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.

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.

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.

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.

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.

Modeling the effect of platelet concentrate supernatants on endothelial cells: focus on endocan/ESM-1.

BACKGROUND: Platelets (PLTs) are prone to activation and the release of biologic response modifiers (BRMs) under storage conditions. The transfusion inflammatory reaction in the vascular compartment involves endothelial cell activation due to cell-cell interactions and BRMs infused with the blood products. Endocan/ESM-1 is a proteoglycan secreted by endothelial cells under the control of proinflammatory cytokines. We aimed to measure endocan activity in supernatants of PLT components (PCs), implicated in serious adverse reactions (SARs) or not (no.AR), sampled at different stages during storage. STUDY DESIGN AND METHODS: PLT function, by quantification of soluble CD62P, and their ability to produce endocan were assessed. Functional testing of PC supernatants was performed on EA.hy926 endothelial cells in vitro by exposing them to PC supernatants from each group (no.AR or SARs); EA.hy926 activation was evaluated by their production of interleukin (IL)-6 and endocan. RESULTS: PLT endocan secretion was not induced in response to PLT surface molecule agonists, and no significant correlation was observed between sCD62P and endocan concentration after PLT activation. However, we observed a significant increase in the secretion of IL-6 and endocan after EA.hy926 activation by all PC supernatants. IL-6 and endocan secretion were significantly higher for cells stimulated with SAR than those stimulated with no.AR PC supernatants, as well as cell apoptosis. CONCLUSION: The correlation between the secretion of endocan and that of IL-6 by endothelial cells suggests that endocan can be used as a predictive marker of inflammation for the quality assessment of transfusion grade PLTs.

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.

Properties of donated red blood cell components from patients with hereditary hemochromatosis.

BACKGROUND: Red blood cells (RBCs) contain large amounts of iron, and periodic therapeutic phlebotomy is thus the main treatment for hereditary hemochromatosis (HH). However, the donation of therapeutic phlebotomy products from asymptomatic patients for transfusion purposes remains controversial. In this study, we compared the quality of RBCs obtained from HH patients with those of non-HH RBCs, within the allowed 42-day storage period. STUDY DESIGN AND METHODS: RBCs were obtained from HH patient donors and random regular blood donors by whole blood collection. RBCs were stored for up to 42 days, according to national regulations and standard blood bank conditions in France. The following variables were assessed: hematologic and biochemical results, RBC membrane and soluble inflammatory markers, and the proinflammatory potential of HH RBC supernatant toward endothelial cells in an in vitro model. RESULTS: There were no major differences between the two groups in terms of biophysical, biochemical, or soluble immunomodulatory factors. However, we observed small but significant differences in changes in RBC membrane proteins during storage, including increased phosphatidylserine expression and decreased hemolysis in HH compared with normal RBCs. However, there were no differences in terms of bioactivity of soluble immunomodulatory factors in the RBC supernatant during storage between HH and control donors, as determined by their effects on endothelial cells in vitro. CONCLUSIONS: These in vitro studies suggest that RBCs from HH patients appear, while exhibiting subtle differences, to be suitable for transfusion purposes according to currently accepted criteria.

Platelet components associated with adverse reactions: predictive value of mitochondrial DNA relative to biological response modifiers.

BACKGROUND: Biological response modifiers (BRMs), secreted by platelets (PLTs) during storage, play a role in adverse events (AEs) associated with transfusion. Moreover, mitochondrial DNA (mtDNA) levels in PLT components (PCs) are associated with AEs. In this study we explore whether there is a correlation between pathogenic BRMs and mtDNA levels and whether these markers can be considered predictors of transfusion pathology. STUDY DESIGN AND METHODS: We investigated a series of reported AEs after PC transfusion, combining clinical observations and mathematical modeling systems. RESULTS: mtDNA was consistently released during the first days of PC storage; however, mtDNA release was earlier in "pathogenic" than in nonpathogenic PCs. PC supernatants with high levels of mtDNA along with soluble CD40 ligand (sCD40L) were significantly associated with occurrences of AEs. The fact that mtDNA did not associate with the 14 BRMs tested suggests the role of mtDNA in PC transfusion-linked inflammation is independent of that of BRMs, known to be associated with AEs. We present evidence that PLTs generate distinct pathogenic secretion profiles of BRMs and mtDNA. The calculated area under the curve for mtDNA was significantly associated with AEs, although less stringently predictive than those of sCD40L or interleukin-13, standard predictors of AE. The established model predicts that distinct subtypes of AEs can be distinguished, dependent on mtDNA levels and PC storage length. CONCLUSIONS: Further work should be considered to test the propensity of mtDNA in PLT concentrates to generate inflammation and cause an AE.

