Functional analysis of thirty-four suspected pathogenic missense variants in ALDH5A1 gene associated with succinic semialdehyde dehydrogenase deficiency.

Deficiency of succinate semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5a1 (ALDH5A1), OMIM 271980, 610045), the second enzyme of GABA degradation, represents a rare autosomal-recessively inherited disorder which manifests metabolically as gamma-hydroxybutyric aciduria. The neurological phenotype includes intellectual disability, autism spectrum, epilepsy and sleep and behavior disturbances. Approximately 70 variants have been reported in the ALDH5A1 gene, half of them being missense variants. In this study, 34 missense variants, of which 22 novel, were evaluated by in silico analyses using PolyPhen2 and SIFT prediction tools. Subsequently, the effect of these variants on SSADH activity was studied by transient overexpression in HEK293 cells. These studies showed severe enzymatic activity impairment for 27 out of 34 alleles, normal activity for one allele and a broad range of residual activities (25 to 74%) for six alleles. To better evaluate the alleles that showed residual activity above 25%, we generated an SSADH-deficient HEK293-Flp-In cell line using CRISPR-Cas9, in which these alleles were stably expressed. This model proved essential in the classification as deficient for one out of the seven studied alleles. For 8 out of 34 addressed alleles, there were discrepant results among the used prediction tools, and/or in correlating the results of the prediction tools with the functional data. In case of diagnostic urgency of missense alleles, we propose the use of the transient transfection model for confirmation of their effect on the SSADH catalytic function, since this model resulted in fast and robust functional characterization for the majority of the tested variants. In selected cases, stable transfections can be considered and may prove valuable.

Optimization of the Pentachlorophenol Adsorption by Organo-Clays Based on Response Surface Methodology.

The aim of this study is to optimize the adsorption of pentachlorophenol (PCP) using an organo-clay under the response surface methodology. The adsorbent was selected from a montmorillonite exchanged by various cations, such as Fe3+, Al3+, Zn2+, Mg2+, Na+, and modified by bromide cetyltrimethylammonium (CTAB) as surfactant. The obtained organo-montmorillonite was characterized using several techniques, such as Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and nitrogen adsorption, performed at -196 °C. The results showed an increase in basal space from 1.65 to 1.88 nm and a decrease in the specific surface and pore volume, with an increase in pore diameter, including the presence of characteristic bands of -CH2- and -CH3- groups at 2926 and 2854 cm-1 in the FTIR spectrum after the modification. The optimization of PCP removal by clay adsorbents is achieved using the response surface methodology (RSM) with a four-factor central composite model, including pH of solution, mass of adsorbent, contact time, and initial concentration. The results proved the validity of the regression model, wherein the adsorption capacity reaches its maximum value of 38 mg/g at a lower adsorbent mass of 20 mg, pH of 6, contact time (tc) of 5 h, and initial concentration of 8 mg/L.

MammaPrint and BluePrint Molecular Diagnostics Using Targeted RNA Next-Generation Sequencing Technology.

Next-generation DNA sequencing is rapidly becoming an indispensable tool for genome-directed cancer diagnostics, but next-generation RNA sequencing (RNA-seq) is currently not standardly used in clinical diagnostics for expression assessment. However, multigene RNA diagnostic assays are used increasingly in the routine diagnosis of early-stage breast cancer. Two of the most widely used tests are currently available only as a central laboratory service, which limits their clinical use. We evaluated the use of RNA-seq as a decentralized method to perform such tests. The MammaPrint and BluePrint RNA-seq tests were found to be equivalent to the clinically validated microarray tests. The RNA-seq tests were highly reproducible when performed in different locations and were stable over time. The MammaPrint RNA-seq test was clinically validated. Our data demonstrate that RNA-seq can be used as a decentralized platform, yielding results substantially equivalent to results derived from the predicate diagnostic device.