Expanding Phenotype of Poirier-Bienvenu Syndrome: New Evidence from an Italian Multicentrical Cohort of Patients.

BACKGROUND: Poirier-Bienvenu Neurodevelopmental Syndrome (POBINDS) is a rare disease linked to mutations of the CSNK2B gene, which encodes for a subunit of caseinkinase CK2 involved in neuronal growth and synaptic transmission. Its main features include early-onset epilepsy and intellectual disability. Despite the lack of cases described, it appears that POBINDS could manifest with a wide range of phenotypes, possibly related to the different mutations of CSNK2B. METHODS: Our multicentric, retrospective study recruited nine patients with POBINDS, detected using next-generation sequencing panels and whole-exome sequencing. Clinical, laboratory, and neuroimaging data were reported for each patient in order to assess the severity of phenotype, and eventually, a correlation with the type of CSNK2B mutation. RESULTS: We reported nine unrelated patients with heterozygous de novo mutations of the CSNK2B gene. All cases presented epilepsy, and eight patients were associated with a different degree of intellectual disability. Other features detected included endocrinological and vascular abnormalities and dysmorphisms. Genetic analysis revealed six new variants of CSNK2B that have not been reported previously. CONCLUSION: Although it was not possible to assess a genotype-phenotype correlation in our patients, our research further expands the phenotype spectrum of POBINDS patients, identifying new mutations occurring in the CSNK2B gene.

Validation of I'screen AFLA M1 Milk for Detection of Aflatoxin M1 in Raw Bovine Milk and Powdered Milk: AOAC Performance Tested MethodSM 072002.

BACKGROUND: Aflatoxin M1 (AFM1) is the major metabolite of Aflatoxin B1 (AFB1) and can be found in the milk of animals fed with feed containing AFB1. The frequency of occurrence of AFM1 in milk has led to the development of specific quantitative methods of analysis to mitigate the risk of adversely affecting human health. OBJECTIVE: To demonstrate that the I'screen AFLA M1 Milk ELISA kit can quantify AFM1 in raw bovine milk and powdered milk. METHOD: Assay performance was evaluated studying lot-to-lot consistency, assay stability, robustness, and possible interferences of related molecules. Raw bovine milk samples spiked at 0, 5.0, 20, 50, 100, and 200 ng/L of AFM1 and powdered milk reference materials and spiked samples at 100 and 200 ng/L were tested to determine recovery, repeatability, and bias. LOD and LOQ were also determined for both matrices. RESULTS: High selectivity for AFM1 was demonstrated and performances were consistent, robust, and stable. The LOQ was validated at 5 ng/L for raw milk and 50 ng/L for powdered milk. Recoveries for spiked raw and powdered milk were 97-122%, with RSDr < 10%, and 106-111% for reference materials, with RSDr < 5%. CONCLUSIONS: The data collected validate the method as a selective, specific, sensitive, accurate, and precise tool for the analysis of AFM1 in raw bovine milk and powdered milk. HIGHLIGHTS: We demonstrated that I'screen AFLA M1 is a reliable kit and a proper screening tool suitable for high analytical throughputs.

Development of a nanoarray capable of the rapid and simultaneous detection of zearalenone, T2-toxin and fumonisin.

Fusarium mycotoxins such as trichothecenes, zearalenone and fumonisins occur on a worldwide basis in cereal grains, animal feeds and forages. Practical solutions for multiple mycotoxin determination in samples are required by industry and regulators for cost effective screening purposes. The feasibility of developing a novel multiplex nanoarray for the simultaneous and semi-quantitative detection of three regulated mycotoxins: zearalenone (ZEA), T2-toxin (T2) and fumonisin B1 (FUM) was examined. Additionally, the assay was also able to detect HT2 toxin and fumonisin B2 and B3 due to the cross reactivity profiles of the antibodies used. Individual mycotoxin conjugates specific to the three mycotoxins were nano-spotted onto wells of a microtitre plate. Optimisation of assay parameters and antibodies was undertaken with both individual and multiplex calibration curves generated. A competitive assay format was employed enabling a calibration curve for concentration analysis and duplicate results for up to 40 samples in 70min for the three target mycotoxins. The characteristics and performance of the nanoarray were evaluated including sensitivity and specificity for each target. Additionally, intra and inter spotting precision, cross reactivity, matrix effects and sample analysis in maize and wheat (n=8) was performed. Sensitivity, determined as the concentration causing 50% inhibition, was 70.1, 2.8 and 90.9ppb in PBS, 172.4, 3.2 and 129.3ppb in methanol, 197.4, 0.7 and 216.7ppb in wheat and 43.6, 0.5 and 25.9ppb in maize for ZEA, T2 and FUM respectively. Intra spotting precision was 6%, 11% and 10% for PBS and 5%, 11% and 12% for methanol for ZEA, T2 and FUM respectively. Inter spotting precision was 4%, 14% and 6% for PBS and 3%, 9% and 16% for methanol for ZEA, T2 and FUM respectively. The feasibility of the nanoarray as an easy to use sensitive screening tool in the 96 well format has been demonstrated for the multiplex detection of three regulated mycotoxins. Improvements in automated image and data analysis software for novice end users are required to improve the overall rapidity of analysis.