Recommendations of the 2006 Human Variome Project meeting.

Lists of variations in genomic DNA and their effects have been kept for some time and have been used in diagnostics and research. Although these lists have been carefully gathered and curated, there has been little standardization and coordination, complicating their use. Given the myriad possible variations in the estimated 24,000 genes in the human genome, it would be useful to have standard criteria for databases of variation. Incomplete collection and ascertainment of variants demonstrates a need for a universally accessible system. These and other problems led to the World Heath Organization-cosponsored meeting on June 20-23, 2006 in Melbourne, Australia, which launched the Human Variome Project. This meeting addressed all areas of human genetics relevant to collection of information on variation and its effects. Members of each of eight sessions (the clinic and phenotype, the diagnostic laboratory, the research laboratory, curation and collection, informatics, relevance to the emerging world, integration and federation and funding and sustainability) developed a number of recommendations that were then organized into a total of 96 recommendations to act as a foundation for future work worldwide. Here we summarize the background of the project, the meeting and its recommendations.

Development of a highly resolutive method, using a double quadruplex tetra-primer-ARMS-PCR coupled with capillary electrophoresis to study CD40LG polymorphisms.

Polymorphisms in the CD40 ligand gene (CD40LG) are associated with various immunological disorders such as tumors, autoimmune and infectious diseases. The aim of this study was to develop a highly optimized double quadruplex tetra-primer amplification refractory mutation system PCR (double quadruplex T-ARMS-PCR) coupled with capillary electrophoresis to allow genotyping of eight relevant candidate CD40LG SNPs and to establish haplotypes. After conducting the double quadruplex T-ARMS-PCR, the genotypes obtained through agarose electrophoresis were compared with those obtained through capillary electrophoresis. This strategy was applied to analyze the genetic patterns of CD40LG in two distinct cohorts of blood donors (211 French and 274 Tunisian). The T-ARMS-PCR method was rapid, inexpensive, reproducible and reliable for SNP determination. Regarding the separation technique, capillary electrophoresis allows traceable and semi-automated analysis while agarose electrophoresis remains a cost-effective technique that does not require specialized or costly equipment. Using these methods, we identified significantly different genetic heterogeneity between the two investigated populations (p ≤ 0.0001) and we also extensively characterized their haplotypes. The obtained genotype distribution and the optimized quadruplex T-ARMS-PCR technique coupled with capillary electrophoresis provides valuable information for studying pathologic inflammation leading to various diseases in which CD40LG might be a candidate gene.

The role of microparticles in inflammation and transfusion: A concise review.

Microparticles are small membrane-bound vesicles found in body fluids including peripheral blood. Microparticles are an intrinsic part of blood labile products delivered to transfused patients and have active roles in inflammation. They are delimited by a lipid bilayer composed mainly of phospholipids, cholesterol, membrane-associated proteins, intracellular components such as metabolic enzymes, proteins-involved in adhesion and fusion, cytoskeletal-associated proteins, surface glycoproteins and/or chemokines. Microparticles can trigger a pro-inflammatory message to neighbouring or target cells. Microparticles originating from platelets, leukocytes, erythrocytes, and endothelial cells are associated with a variety of pathophysiological conditions. This review summarises the role of Microparticles in modulating inflammation.

