of quantitative parameters has shown a certain degree of variability, which deserves further evaluation.

A novel splicing defect (IVS6+1G>T) in a patient with pseudovitamin D deficiency rickets.

A 15-month-old boy with severe rickets, that by clinical analysis was diagnosed as affected by hereditary pseudovitamin D deficiency rickets (PDDR), was evaluated for mutations in the 25OHD3 1alpha-hydroxylase gene. Molecular analysis showed a double heterozygous state for a novel splicing mutation in the invariant dinucleotide of the donor site of IVS6 and a 7 nucleotide insertion in the exon 8, which is common in different ethnical backgrounds.