Identification of mutations that causes glucose-6-phosphate transporter defect in tunisian patients with glycogenosis type 1b.

BACKGROUND: Glycogen storage disease type 1b (GSD1b) is an autosomal recessive lysosomal storage disease caused by defective glucose-6-phosphate transporter encoded by SLC37A4 leading to the accumulation of glycogen in various tissues. The high rate of consanguineous marriages in Tunisian population provides an ideal environment to facilitate the identification of homozygous pathogenic mutations. We aimed to determine the clinical and genetic profiles of patients with GSD1b to evaluate SLC37A4 mutations spectrum in Tunisian patients. METHODS: All exons and flanking intron regions of SLC37A4 gene were screened by direct sequencing to identify mutations and polymorphisms in three unrelated families with GSD1b. Bioinformatics tools were then used to predict the impacts of identified mutations on the structure and function of protein in order to propose a function-structure relationship of the G6PT1 protein. RESULTS: Three patients (MT, MB and SI) in Families I, II and III who had the severe phenotype were homoallelic for the two identified mutations: p.R300H (famillies I, II) and p.W393X (Family III), respectively. One of the alterations was a missense mutation p.R300H of exon 6 in SLC37A4 gene. The analysis of the protein structure flexibility upon p.R300H mutation using DynaMut tool and CABS-flex 2.0 server showed that the reported mutation increase the molecule flexibility of in the cytosol region and would probably lead to significant conformational changes. CONCLUSION: This is the first Tunisian report of SLC37A4 mutations identified in Tunisia causing the glycogenosis type Ib disease. Bioinformatics analysis allowed us to establish an approximate structure-function relationship for the G6PT1 protein, thereby providing better genotype/phenotype correlation knowledge.

Molecular characterization of CTNS mutations in Tunisian patients with ocular cystinosis.

BACKGROUND: Ocular cystinosis is a rare autosomal recessive disorder characterized by intralysosomal cystine accumulation in renal, ophthalmic (cornea, conjunctiva), and other organ abnormalities. Patients with ocular cystinosis are mostly asymptomatic and typically experience mild photophobia due to cystine crystals in the cornea observed accidently during a routine ocular examination. The ocular cystinosis is associated with different mutations in CTNS gene. Cysteamine therapy mostly corrects the organ abnormalities. METHODS: This study was performed in collaboration with the department of ophthalmology of Farhat Hached Hospital. The Optical Coherence Tomography (OCT) of the cornea and retinal photography were used to search cystine crystals within the corneas and conjunctiva in eight Tunisian patients. Screening for the common 57-kb deletion was performed by standard multiplex PCR, followed by direct sequencing of the entire CTNS gene. RESULTS: The studied patients were found to have cystine crystal limited anterior corneal stroma and the conjunctiva associated with retinal crystals accumulation. CTNS gene sequencing disclosed 7 mutations: three missense mutations (G308R, p.Q88K, and p.S139Y); one duplication (C.829dup), one framshift mutation (p.G258f), one splice site mutation (c.681 + 7delC) and a large deletion (20,327-bp deletion). Crystallographic structure analysis suggests that the novel mutation p.S139Y is buried in a first transmembrane helix closed to the lipid bilayer polar region, introducing a difference in hydrophobicity which could affect the hydrophobic interactions with the membrane lipids. The second novel mutation p.Q88K which is located in the lysosomal lumen close to the lipid membrane polar head region, introduced a basic amino acid in a region which tolerate only uncharged residue. The third missense mutation introduces a positive change in nonpolar tail region of the phospholipid bilayer membrane affecting the folding and stability of the protein in the lipid bilayer. CONCLUSIONS: Our data demonstrate that impaired transport of cystine out of lysosomes is the most common, which is obviously associated with the mutations of transmembrane domains of cystinosine resulting from a total loss of its activity.

Fucosidosis in Tunisian patients: mutational analysis and homology-based modeling of FUCA1 enzyme.

