Amel Ben Chehida

Founder effect confirmation of c.241A>G mutation in the L2HGDH gene and characterization of oxidative stress parameters in six Tunisian families with L-2-hydroxyglutaric aciduria

L-2-hydroxyglutaric aciduria (L2HGA) is an autosomal recessive neurometabolic disorder characterized essentially by the presence of elevated levels of L-2-hydroxyglutaric acid (LGA) in plasma, cerebrospinal fluid and urine. L2HGA is caused by a deficiency in the L2-Hydroxyglutaric dehydrogenase (L2HGDH) enzyme involved in the oxidation of LGA to the alpha 2-ketoglutarate. LGA has been proposed as an endo- and exogenous cytotoxic organic acid that induces free radical formation and generation of reactive oxygen species (ROS). In this report, we analyzed 14 L2HGA patients belonging to six unrelated consanguineous families the south of Tunisia. The patients were diagnosed with L2HGA disease confirmed on the presence of high level of LGA in urine. We analyzed the L2HGDH gene in all probands and identified the same c.241A>G homozygous mutation, which was previously reported in Tunisia. We also used intragenic single nucleotide length polymorphisms (SNPs) and two extragenic microsatellites flanking the L2HGDH gene to confirm the founder effect of c.241A>G mutation in the 14 studied cases. In addition, we carried out the measurement of the oxidative stress parameters in the plasma of L2HGA patients which revealed a significant increase in the malondialdehyde levels (MDA), a biomarker of lipid peroxydation, and the reduced glutathione (GSH). A diminution of the antioxidant enzyme activities including superoxide dismutase (SOD), glutathione peroxidase (GPx), was also observed.

Clinical and Genetic Characterization of 26 Tunisian Patients with Allgrove Syndrome

BACKGROUND AND AIMS Allgrove syndrome is characterized by achalasia, alacrima, and adrenal insufficiency as well as being associated with progressive neurological signs. This is an autosomal recessive disorder due to mutations in the AAAS gene located on chromosome 12q13. The AAAS gene encodes a protein of 546 amino acids, ALADIN. Mutations in this genwere reported in families from North Africa and Europe. Our objective is to conduct a clinical, molecular and genetic study of 26 Tunisian patients with Allgrove syndrome. METHODS We report 26 Tunisian patients with between two and four clinical features associated with Allgrove syndrome. Blood samples were collected and isolated DNA derived from subjects was amplified. The entire sequence of the AAAS gene was analyzed by PCR and sequencing. PCR-RFLP method was performed to identify the frequent mutations found. RESULTS Sequencing of the AAAS gene revealed a major homozygous mutation (c.1331+1G>A) in 25 patients and R286X mutation in one patient. The presence of a major mutation in several unrelated affected individuals suggests the presence of a founder effect in Tunisia and allows for a fast and targeted molecular diagnosis. CONCLUSIONS We created an easy and rapid molecular enzymatic protocol based on PCR-RFLP using MvaI restriction enzyme that directly targets this major mutation and can be used for prenatal diagnosis and genetic counseling for Tunisian families at risk. To the best of our knowledge, this is the first major series report of Allgrove syndrome in Tunisia.

Splicing defects in ABCD1 gene leading to both exon skipping and partial intron retention in X-linked adrenoleukodystrophy Tunisian patient

X-linked adrenoleukodystrophy (X-ALD) affects the nervous system white matter and adrenal cortex secondary to mutations in the ABCD1 gene that encodes a peroxisomal membrane protein: the adrenoleukodystrophy protein. The disease is characterized by high concentrations of very long-chain fatty acids in plasma, adrenal, testicular and nervous tissues. Various types of mutations have been identified in the ABCD1 gene: point mutations, insertions, and deletions. To date, more than 40 point mutations have been reported at the splice junctions of the ABCD1 gene; only few functional studies have been performed to explore these types of mutations. In this study, we have identified de novo splice site mutation c.1780+2T>G in ABCD1 gene in an X-ALD Tunisian patient. Sequencing analysis of cDNA showed a minor transcript lacking exon 7 and a major transcript with a partial intron 7 retention due to activation of a new intronic cryptic splice site. Both outcomes lead to frameshifts with premature stop codon generation in exon 8 and intron 7 respectively. To the best of our knowledge, the current study demonstrates that a single splicing mutation affects the ABCD1 transcripts and the ALDP protein function.

