VAPB ER-Aggregates, A Possible New Biomarker in ALS Pathology.

A point mutation (P56S) in the gene-encoding vesicle-associated membrane-protein-associated protein B (VAPB) leads to an autosomal-dominant form of amyotrophic lateral sclerosis (ALS), classified as ALS-8. The mutant VAPB is characterized by ER-associated aggregates that lead to a complete reorganization of ER structures. Growing evidences suggest VAPB involvement in ALS pathomechanisms. In fact, numerous studies demonstrated VAPB alteration also in sporadic ALS (sALS) and showed the presence of its aggregates when others ALS-related gene are mutant. Recently, the identification of new biomarkers in peripheral blood mononuclear cells (PBMCs) has been proposed as a good noninvasive option for studying ALS. Here, we evaluated VAPB as a possible ALS pathologic marker analyzing PBMCs of sALS patients. Immunofluorescence analysis (IFA) showed a peculiar pattern of VAPB aggregates in sALS, not evident in healthy control (HC) subjects and in Parkinson's disease (PD) PBMCs. This specific pattern led us to suppose that VAPB could be misfolded in sALS. The data indirectly confirmed by flow cytometry assay (FCA) showed a reduction of VAPB fluorescent signals in sALS. However, our observations were not associated with the presence of a genetic mutation or altered gene expression of VAPB. Our study brings further evidences of the VAPB role in ALS as a diagnostic biomarker.

Intrahepatic bile duct primary cilia in biliary atresia.

AIM: The etiopathogenesis of non-syndromic biliary atresia (BA) is obscure. The primary aim was to investigate intrahepatic bile duct cilia (IHBC) in BA at diagnosis and its correlation with clinical outcome. The secondary aim was to analyze IHBC in routine paraffin-embedded liver biopsies using conventional scanning electron microscopy (SEM). METHODS: Surgical liver biopsies taken at diagnosis from 22 BA infants (age range, 39-116 days) and from eight children with non-BA chronic cholestasis (age range, 162 days -16.8 years) were evaluated for IHBC by immunofluorescence (IF) and SEM. A minimum 18-month follow-up after surgery was available for all patients. RESULTS: By IF, cilia were present in 6/8 (75%) non-BA but only in 3/22 (14%) BA cases, and cilia were reduced or absent in 19/22 (86%) BA and 2/8 (25%) non-BA livers (P < 0.01). In BA, cilia presence was found to be associated with clearance of jaundice at 6-month follow-up (P < 0.05). However, high overall survival rates with native liver, >90% at 12 months, and >70% at 24 months post-surgery, were recorded regardless of cilia presence/absence at diagnosis. Electron microscopy was able to detect bile ducts and cilia in routine liver biopsies, revealing significant abnormalities in 100% BA livers. CONCLUSIONS: The presence of IHBC in BA livers at the diagnosis was associated with resolution of cholestasis, although was not predictive of short-term survival with native liver. Scanning electron microscopy represents a powerful new tool to study routine liver biopsies in biliary disorders. Cilia dysfunction in BA pathogenesis and/or disease progression warrants further investigation.

Organ-specific antibodies in LADA patients for the prediction of insulin dependence.

AIM OF THE STUDY: The aim of the present study was to define the frequency of organ-specific and non-organ-specific autoantibodies in a cohort of Latent Autoimmune Diabetes in Adults (LADA) patients and to test whether multiple antibodies positivity could be a predictor of early insulin dependence. MATERIALS AND METHODS: We enrolled 210 LADA and 210 type 2 diabetes mellitus (T2D) patients. In all subjects anti-islet antigen-2 (IA-2Ab), anti-thyroperoxidase (TPOAb), anti-zinc transporter 8 (ZnT8Ab), anti-nuclear (ANA), anti-parietal cell (APCA), anti-smooth muscle (ASMA), anti-mitochondrial (AMA), anti-liver kidney microsomes (LKM), and anti-reticulin (ARA) circulating antibodies were assessed. RESULTS: The frequency of TPOAb, ZnT8Ab, APCA, and IA-2Ab positivity was, respectively, detected in 40.0%, 32.4%, 24.7%, and 9.5% of LADA patients, whereas their frequency was significantly lower in T2D patients (11.4%, 1.9%, 9.5%, and 0.0%, respectively, p < 0.001). The frequency of ANA was the same in both groups whereas the frequency of ASMA, ARA, AMA, and LKM was very low (range 0.0-3.3%). The presence of TPOAb associated with ZnT8Ab, IA-2Ab, or APCA allows one to predict the progression of disease with a high specificity but low sensibility. CONCLUSIONS: LADA patients show an increased frequency of organ- and non-organ-specific antibodies. Consequently, a screening is worthwhile in these patients. The simultaneous presence of TPOAb with ZnT8, IA-2Ab, or APCA may help differentiate clinical phenotypes and predict faster insulin dependence in LADA patients.