BACKGROUND: Fucosidosis is an autosomal recessive lysosomal storage disease caused by defective alpha-L-fucosidase (FUCA1) activity, leading to the accumulation of fucose-containing glycolipids and glycoproteins in various tissues. Clinical features include angiokeratoma, progressive psychomotor retardation, neurologic signs, coarse facial features, and dysostosis multiplex. METHODS: All exons and flanking intron regions of FUCA1 were screened by direct sequencing to identify mutations and polymorphisms in three unrelated families with fucosidosis. Bioinformatics tools were then used to predict the impacts of novel alterations on the structure and function of proteins. Furthermore, the identified mutations were localized onto a 3D structure model using the DeepView Swiss-PdbViewer 4.1 software, which established a function-structure relationship of the FUCA1 proteins. RESULTS: Four novel mutations were identified in this study. Two patients (P1 and P2) in Families 1 and 2 who had the severe phenotype were homoallelic for the two identified frameshift mutations p.K57Sfs*75 and p.F77Sfs*55, respectively. The affected patient (P3) from Family 3, who had the milder phenotype, was heterozygous for the novel missense mutation p.G332E and the novel splice site mutation c.662+5g>c. We verified that this sequence variation did not correspond to a polymorphism by testing 50 unrelated individuals. Additionally, 16 FUCA1 polymorphisms were identified. The structure prediction analysis showed that the missense mutation p.G332E would probably lead to a significant conformational change, thereby preventing the expression of the FUCA1 protein indeed; the 3D structural model of the FUCA1 protein reveals that the glycine at position 332 is located near a catalytic nucleophilic residue. This makes it likely that the enzymatic function of the protein with p.G332E is severely impaired. CONCLUSION: These are the first FUCA1 mutations identified in Tunisia that cause the fucosidosis disease. Bioinformatics analysis allowed us to establish an approximate structure-function relationship for the FUCA1 protein, thereby providing better genotype/phenotype correlation knowledge.

Effects and Side Effects of Platelet Transfusion.

Aside from their canonical role in hemostasis, it is increasingly recognized that platelets have inflammatory functions and can regulate both adaptive and innate immune responses. The main topic this review aims to cover is the proinflammatory effects and side effects of platelet transfusion. Platelets prepared for transfusion are subject to stress injury upon collection, preparation, and storage. With these types of stress, they undergo morphologic, metabolic, and functional modulations which are likely to induce platelet activation and the release of biological response modifiers (BRMs). As a consequence, platelet concentrates (PCs) accumulate BRMs during processing and storage, and these BRMs are ultimately transfused alongside platelets. It has been shown that BRMs present in PCs can induce immune responses and posttransfusion reactions in the transfusion recipient. Several recent reports within the transfusion literature have investigated the concept of platelets as immune cells. Nevertheless, current and future investigations will face the challenge of encompassing the immunological role of platelets in the scope of transfusion.

Platelet depletion limits the severity but does not prevent the occurrence of experimental transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a severe pulmonary reaction due to blood transfusions. The pathophysiology of this complication is still not widely elucidated by the scientific community, especially regarding the direct role of blood platelets within the cellular mechanism responsible for the development of TRALI. STUDY DESIGN AND METHODS: In this study, a mouse model was used to induce the development of antibody-mediated acute lung injury through injections of lipopolysaccharide and an anti-major histocompatibility complex Class I antibody. BALB/c mice were pretreated with an anti-GPIbα antibody, which induces platelet depletion, or ML354, a protease receptor 4 pathway inhibitor, 30 minutes before TRALI induction. RESULTS: Depletion of platelets before TRALI induction appeared to reduce the severity of TRALI without completely inhibiting its development. Also, inhibition of platelet activation by ML354 did not prevent the onset of TRALI. Finally, the stimuli used for TRALI induction also triggered specific platelet activation upon ex vivo stimulation. CONCLUSIONS: This study suggests that blood platelets are not critically required for TRALI induction, although they are to some extent involved in its pathophysiology.

Dysregulated pathways and differentially expressed proteins associated with adverse transfusion reactions in different types of platelet components.

