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.

Novel RAB3GAP1 Mutation in the First Tunisian Family With Warburg Micro Syndrome.

BACKGROUND AND PURPOSE: Warburg Micro syndrome (WARBM) is a rare autosomal recessive genetic disease characterized by ocular, neurologic, and endocrine anomalies. WARBM is a phenotypically and genetically heterogeneous syndrome caused by mutations in RAB3GAP1, RAB3GAP2, RAB18, and TBC1D20. Here we present the clinical and genetic characterization of a consanguineous Tunisian family with a WARBM phenotype presenting two pathogenic variations, one of which is on RAB3GAP1. METHODS: We applied whole-exome sequencing (WES) to two affected young males presenting a WARBM-compatible phenotype. RESULTS: We reveal a new variation in RAB3GAP1 (NM_012233.3: c.297del, p.Gln99fs) and another variation in ABCD1 (NM_000033: c.896A>G, p.His299Arg). Each of these mutations, which in silico predictions concluded as being pathogenic variations, affects a critical protein region. Both affected males presented a WARBM-compatible phenotype, with severe intellectual disability, severe developmental delay, postnatal growth delay, postnatal microcephaly, congenital bilateral cataracts, general hypotonia, and a thin corpus callosum without a splenium. However, intrafamilial clinical heterogeneity was present, since only the oldest child had large ears, microphthalmia, foot deformities, and a genital anomaly, and only the youngest child had microcornea. Despite the mutation identified in ABCD1, our patients did not have any X-linked symptoms of adrenoleukodystrophy disorder that are usually caused by ABCD1 mutations, which prompted our interest in clinical monitoring. CONCLUSIONS: WES analysis of a consanguineous Tunisian family with WARBM revealed a novel variation in RAB3GAP1 (NM_012233.3: c.297del, p.Gln99fs) that is most likely pathogenic and allowed us to confirm the diagnosis of WARBM.

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.

Large-scale screening of lipase acid deficiency in at risk population.

BACKGROUND: Lysosomal acid lipase deficiency (LALD, OMIM#278000) is a rare lysosomal disorder with an autosomal recessive inheritance. The main clinical manifestations are related to a progressive accumulation of cholesteryl esters, triglycerides or both within the lysosome in different organs such as the liver, spleen, and cardiovascular system. A wide range of clinical severity is associated with LALD including a severe very rare antenatal/neonatal/infantile phenotype named Wolman disease and a late-onset form named cholesteryl ester storage disease (CESD). METHODS: This study aimed to investigate a cohort of at-risk patients (4174) presenting with clinical or biological signs consistent with LALD using the assessment of LAL activity on dried blood spots. RESULTS: LAL activity was lower than 0.05 nmol/punch/L (cut-off: 0.12) in 19 patients including 13 CESD and 6 Wolman. Molecular study has been conducted in 17 patients and succeeded in identifying 34 mutated alleles. Fourteen unique variants have been characterized, 7 of which are novel. CONCLUSION: This study allowed to identify a series of patients and expanded the molecular spectrum knowledge of LALD. Besides, a new screening criteria grid based on the clinical/biological data from our study and the literature has been proposed in order to enhance the diagnosis rate in at risk populations.

Homozygous pArg610del Mutation Unusually Associated With Severe Delay of Growth in 2 Acid Sphingomyelinase Deficiency-affected Sibs.

BACKGROUND: Typically, patients with Acid Sphingomyelinase Deficiency (ASMD) because of p.Arg610del mutation, have mild phenotype with normal linear growth. OBSERVATION: We reported the case of 2 Tunisian brothers who have been referred for splenomegaly, polyadenopathies, pubertal, and growth delay. Molecular testing of SMPD1 gene revealed the presence of a homozygous p.Arg610del mutation. Lysosphingomyelin and its isoform-509 were both increased confirming ASMD for both cases. Growth hormone deficiency was highly suspected but growth hormone response after stimulating tests was acceptable for both patients. CONCLUSIONS: There is no correlation between phenotype-genotype in case of p.Arg610del mutation that could be associated to a severe delay of growth.

Role play for genetic counseling learning: Value and students perceptions.

BACKGROUND: Performing genetic counseling is one of the tasks of every paediatrician. This assumes prior training during the residency. AIM: To assess the impact of role-play (RP) for training of paediatric residents in genetic counseling and participants' perception. METHODS: Repetitive cross-sectional evaluation study. During two RP sessions, two residents played the role of the parents of a patient with cystic fibrosis, and another the role of the doctor. Residents had an evaluation by standardized patient exercises immediately before and after the session. Test scores were compared by the Wilcoxon rank test for associated samples. A satisfaction questionnaire was completed by the participants anonymously. RESULTS: Post-test scores were better than pre-test scores overall (p = 0.002) and for items in the cognitive domain (p = 0.002). Of the 12 participants, only one had had previous training in genetic counseling. All participants were satisfied with the learning and felt that it would change the way they practice. All participants thought they could do genetic counseling autonomously, but nine of them wanted to have other RP sessions on the same theme. Only one participant found the session stressful and all wanted to multiply this type of sessions for other learning. CONCLUSION: RP is an effective and well-accepted means for genetic counseling training. It should be integrated with paediatric resident training.