Molecular Characterization of X-Linked Adrenoleukodystrophy in a Tunisian Family: Identification of a Novel Missense Mutation in the ABCD1 Gene

Background: X-linked adrenoleukodystrophy (X-ALD) is a recessive neurodegenerative disorder that affects the brains white matter and is associated with adrenal insufficiency. It is characterized by an abnormal function of the peroxisomes, which leads to an accumulation of very long-chain fatty acids (VLCFA) in plasma and tissues, especially in the cortex of the adrenal glands and the white matter of the central nervous system, causing demyelinating disease and adrenocortical insufficiency (Addisons disease). X-ALD is caused by a mutation in the ABCD1 gene (ATP-binding cassette, subfamily D, member 1), which encodes the adrenoleukodystrophy protein involved in the transport of fatty acids into the peroxisome for degradation. Objective: We report here a disease-related variant in the ABCD1 gene in a 19-year-old Tunisian boy with childhood cerebral adrenoleukodystrophy. Methods: The diagnosis was based on clinical symptoms, high levels of VLCFA in plasma, typical MRI pattern and molecular analysis. Results: Molecular analysis by direct sequencing of the ABCD1 gene showed the presence of a novel missense mutation c.284C>A (p.Ala95Asp) occurring in the transmembrane domain in the proband, his mother and his sister. Conclusion: Using bioinformatic tools we suggest that this novel variant may have deleterious effects on adrenoleukodystrophy protein structure and function.

Complications rénales dans la glycogénose de type 1 : quelles implications pratiques ?

AIM To investigate risk factors of renal complications in glycogen storage disease type I, in order to identify practical implications for renal preservation. METHODS A retrospective study of 38 patients with glycogen storage disease type I. RESULTS The patients studied were 8.6 years old in average (1.5 to 22 years) and were followed during 7.4 ± 4.5 years. Hypercalciuria was detected in 23 patients and was related to acidosis (P=0.028), higher lactate levels (5.9 ± 3.5 versus 3.7 ± 1.7 mmol/L; P=0.013) and smaller height (-2.1 ± 1.5 SD versus -0.8 ± 1.5 SD; P=0.026). Urolithiasis was diagnosed in 7 cases. Glomerular disease (19/38) was more frequent in cases with severe hypertriglyceridemia (P=0.042) and occurred at an older age (P=0.007). Microalbuminuria occurred in 15/31 cases; ACE inhibitors were prescribed in only 8 cases. The frequency of renal complications did not differ according to the diet group (continuous enteral feeding or uncooked starch). Logistic regression concluded as risk factors: lactic acidosis for tubular disease and age>10 years for glomerular disease. CONCLUSIONS Renal involvement is common in glycogen storage disease type I patients. Tubular abnormalities are precocious, related to lactic acidosis and may be detected by monitoring of urinary calcium. Glomerular hyperfiltration is the first stage of a progressive glomerular disease and is related to age. Practical implications for renal preservation are discussed based on our results and literature.

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

Abstract 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.

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 de-novo large deletion of 2.8 kb produced in the ABCD1 gene causing adrenoleukodystrophy disease

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder caused by mutations in the ABCD1 gene, which encodes an ATP-binding cassette transporter protein, ALDP. The disease is characterized by increased concentrations of very long chain fatty acids (VLCFAs) in plasma, adrenal, testicular, and nerve tissues. For this study, our objective was to conduct clinical, molecular, and genetic studies of a Tunisian patient with X-ALD. The diagnosis was based on clinical indications, biochemical analyses, typical brain-scan patterns, and molecular biology; the molecular analyses were based on PCR, long-range PCR, and sequencing. The molecular analysis by long-range PCR and direct sequencing of the ABCD1 gene showed the presence of a de-novo 2794 bp deletion covering the whole of exon 2. Using bioinformatics tools, we demonstrate that the large deletion is located in a region rich with Alu sequences. Furthermore, we suggest that the AluJb sequence could be the cause of the large deletion of intron 1, exon 2, and intron 2, and the creation of a premature stop codon within exon 3. This report is the first report in which we demonstrate the breakpoints and the size of a large deletion in a Tunisian with X-ALD.