Platelet components (PCs) are occasionally associated with adverse transfusion reactions (ATRs). ATRs can occur regardless of the type of PC being transfused, whether it is a single-donor apheresis PC (SDA-PC) or a pooled PC (PPCs). The purpose of this study was to investigate the proteins and dysregulated pathways in both of the main types of PCs. The proteomic profiles of platelet pellets from SDA-PCs and PPCs involved in ATRs were analysed using the label-free LC-MS/MS method. Differentially expressed proteins with fold changes >|1.5| in clinical cases versus controls were characterised using bioinformatic tools (RStudio, GeneCodis3, and Ingenuity Pathways Analysis (IPA). The proteins were confirmed by western blotting. The common primary proteins found to be dysregulated in both types of PCs were the mitochondrial carnitine/acylcarnitine carrier protein (SLC25A20), multimerin-1 (MMRN1), and calumenin (CALU), which are associated with the important enrichment of platelet activation, platelet degranulation, and mitochondrial activity. Furthermore, this analysis revealed the involvement of commonly dysregulated canonical pathways, particularly mitochondrial dysfunction, platelet activation, and acute phase response. This proteomic analysis provided an interesting contribution to our understanding of the meticulous physiopathology of PCs associated with ATR. A larger investigation would assist in delineating the most relevant proteins to target within preventive transfusion safety strategies. BIOLOGICAL SIGNIFICANCE: Within platelet transfusion strategies, the two primary types of PCs predominantly processed in Europe, include (i) single donor apheresis PCs (SDA-PCs) from one donor and (ii) pooled PCs (PPCs). The current study used PCs from five buffy coats derived from five whole blood donations that were identical in ABO, RH1 and KEL1 groups. Both PC types were shown to be associated with the onset of an ATR in the transfused patient. Several common platelet proteins were found to be dysregulated in bags associated with ATR occurrences regardless of the type of PCs transfused and of their process. The dysregulated proteins included mitochondrial carnitine/acylcarnitine carrier protein (SLC25A20), which is involved in a fatty acid oxidation disorder; calumenin (CALU); and multimerin-1 (MMRN1), which is chiefly involved in platelet activation and degranulation. Dysregulated platelet protein pathways for ATRs that occurred with SDA-PCs and PPCs could support the dysregulated functions found in association with those three proteins. Those common platelet proteins may become candidates to define biomarkers associated with the onset of an ATR from PC transfusions, including monitoring during the quality steps of PC manufacturing, provided that the results are confirmed in larger cohorts. This study enriches our knowledge of platelet proteomics in PCs under pathological conditions.

Evidence of CD40L/CD40 pathway involvement in experimental transfusion-related acute lung injury.

Platelet transfusions can cause adverse reactions in their recipients, including transfusion-related acute lung injury (TRALI). The pathophysiology of TRALI depends on a number of signaling pathways and the inflammatory role played by blood platelets remains controversial. Platelets are important in inflammation, particularly via the immunomodulator complex CD40/CD40L. We studied the specific function of the CD40/CD40L interaction in regulating an experimental TRALI Two-hit model. A mouse model of immune TRALI was triggered by injection of LPS and an anti-MHC I antibody, and the effect of injection of a neutralizing anti-CD40L antibody before induction of TRALI investigated. The characteristics of TRALI were decreased body temperature, pulmonary lesions, and immune cell infiltration into the alveolar space. Pulmonary infiltration was evaluated by blood counts of specific immune cells and their detection in lung sections. Inhibition of the CD40/CD40L immunomodulator interaction significantly reduced communication between immune and/or endothelial cells and the development of pulmonary edema. Hence, our results indicate that targeting of the CD40/CD40L interaction could be an important method to prevent TRALI. While considering that our work concerned a mouse model, we postulate that improvement of the conditions under which platelet concentrates are prepared/stored would assist in alleviating the risk of TRALI.

Platelet Inflammatory Response to Stress.