High Frequency of Cardiovascular Complications in Tunisian Kawasaki Disease Patients: Need for a Further Awareness.

BACKGROUND: The outcome of Kawasaki disease (KD) depends on cardiovascular complications (CVCs). OBJECTIVES: This study aimed to explore diagnostic features and CVCs in Tunisian patients with KD. METHODS: In total, 33 Tunisian patients (age, 2.9 ± 2.2 years) fulfilling the diagnosis criteria of KD, were retrospectively reviewed. Nonparametric tests were used to compare the two groups with regards to coronary complications (CCs). RESULTS: Diagnosis of KD was established at day 11 ± 5.1 from the beginning of the fever. Apyrexia was obtained in an average of 29 h after completion of intravenous immunoglobulin. CVCs were identified in 52% of cases: CC in 15 patients (giant aneurysm >8 mm in five patients) and non-CCs in 6 patients (severe in three patients). CCs were more frequently associated with the male sex (p = 0.037), fever lasting >8 days (p = 0.028) and longer time to apyrexia (p = 0.031). CONCLUSION: In Tunisia, better knowledge and monitoring of KD are warranted.

A Novel Mutation c.153 C>A in a Tunisian Girl With Wolman Disease and Unusual Presentation: Hemophagocytic Lymphohistiocytosis.

Wolman disease is an ultrarare lysosomal storage disease caused by a mutation in the LIPA gene. The clinical features of Wolman disease include early onset of vomiting, diarrhea, failure to thrive, hepatosplenomegaly, and bilateral adrenal calcification. We report the case of a 3-month-old infant who presented clinical features of hemophagocytic lymphohistiocytosis. Genetic sequence analysis of the LIPA gene revealed homozygous mutation c.153 C>A (p.Tyr51*). The parents were heterozygous for this mutation. Prenatal diagnosis has been carried out in the next pregnancy. To our knowledge, this mutation has never been reported before, and this is an unusual case of secondary hemophagocytic lymphohistiocytosis complicating Wolman disease.

Neuromuscular Involvement in Glycogen Storage Disease Type III in Fifty Tunisian Patients: Phenotype and Natural History in Young Patients.

BACKGROUND: Our aim was to describe the natural history of neuromuscular involvement (NMI) in glycogen storage disease type III (GSDIII). METHODS: We conducted a longitudinal study of 50 Tunisian patients, 9.87 years old in average. RESULTS: NMI was diagnosed at an average age of 2.66 years and was clinically overt in 85% of patients. Patients with clinical features were older (p = 0.001). Complaints were dominated by exercise intolerance (80%), noticed at 5.33 years in average. Physical signs, observed at 6.75 years in average, were dominated by muscle weakness (62%). Functional impairment was observed in 64% of patients, without any link with age (p = 0.255). Among 33 patients, 7 improved. Creatine kinase (CK) and aspartate aminotransferase (AST) levels were higher with age.Electrophysiological abnormalities, diagnosed in average at 6.5 years, were more frequent after the first decade (p = 0.0005). Myogenic pattern was predominant (42%). Nerve conduction velocities were slow in two patients. Lower caloric intake was associated with more frequent clinical and electrophysiological features. Higher protein intake was related to fewer complaints and physical anomalies. CONCLUSION: Neuromuscular investigation is warranted even in asymptomatic patients, as early as the diagnosis of GSDIII is suspected. Muscle involvement can be disabling even in children. Favorable evolution is possible in case of optimal diet.

Patient-management Problem (PMP) for paediatrics learning: Value and students perceptions.

