Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .     

Lab-on-a-chip: Emerging analytical platforms for immune-mediated diseases

Miniaturization of analytical procedures has a significant impact on diagnostic testing since it provides several advantages such as: reduced sample and reagent consumption, shorter analysis time and less sample handling. Lab-on-a-chip (LoC), usually silicon, glass, or silicon-glass, or polymer disposable cartridges, which are produced using techniques inherited from the microelectronics industry, could perform and integrate the operations needed to carry out biochemical analysis through the mechanical realization of a dedicated instrument.Analytical devices based on miniaturized platforms like LoC may provide an important contribution to the diagnosis of high prevalence and rare diseases. In this paper we review some of the uses of Lab-on-a-chip in the clinical diagnostics of immune-mediated diseases and we provide an overview of how specific applications of these technologies could improve and simplify several complex diagnostic procedures.     

The p.A382T TARDBP gene mutation in Sardinian patients affected by Parkinson's disease and other degenerative parkinsonisms

Based on our previous finding of the p.A382T founder mutation in ALS patients with concomitant parkinsonism in the Sardinian population, we hypothesized that the same variant may underlie Parkinson's disease (PD) and/or other forms of degenerative parkinsonism on this Mediterranean island. We screened a cohort of 611 patients with PD (544 cases) and other forms of degenerative parkinsonism (67 cases) and 604 unrelated controls for the c.1144G > A (p.A382T) missense mutation of the TARDBP gene. The p.A382T mutation was identified in nine patients with parkinsonism. Of these, five (0.9 % of PD patients) presented a typical PD (two with familiar forms), while four patients (6.0 % of all other forms of parkinsonism) presented a peculiar clinical presentation quite different from classical atypical parkinsonism with an overlap of extrapyramidal–pyramidal–cognitive clinical signs. The mutation was found in eight Sardinian controls (1.3 %) consistent with a founder mutation in the island population. Our findings suggest that the clinical presentation of the p.A382T TARDBP gene mutation may include forms of parkinsonism in which the extrapyramidal signs are the crucial core of the disease at onset. These forms can present PSP or CBD-like clinical signs, with bulbar and/or extrabulbar pyramidal signs and cognitive impairment. No evidence of association has been found between TARDBP gene mutation and typical PD.     

Low renin-angiotensin system activity gene polymorphism and dysplasia associated with posterior urethral valves

PURPOSE: Obstructive uropathies, including posterior urethral valves (PUVs) and kidney hypodysplasia, are the most frequent cause of renal failure in children. The role of renin-angiotensin system genes in renal and urinary tract development has been observed in experimental models. The aim of this study was to investigate the distribution of angiotensin converting enzyme (ACE), angiotensinogen (AGT) and angiotensin receptor type 1 (ATR1) genetic polymorphisms in children affected by chronic renal failure due to renal hypodysplasia associated with posterior urethral valves or without urethral abnormalities. MATERIALS AND METHODS: The study included 50 children (21 with hypodysplasia associated with PUVs, 7 with obstructive uropathy and 22 with pure hypodysplasia) and 50 healthy subjects matched for sex and geographic origin. ACE ID, AGT TC and ATR1 AC gene polymorphisms were assayed in all patients with standard polymerase chain reaction techniques. RESULTS: ACE II was expressed more in patients with PUVs compared to those with other dysplasias and controls (43% vs 7% and 10%, respectively, chi-square test p <0.05), while ATR1 AA was significantly less represented in patients with hypodysplasia compared to controls (38% vs 56%, chi-square test p <0.05). ACE DD and AGT genotypes were not distributed differently in patients with PUVs compared to those with other dysplasias and controls. CONCLUSIONS: To our knowledge this is the first report associating severe congenital uropathies and renal hypodysplasia with decreased renin-angiotensin system activity associated with the ACE II genotype and a possible functional imbalance among ATR1 receptors.     

FSH-receptor <Emphasis Type="Italic">Ala307Thr</Emphasis> polymorphism is associated to polycystic ovary syndrome and to a higher responsiveness to exogenous FSH in Italian women

Objective  

Herein we analyzed FSH-R polymorphism at position 307 aiming (a) to assess the prevalence of the three allelic variants (Ala307Ala, Ala307Thr and Thr307Thr) in relation to the type of ovary and (b) to clarify if the allelic variant could influence the responsiveness to exogenous FSH.     