Blood platelets play a central hemostatic role, (i) as they repair vascular epithelial damage, and (ii) they play immune defense roles, as they have the capacity to produce and secrete various cytokines, chemokines, and related products. Platelets sense and respond to local dangers (infectious or not). Platelets, therefore, mediate inflammation, express and use receptors to bind infectious pathogen moieties and endogenous ligands, among other components. Platelets contribute to effective pathogen clearance. Damage-associated molecular patterns (DAMPs) are danger signals released during inflammatory stress, such as burns, trauma and infection. Each pathogen is recognized by its specific molecular signature or pathogen-associated molecular pattern (PAMP). Recent data demonstrate that platelets have the capacity to sense external danger signals (DAMPs or PAMPs) differentially through a distinct type of pathogen recognition receptor (such as Toll-like receptors). Platelets regulate the innate immune response to pathogens and/or endogenous molecules, presenting several types of "danger" signals using a complete signalosome. Platelets, therefore, use complex tools to mediate a wide range of functions from danger sensing to tissue repair. Moreover, we noted that the secretory capacity of stored platelets over time and the development of stress lesions by platelets upon collection, processing, and storage are considered stress signals. The key message of this review is the "inflammatory response to stress" function of platelets in an infectious or non-infectious context.

Data from differentially expressed proteins in platelet components associated with adverse transfusion reactions.

The presented dataset was used for the study focused on the search for differentially expressed proteins in blood platelet components (PCs) associated with adverse transfusion reactions (ATRs). Pellets of ATR platelet components and their controls were subjected to high-throughput proteomics analysis using a Q Exactive high-resolution tandem mass spectrometer. The data reported here constitutes an extension of "Differential protein expression of blood platelet components associated with adverse transfusion reactions" article Aloui et al., 2018. The reported data herein have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD003510 for the pooled platelet components (PPCs) and PXD008886 for the apheresis platelet components (SDA-PCs) associated with ATRs.

Platelet-derived extracellular vesicles convey mitochondrial DAMPs in platelet concentrates and their levels are associated with adverse reactions.

BACKGROUND: Whereas platelet transfusion is a common medical procedure, inflammation still occurs in a fraction of transfused individuals despite the absence of any apparent infectious agents. Platelets can shed membrane vesicles, called extracellular vesicles (EVs), some of which contain mitochondria (mito+EV). With its content of damage-associated molecular pattern (DAMP), the mitochondrion can stimulate the innate immune system. Mitochondrial DNA (mtDNA) is a recognized DAMP detected in the extracellular milieu in numerous inflammatory conditions and in platelet concentrates. We hypothesized that platelet-derived mitochondria encapsulated in EVs may represent a reservoir of mtDNA. STUDY DESIGN AND METHODS: Herein, we explored the implication of mito+EVs in the occurrence of mtDNA quantified in platelet concentrate supernatants that induced or did not induce transfusion adverse reactions. RESULTS: We observed that EVs were abundant in platelet concentrates, and platelet-derived mito+EVs were more abundant in platelet concentrates that induced adverse reactions. A significant correlation (rs = 0.73; p < 0.0001) between platelet-derived mito+EV levels and mtDNA concentrations was found. However, there was a nonsignificant correlation between the levels of EVs without mitochondria and mtDNA concentrations (rs = -0.11; p = 0.5112). The majority of the mtDNA was encapsulated into EVs. CONCLUSION: This study suggests that platelet-derived EVs, such as those that convey mitochondrial DAMPs, may be a useful biomarker for the prediction of potential risk of adverse transfusion reactions. Moreover, our work implies that investigations are necessary to determine whether there is a causal pathogenic role of mitochondrial DAMP encapsulated in EVs as opposed to mtDNA in solution.

Inhibition of the CD40/CD40L complex protects mice against ALI-induced pancreas degradation.

BACKGROUND: Acute lung injury (ALI) is a severe complication of transfusion. In a previous study, we saw that inhibition of the CD40/CD40L complex allowed restoration of ALI lesions in an experimental mouse model. OBJECTIVES: This study focused on pancreas-associated injury development during experimental ALI pathogenesis and its limitation through CD40/CD40L complex inhibition. MATERIALS AND METHODS: An ALI mouse model was established through intraperitoneal lipopolysaccharide and intravenous anti-major histocompatibility complex class I monoclonal antibody injection. Preemption of lesions was achieved with intravenous injection of neutralizing anti-CD40L monoclonal antibody 30 minutes before the trigger, that is, anti-major histocompatibility complex class I monoclonal antibody administration. Histology and immunoassay analyses were used to evaluate pancreatic lesions. RESULTS: ALI development induced significant degradation of the lungs and pancreas and was associated with pancreatic lesions. Different scores were established showing more severe injury to the pancreas in ALI conditions; however, injury was significantly reduced through CD40/CD40L complex inhibition. CONCLUSION: This study supports the idea that several organs are exposed during ALI development, and particularly when such experimental ALI aims at mimicking transfusion-associated ALI; nevertheless, preventive treatment inhibiting CD40/CD40L (sCD40L) complex formation provides protection from lung disease as well as disease of other organs.

