A case report of two Moroccan patients with hereditary neurological disorders and molecular modeling study on the S72L de novo PMP22 variant.

Hereditary neurological disorders represent a wild group of hereditary illnesses affecting mainly the nervous system, the majority of which have a Mendelian inheritance pattern. Here we present the case of two Moroccan patients each affected by a different hereditary neurological disorder. In the first patient WES analysis revealed the presence of the p.Ser72Leu de novo mutation in the PMP22 gene reported for the first time in Africa, specifically in Morocco. This variant is predicted to be in a mutation "hot-spot" region causing Dejerine-Sottas syndrome called also Charcot-Marie-Tooth type 3. The molecular modeling study suggests an important alteration of hydrogen and hydrophobic interactions between the residue in position 72 of the PMP22 protein and its surrounding amino acids. On the other hand, the p.Ala177Thr mutation on the RNASEH2B gene, responsible of Aicardi-Goutières syndrome 2, was carried in a homozygous state by the second patient descending from a consanguineous family. This mutation is common among the Moroccan population as well as in other North African countries. The present results contributed to a better follow-up of both cases allowing better symptom management with convenient treatments.

Polystyrene derivatives as candidates for extracorporeal adsorption of Factor VIII antibodies in the management of haemophiliac patients.

BACKGROUND AND OBJECTIVES: This article presents a new approach for removing the Factor VIII inhibitors (anti-FVIII) in haemophiliac patients by immunoadsorption using an affinity matrix. MATERIALS AND METHODS: Ten blood samples from haemophiliac patients with anti-FVIII were assayed for antibodies, total immunoglobulins, procoagulant proteins and complement C3 protein after circulation over one or two columns filled with the polymers under investigation. RESULTS: These new synthetic sorbents are able to remove in vitro 90% of anti-FVIII from haemophiliac plasma with inhibitors (up to 540 Bethesda Units/ml). Neither coagulation factor adsorption nor effects on complement system activation were observed. CONCLUSIONS: The data presented clearly show that these polymers allow a rapid and efficient reduction of inhibitor titre. In view of the parameters studied, these polymers fulfil the requirements for use in a blood purification process to decrease high inhibitor titres without losing essential proteins.

Temperature-responsive size-exclusion chromatography using poly(N-isopropylacrylamide) grafted silica.

Silica-based packing materials induce non-specific interactions with proteins in aqueous media because of the nature of their surface, mainly silanol groups. Therefore, the silica surface has to be modified in order to be used as stationary phase for the High Performance Size-Exclusion Chromatography (HPSEC) of proteins. For this purpose, porous silica beads were coated with hydrophilic polymer gels (dextrans of different molecular weights) carrying a calculated amount of diethyl-aminoethyl groups (DEAE). Actually, as shown by HPSEC, these dextran modified supports minimize non-specific adsorption for proteins and pullulans in aqueous solution. Then, in order to change the pore size in response to temperature, temperature responsive polymer of poly(N-isopropylacrylamide) (PIPAAm) was introduced into the surface of dextran-DEAE on porous silica beads. The structure of these supports before and after modification was alternately studied by Scanning Electronic Microscopy (SEM) and Scanning Force Microscopy (SFM). An adsorption of radiolabelled albumin was performed to complete our study. Silica modifications by dextran-DEAE and PIPAAm improve the neutrality of the support and minimize the non-specific interactions between the solid support and proteins in solution. At low temperature, the support having PIPAAm exhibits a high resolution domain in HPSEC and finally permits a better resolution of proteins and pullulans. At higher temperature, hydrophobic properties of PIPAAm produce interactions with some proteins and trigger off a slight delay of their elution time.

Influence of the nature of coupling agents on insulin adsorption on supports grafted with sialic acid for high-performance affinity chromatography.

Porous silica exhibits excellent mechanical properties for use as a stationary phase for high-performance liquid chromatography. However, negative surface charges make it unusable in its native state. For this reason, silica beads are coated with dextran polymers carrying a calculated amount of diethylaminoethyl groups. Both the minimization of non-specific interactions and the hydrophilic character of such supports allow their functionalization with biospecific ligands and finally their use in high-performance affinity chromatography of biological products. The use of these modified supports in high-performance affinity chromatography requires a better understanding of various characteristics of stationary phases. For this purpose, several techniques were utilized, in particular, size-exclusion chromatography and adsorption of radiolabelled albumin. These methods provided complementary information on the structure of these supports. Coated silica-based supports were functionalized with sialic acid by means of different coupling agents. The affinity of these supports for insulin was determined by the establishment of adsorption isotherms and by high-performance affinity chromatography, to evidence the relationships between structural characteristics of the supports and their separation properties. The study of interactions between these supports and insulin allowed us to show the importance of the coupling method on the performances of supports in affinity chromatography.

Interaction mechanisms between insulin and N-acetylneuraminic acid in affinity chromatography.

Silica beads are coated with dextran carrying a calculated amount of positively charged diethylaminoethyl (DEAE) groups in order to neutralize negatively charged silanol groups at the silica surface and in this way to minimize non-specific interactions between silica and proteins in solution. Dextran-coated silica supports are potentially excellent stationary phases for high-performance liquid chromatography of proteins. These supports combine the advantages of polysaccharide phases with the excellent mechanical characteristics of silica. These supports [silica-dextran-DEAE (SID)] are easily functionalized by grafting N-acetylneuramic acid (NANA), extracted from edible birds' nests, using conventional coupling methods. The performance of supports bearing NANA was studied by high-performance liquid affinity chromatography of insulin, the hypoglycaemic peptide hormone of the human organism. The study showed that these supports exhibit a reversible and specific affinity towards insulin and allow separations with high purification yields. The influence of different physico-chemical parameters (pH, temperature and insulin concentration) on insulin retention on the support was studied. This allowed the optimization of the conditions of adsorption and a better understanding of the interaction mechanisms between insulin and NANA as a biospecific ligand.

High-performance affinity chromatography of insulin on coated silica grafted with sialic acid.

High-performance liquid affinity chromatography (HPLAC) is a powerful method for the purification of biological compounds, owing to its specificity, speed and high resolution. We developed new chromatographic supports based on porous silica beads. In order to minimize non-specific interaction between the silanol groups at the silica surface and biological molecules, the beads are coated with dextran carrying a calculated amount of positively charged functions. Such supports have the mechanical properties of the starting inorganic material. Moreover, they can be easily activated and functionalized by active ligands using conventional coupling methods. In the present study, N-acetylneuraminic acid (NANA), a member of the sialic acid family, is coupled to dextran coated silica beads to obtain affinity supports. This class of compounds seems to play an important role in the cell recognition mechanism. In particular, sialic acids are present in the structure of the cellular receptors for insulin. By HPLAC, we can study the interactions between coated silica grafted with NANA and insulin. It is also possible to use these active supports to purify the compounds by affinity chromatography. However, it is important to determine and optimize the conditions for adsorption and desorption of insulin on supports grafted with sialic acid and to estimate the chromatographic performances of these active phases.