A new device for continuous assessment of gut perfusion: proof of concept on a porcine model of septic shock

Introduction

We evaluate an innovative device consisting of an enteral feeding tube equipped with a photoplethysmography (PPG) sensor in contact with the duodenal mucosa. This study aims to determine if the PPG signal, composed of a continuous (PDC) and a pulsatile part (PAC), is a reliable method to assess gut perfusion in a porcine model of septic shock.

Method

Fourteen piglets were anesthetized and mechanically ventilated. They were randomly assigned to two groups: the nonseptic (NS) group received an infusion of Ringer’s lactate solution (RL) alone, the septic (S) group received in addition a suspension of live Pseudomonas aeruginosa. Heart rate (HR), pulse oximetry (SpO₂), mean arterial pressure (MAP), cardiac index (CI) and serum lactates were recorded and gut microcirculation (GM) was monitored with a laser Doppler probe applied on the duodenal serosa. PDC and PAC were given by the PPG probe inserted in the duodenum. Data was collected every 15 minutes (t₀, t₁₅…) during 150 minutes (t₁₅₀). After administration of the bacteria suspension (t₀), resuscitation maneuvers were performed following a defined algorithm. GM PAC, and PDC were expressed as variation from baseline (GM_var, PAC_var, PDC_var). Analysis of variance (ANOVA) with repeated measures was performed to compare hemodynamic variables, with Bonferroni correction as post hoc analysis on t₀, t₆₀ and t₁₅₀.

Results

One piglet was withdrawn from analysis due to a defective probe. S group (six piglets) received resuscitation therapy while NS group (seven piglets) did not. A significant group effect was found for the all parameters except HR. Post hoc analysis found a significant decrease for GM and PAC at t₆₀. The correlation between PAC, PDC and microcirculatory parameters were as follows: r_PACvar-GMvar = 0.496, P <0.001, r_PDCvar-GMvar = 0.244; P = 0.002. In the septic group, correlations were as follows: r_PAC-lactate = -0.772, P <0.001; r_PDC-lactate = -0.681, P <0.01). At the onset of shock, a decrease of PAC, PDC and GM occurred before the alteration of MAP.

Conclusions

PAC and PDC decreased at the onset of shock and were correlated with GM and lactate. These results confirm that PPG signal reliably reflects the early perfusion alteration of the gut. Further studies should assess the clinical use of this device.     

An ALS case with a novel D90N-SOD1 heterozygous missense mutation

Abstract Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease. We describe the case of a patient with a rapidly progressive form of ALS characterized by both upper and lower motor neuron impairment, no early bulbar signs and severe pain in all four extremities. The patient had a heterozygous c.271G > A mutation in SOD1, leading to an amino acids substitution of asparagine to aspartate at position 90 of the protein chain (p.D90N). Our report confirms that ALS patients with D90 codon heterozygous mutations may be associated with rapid progression and a prominent pain syndrome.     

Replication of association of CHRNA4 rare variants with sporadic amyotrophic lateral sclerosis: The Italian multicentre study

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels widely expressed throughout the mammalian brain, including bulbar and spinal motor neurons. They are involved in neuroprotection and in control of release of many neurotransmitters, including glutamate. Previous data raised the hypothesis that rare variants in the region coding the intracellular loop subunits of nAChRs might represent one of several genetic risk factors for SALS. The aim of present study was to replicate the study in an independent cohort of ALS patients. We analysed 718 sporadic ALS patients from five Italian ALS centres and 1300 ethnically matched controls. We focused primarily on CHRNA4, encoding α4 subunit, since most mutations were previously detected in this gene. We observed a significant association between CHRNA4 mutations and ALS (OR 2.91; 95% CI 1.4080-6.0453; p =?0.0056). Most mutations detected in patients were not present in the dbSNP134 and in 3500 ethnically matched control chromosomes and affected evolutionary conserved amino acid residues. In conclusion, the present data confirm that CHRNA4 variants are overrepresented in SALS strengthening the hypothesis can they act as predisposing genetic factors for SALS.     

Analysis of CA/GT microsatellite polymorphism in IVS8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene: a study of Italian CF families

Forty-six CF Italian patients and their parents were screened for a highly polymorphic microsatellite consisting of a variable number of CA/GT repeats in intron 8 of the CFTR gene. A strong degree of association was found between alleles 2 and 6 and the CF mutation delta F508. Moreover, considering the haplotypes at the closely linked locus D7S23 and the microsatellite's alleles, a strong linkage disequilibrium was again found for delta F508 and also for non-delta F508 CF chromosomes and the eight commonest haplotypes (B2, B6, C7, A6, A7, B7, D2 and D7). These data, compared with those described in the Spanish population, further support the common origin of the delta F508 mutation in Southern European populations.