Soluble CD40L and CD62P levels differ in single-donor apheresis platelet concentrates and buffy coat-derived pooled platelet concentrates.

BACKGROUND: Platelet storage lesions are structural and biochemical changes in platelet concentrates (PCs), and depend on variables in collection and processing, as well as secondary procedures and storage conditions; such lesions can be mitigated by the use of platelet additive solutions (PASs). STUDY DESIGN AND METHODS: This study investigated release of the inflammatory markers sCD40L and sCD62P by single-donor apheresis platelet concentrates (SDA-PCs) and buffy coat-derived pooled platelet concentrates (PPCs) before and after storage. SDA-PC and PPC samples (n = 9089) processed by various methods and stored for different durations were obtained following production in one regional setting, the French National Blood Service. Soluble factors were quantified in PC supernatants immediately after processing and at the time of delivery, using biological testing technology (Luminex). RESULTS: SDA-PCs appeared more activated than PPCs at the end of the production step (i.e., prior to storage); however, proinflammatory soluble factors exhibited greater increases in PPCs than in SDA-PCs during storage. In SDA-PCs, PAS-D (65%) led to reduced secretion of sCD62P, but favored secretion of sCD40L, compared with the alternative PAS-E. CONCLUSION: These data stress the importance of the production (processing) steps of PC manufacture and of storage. The extent to which they affect patient outcomes awaits further investigation in clinical studies.

Differential protein expression of blood platelet components associated with adverse transfusion reactions.

Platelets found within platelet components (PCs) intended for transfusion release inflammatory molecules. Despite the implementation of leukoreduction, some of these PCs are occasionally associated with adverse transfusion reactions (ATRs). The aim of this study was to decipher the platelet proteome in two types of PCs, buffy-coat-derived pooled PCs (PPCs) and single-donor apheresis PCs (SDA-PCs), associated with ATRs. A label-free LC-MS/MS method was used for the proteomic analysis of washed platelet pellets from 3 PPCs and 3 SDA-PCs associated with ATRs, compared to matched controls. Bioinformatics tools allowed us to characterise the differentially expressed (DE) proteins between cases (ATR-PCs) and controls (no.ATR-PCs). From the PPCs and SDA-PCs, 473 and 146 proteins were DE, respectively. The functional interpretation of these proteins revealed enrichment in platelet activation and degranulation as the most important biological process. The most dysregulated pathways were integrin signaling for PPCs and acute phase response signaling for SDA-PCs. Interestingly, inflammatory disorders were found to be enriched in both PC types. Profound proteome changes were found in the platelets of PCs that led to clinical ATRs in patients. This study presents the first exploration of the platelet proteomic signature associated with ATRs and could provide clues to improving transfusion medicine. BIOLOGICAL SIGNIFICANCE: Adverse transfusion reactions (ATRs) can still occur after transfusion of platelet components (PC). This is the first report on the proteomic analysis of PCs associated with ATR. In this study, the contents of PC bags implicated in ATRs were examined. The aims of this study were to characterise molecules that could be central to the inflammation of ATRs and to highlight dysregulated mechanisms to explain the onset of ATRs. Two types of PCs were used: 3 PPCs (each from 5 donors) and 3 SDA-PCs (each from one donor). We have shown that the two types of PCs, from bags undergoing different processing (i.e., sampling, preparation), involve two types of dysregulated - pathophysiological mechanisms associated with the onset of ATRs. The most dysregulated signaling pathways were cytoskeleton and integrin regulation for PPCs, acute phase response signaling and remodelling of adherens junctions for SDA-PCs. Inflammation, platelet activation and degranulation processes were present in both PC types but were more important for PPCs. This proteomics analysis provides a better understanding of the pathophysiological mechanisms involved in ATRs and may lead to novel steps to ensure safe PC transfusion.