BACKGROUND: The use of the pedagogic tool Patient-Management Problem (PMP) for medical teaching and evaluation remains limited in Tunisia. AIM: to evaluate the value of PMP learning sessions in pediatrics and students' perception of the use of PMP for learning and evaluation. METHODS: We conducted a cross-sectional evaluative study in four pediatric departments in Tunis. Students had a learning session with an electronic PMP. Their knowledge was assessed using a pre-test and a post-test. Their perception of the learning was assessed using a questionnaire. RESULTS: Forty-four students participated. The post-test scores were statistically higher than those of the pre-test (p <0.001). More than 90% of the students, found that the PMP was a useful learning tool, which would change their way of thinking and agreed to its regular use for teaching. 86% of students declared that the PMP were better than other means of learning and 79% that PMP was a reliable assessment tool, but 75% believed it was more stressful than other means of assessment. The degree of satisfaction with previous PMP experience was negatively correlated with perception of reliability (p = 0.043), impact on clinical reasoning (p = 0.044), and PMP being better than the other learning means (p = 0.044). CONCLUSION: The PMP is an effective learning tool and is well accepted by students. Its use should be generalized to all disciplines for teaching and evaluation. Further trainings are necessary for medical teachers to guarantee quality PMPs.

Correction to: Full title: peripheral venous catheter complications in children: predisposing factors in a multicenter prospective cohort study.

Following publication of the original article [1], one of the authors flagged that the title of the article was submitted (incorrectly) with "Full title:" at the beginning.

A lower energetic, protein and uncooked cornstarch intake is associated with a more severe outcome in glycogen storage disease type III: an observational study of 50 patients.

Background Glycogen storage disease type III (GSDIII), due to a deficiency of glycogen debrancher enzyme (GDE), is particularly frequent in Tunisia. Phenotypic particularities of Tunisian patients remain unknown. Our aim was to study complications of GSDIII in a Tunisian population and to explore factors interfering with its course. Methods A retrospective longitudinal study was conducted over 30 years (1986-2016) in the referral metabolic center in Tunisia. Results Fifty GSDIII patients (26 boys), followed for an average 6.75 years, were enrolled. At the last evaluation, the median age was 9.87 years and 24% of patients reached adulthood. Short stature persisted in eight patients and obesity in 19 patients. Lower frequency of hypertriglyceridemia (HTG) was associated with older patients (p<0.0001), higher protein diet (p=0.068) and lower caloric intake (p=0.025). Hepatic complications were rare. Cardiac involvement (CI) was frequent (91%) and occurred early at a median age of 2.6 years. Severe cardiomyopathy (50%) was related to lower doses of uncooked cornstarch (p=0.02). Neuromuscular involvement (NMI) was constant, leading to a functional discomfort in 64% of cases and was disabling in 34% of cases. Severe forms were related to lower caloric (p=0.005) and protein intake (p<0.015). Conclusions A low caloric, protein and uncooked cornstarch intake is associated with a more severe outcome in GSDIII Tunisian patients. Neuromuscular and CIs were particularly precocious and severe, even in childhood. Genetic and epigenetic factors deserve to be explored.

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.

Peripheral venous catheter complications in children: predisposing factors in a multicenter prospective cohort study.

BACKGROUND: Peripheral venous catheterization (PVC) is frequently used in children. This procedure is not free from potential complications. Our purpose was to identify the types and incidences of PVC complications in children and their predisposing factors in a developing country. METHODS: We conducted a prospective observational multicenter study in five pediatric and pediatric surgery departments over a period of 2 months. Two hundred fifteen PVC procedures were conducted in 98 children. The times of insertion and removal and the reasons for termination were noted, and the lifespan was calculated. Descriptive data were expressed as percentages, means, standard deviations, medians and interquartile ranges. The Chi2 test or the Fisher test, with hazard ratios and 95% confidence intervals (CI95%), as well as Student's t test or the Mann-Whitney U test were used to compare categorical and quantitative variables, respectively, in groups with and without complications. The Spearman test was used to determine correlations between the lifespan and the quantitative variables. The Kruskal Wallis test was used to test for differences in the median lifespan within 3 or more subgroups of a variable. Linear regression and logistic binary regression were used for multivariate analysis. A p-value <0.05 was considered significant. RESULTS: The mean lifespan was 68.82 ± 35.71 h. A local complication occurred in 111 PIVC (51.9%) cases. The risk factors identified were a small catheter gauge (24-gauge) (p = 0.023), the use of a volume-controlled burette (p = 0.036), a longer duration of intravenous therapy (p < 0.001), a medical diagnosis of respiratory or infectious disease (p = 0.047), the use of antibiotics (p = 0.005), including cefotaxime (p = 0.024) and vancomycin (p = 0.031), and the use of proton pump inhibitors (p = 0.004).The lifespan of the catheters was reduced with the occurrence of a complication (p < 0.001), including the use of 24-gauge catheters (p = 0.001), the use of an electronic pump or syringe(p = 0.036) and a higher rank of the intravenous device in each patient (p = 0.010). CONCLUSIONS: PVC complications were frequent in our pediatric departments and are often associated with misuse of the device. These results could engender awareness among both doctors and nurses regarding the need for rationalization of the use of PVC and better adherence to the recommendations for the use of each drug and each administration method.