Specific activation, signalling and secretion profiles of human platelets following PAR-1 and PAR-4 stimulation.

Blood platelets play a central haemostatic function; however, they also play a role in inflammation and are capable of secreting various cytokines, chemokines and related products. The purpose of this study was to identify subtle variations in platelet physiology using proteomics. We compared the levels of membrane proteins (n = 3), α and δ granule proteins (n = 18), and signalling proteins (n = 30) from unstimulated platelets with those of protease-activated receptor (PAR)-1- and PAR-4-stimulated platelets (n = 10). The vast majority of these proteins responded similarly to PAR-1 or PAR-4 engagement. However, differences were observed within membrane CD40L expressed, and α granule GRO-α and MDC secreted proteins.

The signaling role of CD40 ligand in platelet biology and in platelet component transfusion.

The CD40 ligand (CD40L) is a transmembrane molecule of crucial interest in cell signaling in innate and adaptive immunity. It is expressed by a variety of cells, but mainly by activated T-lymphocytes and platelets. CD40L may be cleaved into a soluble form (sCD40L) that has a cytokine-like activity. Both forms bind to several receptors, including CD40. This interaction is necessary for the antigen specific immune response. Furthermore, CD40L and sCD40L are involved in inflammation and a panoply of immune related and vascular pathologies. Soluble CD40L is primarily produced by platelets after activation, degranulation and cleavage, which may present a problem for transfusion. Soluble CD40L is involved in adverse transfusion events including transfusion related acute lung injury (TRALI). Although platelet storage designed for transfusion occurs in sterile conditions, platelets are activated and release sCD40L without known agonists. Recently, proteomic studies identified signaling pathways activated in platelet concentrates. Soluble CD40L is a good candidate for platelet activation in an auto-amplification loop. In this review, we describe the immunomodulatory role of CD40L in physiological and pathological conditions. We will focus on the main signaling pathways activated by CD40L after binding to its different receptors.

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.

Xmn I polymorphism associated with concomitant activation of Gγ and Aγ globin gene transcription on a ß0-thalassemia chromosome.

The -158 (C→T) nucleotide change, known as Xmn I polymorphism, occurs in (G)γ-globin gene promoter, and results in elevated fetal hemoglobin (HbF). We found this mutation in cis of a ß(0)-thalassemia splicing mutation. Despite the complete absence of adult HbA, the phenotype was only moderately severe with no detectable alteration of α-globin gene expression. Interestingly, the ß-globin locus haplotype has not been described to bear the (G)γ promoter mutation. Using a gene-specific real-time RT-PCR approach, we found a dramatic increase of both (G)γ and (A)γ mRNA accumulated in the reticulocytes, suggesting that the (G)γ-promoter mutation, alone or in association with another genetic modification, alters in concert the transcription of both (G)γ and (A)γ. This observation is discussed in light of recent regulatory model for ß-globin locus.

[Morquio A disease: clinical and molecular study of Tunisian patients]. / La maladie de Morquio A : étude clinique et moléculaire des patients tunisiens.

Type IVA mucopolysaccharidosis or Morquio A disease is a lysosomal storage disease, autosomal recessive, caused by deficiency of the N-acetylgalactosamine-6-sulfate sulfatase or GALNS. The severe phenotype is characterized by a severe skeletal dysplasia without any mental retardation. The aim of this study was to propose a strategy of molecular and prenatal diagnosis of this pathology. A molecular study was carried out on 7 patients MPS IVA issued from 5 unrelated families recruited from different Tunisian regions. All the patients were offspring of consanguineous marriages. The clinical and biologic study confirmed the diagnosis of MPS IVA within the 7 studied patients. Three GALNS mutations were identified by molecular analysis: IVS1+1G>A, G66R and A85T. The unions between Tunisian relatives are important and increase the Morquio A incidence in Tunisia. The identification of GALNS mutations in the Tunisian population permits better understanding of the Morquio A phenotype, a reliable genetic counselling and a molecular prenatal diagnosis to Tunisian at-risk relatives.

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.