Platelet concentrate supernatants alter endothelial cell mRNA and protein expression patterns as a function of storage length.

BACKGROUND: Platelet transfusions are safe but can nevertheless cause serious adverse reactions (SARs). This study investigated the effects of platelet biological response modifiers (BRMs) that accumulate during storage and are commonly associated with transfusion adverse reactions. STUDY DESIGN AND METHODS: Endothelial cells (ECs), that is, EA.hy926, were exposed in vitro to supernatants of platelet components (PCs) that had been either implicated or not in SARs. The EC Biology RT2 Profiler PCR Array was used at the same time to study 84 genes related to functions of ECs. Soluble cytokines and surface expression of EC markers were determined by Luminex/enzyme-linked immunosorbent assay technology and flow cytometry, respectively. Apoptosis and scratch wound assays were performed using IncuCyte technology. RESULTS: In vitro exposure of EA.hy926 monolayers with Day 0, 1-2, and 3-4 stored PC supernatants resulted in decreases in surface expression of markers of ECs. There was differential production of soluble BRMs in the tested cell line. Exposure to the supernatants of PCs that had been implicated in SARs showed a significant difference in the expression of the EC surface markers. EC mediators also responded differently when exposed to PC supernatants of different storage times and PCs involved in SARs. CONCLUSION: PC supernatants collected at Day 1-2 activate fewer cell lines of ECs compared with supernatants collected at Day 3-4. Moreover, PC supernatants involved in SARs appear to alter EC activation compared with the control and storage length.

Molecular genetic diagnosis of Tunisian Glanzmann thrombasthenia patients reveals a common nonsense mutation in the ITGA2B gene that seems to be specific for the studied population.

: Glanzmann thrombasthenia is an inherited severe bleeding disease. Mutations associated with Glanzmann thrombasthenia are highly heterogeneous and occur across the two genes coding for the platelet αIIbß3 integrin. This study was aimed at identifying Glanzmann thrombasthenia-associated novel mutations in Tunisian patients. Seven unrelated Glanzmann thrombasthenia patients issued from high consanguineous families (86%; 6/7 of the patients) were studied. Glanzmann thrombasthenia diagnoses were based on patients' bleeding histories and platelet aggregation tests. Screening of ITGA2B and ITGB3 genes was performed by denaturing high-performance liquid chromatography (DHPLC) analysis. Amplicons with abnormal elution profiles were subjected to direct sequencing. DHPLC/sequencing analysis identified a pathogenic homozygous mutation in exon 26 at position c.2702C>A, inducing a substitution of a serine to a stop codon (p.S901*) in the ITGA2B gene, in three patients. This mutation was only previously reported in a Glanzmann thrombasthenia patient of a Tunisian origin and not in other populations. We diagnosed a pathogenic Glanzmann thrombasthenia mutation in ITGA2B screened by DHPLC that appears to be specific to individuals of Tunisian heritage and that deserves to be investigated in first intention. As a result, we determined that performing prenatal diagnosis and setting a prevention strategy via counselling for affected heterozygote individuals will be helpful for Tunisian Glanzmann thrombasthenia families where there is still a high rate of consanguinity.

Assessment of soluble platelet CD40L and CD62P during the preparation process and the storage of apheresis platelet concentrates: Absence of factors related to donors and donations.

Platelet transfusions may be associated with certain adverse effects in recipients, potentially caused by the presence of biological response modifiers contained in the platelet concentrates. The aim of this study is to identify the parameters that reflect platelet activation during both the preparation process and the storage of platelet concentrates. A total of 3,949apheresis platelet concentrate samples were studied with regard to parameters related to the donor as well as to the preparation process and their storage. Key glycoproteins characteristic of platelet activation, i.e. soluble CD40L and CD62P, were quantified in platelet concentrate supernatants on completion of their processing and during storage, using Luminex technology. We observed an increase in soluble factors over time. However, the different parameters studied in connection either with the donors or with the donations, such as (i) donor gender, (ii) donor blood group, (iii) time of collection and (iv) type of apheresis separator, do not seem to have any effect on platelet activation or the release of soluble CD40L and CD62P.