Mendelian breeding units versus standard sampling strategies: Mitochondrial DNA variation in southwest Sardinia.

We report a sampling strategy based on Mendelian Breeding Units (MBUs), representing an interbreeding group of individuals sharing a common gene pool. The identification of MBUs is crucial for case-control experimental design in association studies. The aim of this work was to evaluate the possible existence of bias in terms of genetic variability and haplogroup frequencies in the MBU sample, due to severe sample selection. In order to reach this goal, the MBU sampling strategy was compared to a standard selection of individuals according to their surname and place of birth. We analysed mitochondrial DNA variation (first hypervariable segment and coding region) in unrelated healthy subjects from two different areas of Sardinia: the area around the town of Cabras and the western Campidano area. No statistically significant differences were observed when the two sampling methods were compared, indicating that the stringent sample selection needed to establish a MBU does not alter original genetic variability and haplogroup distribution. Therefore, the MBU sampling strategy can be considered a useful tool in association studies of complex traits.

Mendelian breeding units versus standard sampling strategies: Mitochondrial DNA variation in southwest Sardinia

We report a sampling strategy based on Mendelian Breeding Units (MBUs), representing an interbreeding group of individuals sharing a common gene pool. The identification of MBUs is crucial for case-control experimental design in association studies. The aim of this work was to evaluate the possible existence of bias in terms of genetic variability and haplogroup frequencies in the MBU sample, due to severe sample selection. In order to reach this goal, the MBU sampling strategy was compared to a standard selection of individuals according to their surname and place of birth. We analysed mitochondrial DNA variation (first hypervariable segment and coding region) in unrelated healthy subjects from two different areas of Sardinia: the area around the town of Cabras and the western Campidano area. No statistically significant differences were observed when the two sampling methods were compared, indicating that the stringent sample selection needed to establish a MBU does not alter original genetic variability and haplogroup distribution. Therefore, the MBU sampling strategy can be considered a useful tool in association studies of complex traits.

The Trp64Arg beta3-adrenergic receptor amino acid variant confers increased sensitivity to the pressor effects of noradrenaline in Sardinian subjects.

The beta(3)-adrenergic receptor (beta(3)AR) plays a critical role in lipid metabolism, and thus alterations in its function may be involved in the metabolic syndrome. Indeed, we have found previously that the Trp64Arg amino acid variant of the beta(3)AR is associated with hypertension and higher serum triacylglycerol levels in the Sardinian population. The aim of the present study was to evaluate the effect of the Trp64Arg beta(3)AR variant on the regulation of triacylglycerol levels and blood pressure during the exogenous infusion of noradrenaline. We studied groups of non-diabetic normotensive subjects: eight with the wild-type Trp64Trp beta(3)AR and eight with the Trp64Arg variant. The subjects each received, on two different days (randomized, double-blind fashion), a 4 h infusion of either noradrenaline (0.147 nmol.min(-1).kg(-1)) or Emagel (subjects had fasted for at least 12 h). The only available subject with a homozygous mutant Arg64Arg beta(3)AR was also studied. Blood pressure was measured every 10 min using a sphygmomanometer, and blood samples were taken every 30 min from the contralateral vein for biochemical determinations. After a 4 h noradrenaline infusion, significant increases in diastolic blood pressure (from 83+/-2 to 91+/-3 mmHg; P <0.01) and serum triacylglycerol levels (from 1.69+/-0.4 to 1.79+/-0.6 mmol/l; P <0.05) were observed compared with basal values in subjects with the Trp64Arg beta(3)AR variant, whereas subjects with the Trp64Trp beta(3)AR did not show any significant change over the infusion period. Glycaemia had increased significantly only at the end of the first 1 h of infusion in subjects with the Trp64Arg variant (from 5.0+/-0.1 to 5.8+/-0.3 mmol/l; P <0.05), with no significantly different behaviour compared with those subjects with the Trp64Trp beta(3)AR during the remaining infusion period. The effects of noradrenaline infusion were more marked in the subject with the Arg64Arg variant. In conclusion, our data indicate that the Trp64Arg amino acid variant of the beta(3)AR confers increased sensitivity to the pressure effect of noradrenaline. Moreover, this variant also influences blood triacylglycerol levels and, to a degree, glucose metabolism.