Novel splice site IDUA gene mutation in Tunisian pedigrees with hurler syndrome.

BACKGROUND: The mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease resulting from the defective activity of the enzyme α-L-iduronidase (IDUA). The disease has three major clinical subtypes (severe Hurler syndrome, intermediate Hurler-Scheie syndrome and attenuated Scheie syndrome). We aim to identify the genetic variants in MPS I patients and to investigate the effect of the novel splice site mutation on splicing of IDUA- mRNA variability using bioinformatics tools. METHODS: The IDUA mutations were determined in four MPS I patients from four families from Northern Tunisia, by amplifying and sequencing each of the IDUA exons and intron-exon junctions. RESULTS: One novel splice site IDUA mutation, c.1650 + 1G > T in intron 11 and two previously reported mutations, p.A75T and p.R555H, were detected. The patients in families 1 and 2 who have the Hurler phenotype were homozygotes for the novel splice site mutation c.1650 + 1G > T. The patient in family 3, who also had the Hurler phenotype, was a compound heterozygote for the novel splice site mutation c.1650 + 1G > T and for the previously reported missense mutation p.A75T. The patient in family 4 who had the Hurler-Scheie phenotype was a compound heterozygote for the novel splice site mutation c.1650 + 1G > T and for the previously reported missense mutation p.R555H. In addition, four known IDUA polymorphisms were identified. Bioinformatics tools allowed us to associate the variant c.1650 + 1G > T with the severe clinical phenotype of MPS I. This variant affects the essential nucleotide + 1 (G to T) of the donor splice site of IDUA intron 11. The G > T in intron 11 leads to wild type donor site broken with minus 19.97% value compared to normal value with 0%, hence the new splice site acceptor has plus 5.59%. CONCLUSIONS: The present findings indicate that the identified mutations facilitate the accurate carrier detection (genetic counseling of at-risk relatives) and the molecular prenatal diagnosis in Tunisia.

The Non-Hemostatic Aspects of Transfused Platelets.

Platelets transfusion is a safe process, but during or after the process, the recipient may experience an adverse reaction and occasionally a serious adverse reaction (SAR). In this review, we focus on the inflammatory potential of platelet components (PCs) and their involvement in SARs. Recent evidence has highlighted a central role for platelets in the host inflammatory and immune responses. Blood platelets are involved in inflammation and various other aspects of innate immunity through the release of a plethora of immunomodulatory cytokines, chemokines, and associated molecules, collectively termed biological response modifiers that behave like ligands for endothelial and leukocyte receptors and for platelets themselves. The involvement of PCs in SARs-particularly on a critically ill patient's context-could be related, at least in part, to the inflammatory functions of platelets, acquired during storage lesions. Moreover, we focus on causal link between platelet activation and immune-mediated disorders (transfusion-associated immunomodulation, platelets, polyanions, and bacterial defense and alloimmunization). This is linked to the platelets' propensity to be activated even in the absence of deliberate stimuli and to the occurrence of time-dependent storage lesions.

Platelet toll-like receptors are crucial sensors of infectious danger moieties.

In addition to their haemostatic role and function in the repair of damaged vascular epithelium, platelets play a defensive role in innate immunity, having the capacity to produce and secrete various anti-infectious factors, as well as cytokines, chemokines and related products, to interact with other immune cells to modulate immune responses to pathogens. Thus, it is now widely acknowledged that platelets participate in inflammatory processes and infection resolution, most notably by expressing and using receptors to bind infectious pathogen moieties and contributing to pathogen clearance. The ability of platelets to sense external danger signals relates to the expression of certain innate immunity receptors, such as toll-like receptors (TLRs), and the activation of efficient cell signalling machinery. TLR engagement triggers platelet response, which results in adapted degranulation according to: the type of TLR engaged, the nature of the ligand and the milieu; together, the TLR-mediated event and other signalling events may be followed by aggregation. Platelets thus use complex tools to mediate a whole range of functions upon sensing danger. By linking the inflammatory and haemostatic platelet response to infection, TLRs play a central role. The extent of the inflammatory response to pathogen clearance is still a debatable issue and is discussed in this short review.