Fakhri Kallabi

Polymorphisms of glutathione S-transferases M1, T1, P1 and A1 genes in the Tunisian population: an intra and interethnic comparative approach.

Genetic polymorphisms in glutathione S-transferases (GSTs) genes might influence the detoxification activities of the enzymes predisposing individuals to cancer risk. Owing to the presence of these genetic variants, inter-individual and ethnic differences in GSTs detoxification capacity have been observed in various populations. Therefore, the present study was performed to determine the prevalence GSTM1 0/0, GSTT1 0/0, GSTP1 Ile(105)Val, and GSTA1 A/B polymorphisms in 154 healthy individuals from South Tunisia, and to compare them with those observed in North and Centre Tunisian populations and other ethnic groups. GSTM1 and GSTT1 polymorphisms were analyzed by a Multiplex-PCR approach, whereas GSTP1 and GSTA1 polymorphisms were examined by PCR-RFLP. The frequencies of GSTM10/0 and GSTT1 0/0 genotypes were 53.9% and 27.9%, respectively. The genotype distribution of GSTP1 was 47.4% (Ile/Ile), 40.9% (Ile/Val), and 11.7% (Val/Val). For GSTA1, the genotype distribution was 24.7% (A/A), 53.9% (A/B), and 21.4% (B/B). The combined genotypes distribution of GSTM1, GSTT1, GSTP1 and GSTA1 polymorphisms showed that thirty one of the 36 possible genotypes were present in our population; eight of them have a frequency greater than 5%. To the best of our knowledge, this is the first report of GSTs polymorphisms in South Tunisian population. Our findings demonstrate the impact of ethnicity and reveal a characteristic pattern for Tunisian population. The molecular studies in these enzymes provide basis for further epidemiological investigations in the population where these functional polymorphisms alter therapeutic response and act as susceptibility markers for various clinical conditions.

A novel mutation MT-COIII m.9267G>C and MT-COI m.5913G>A mutation in mitochondrial genes in a Tunisian family with maternally inherited diabetes and deafness (MIDD) associated with sever nephropathy

Mitochondrial diabetes (MD) is a heterogeneous disorder characterized by a chronic hyperglycemia, maternal transmission and its association with a bilateral hearing impairment. Several studies reported mutations in mitochondrial genes as potentially pathogenic for diabetes, since mitochondrial oxidative phosphorylation plays an important role in glucose-stimulated insulin secretion from beta cells. In the present report, we studied a Tunisian family with mitochondrial diabetes (MD) and deafness associated with nephropathy. The mutational analysis screening revealed the presence of a novel heteroplasmic mutation m.9276G>C in the mitochondrial COIII gene, detected in mtDNA extracted from leukocytes of a mother and her two daughters indicating that this mutation is maternally transmitted and suggest its implication in the observed phenotype. Bioinformatic tools showed that m.9267G>C mutation (p.A21P) is « deleterious » and it can modify the function and the stability of the MT-COIII protein by affecting the assembly of mitochondrial COX subunits and the translocation of protons then reducing the activity of the respective OXPHOS complexes of ATP synthesis. The nonsynonymous mutation (p.A21P) has not been reported before, it is the first mutation described in the COXIII gene which is related to insulin dependent mitochondrial diabetes and deafness and could be specific to the Tunisian population. The m.9267G>C mutation was present with a nonsynonymous inherited mitochondrial homoplasmic variation MT-COI m.5913 G>A (D4N) responsible of high blood pressure, a clinical feature detected in all explored patients.

Novel cases of Tunisian patients with mutations in the gene encoding 17β-hydroxysteroid dehydrogenase type 3 and a founder effect.

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3) is expressed almost exclusively in the testis and converts Δ4-androstene-3,17-dione to testosterone. Mutations in the HSD17B3 gene causing 17β-HSD3 deficiency are responsible for a rare recessive form of 46, XY Disorders of Sex Development (46, XY DSD). We report novel cases of Tunisian patients with 17β-HSD3 deficiency due to previously reported mutations, i.e. p.C206X and p.G133R, as well as a case with the novel compound heterozygous mutations p.C206X and p.Q176P. Moreover, the previously reported polymorphism p.G289S was identified in a heterozygous state in combination with a novel non-coding variant c.54G>T, also in a heterozygous state, in a male patient presenting with micropenis and low testosterone levels. The identification of four different mutations in a cohort of eight patients confirms the generally observed genetic heterogeneity of 17β-HSD3 deficiency. Nevertheless, analysis of DNA from 272 randomly selected healthy controls from the same geographic area (region of Sfax) revealed a high carrier frequency for the p.C206X mutation of approximately 1 in 40. Genotype reconstruction of the affected pedigree members revealed that all p.C206X mutation carriers harbored the same haplotype, indicating inheritance of the mutation from a common ancestor. Thus, the identification of a founder effect and the elevated carrier frequency of the p.C206X mutation emphasize the importance to consider this mutation in the diagnosis and genetic counseling of affected 17β-HSD3 deficiency pedigrees in Tunisia.

A novel frameshift mutation in BLM gene associated with high sister chromatid exchanges (SCE) in heterozygous family members

The Bloom syndrome (BS) is an autosomic recessive disorder comprising a wide range of abnormalities, including stunted growth, immunodeficiency, sun sensitivity and increased frequency of various types of cancer. Bloom syndrome cells display a high level of genetic instability, including a 10-fold increase in the sister chromatid exchanges (SCE) level. Bloom syndrome arises through mutations in both alleles of the BLM gene, which was identified as a member of the RecQ helicase family. In this study, we screened a Tunisian family with three BS patients. Cytogenetic analysis showed several chromosomal aberrations, and an approximately 14-fold elevated SCE frequency in BS cells. A significant increase in SCE frequency was observed in some family members but not reaching the BS patients values, leading to suggest that this could be due to the heterozygous profile. Microsatellite genotyping using four fluorescent dye-labeled microsatellite markers revealed evidence of linkage to BLM locus and the healthy members, sharing higher SCE frequency, showed heterozygous haplotypes as expected. Additionally, the direct BLM gene sequencing identified a novel homozygous frameshift mutation c.3617–3619delAA (p.K1207fsX9) in BS patients and a heterozygous BLM mutation in the family members with higher SCE frequency. Our findings suggest that this latter mutation likely leads to a reduced BLM activity explaining the homologous recombination repair defect and, therefore, the increase in SCE. Based on the present data, the screening of this mutation could contribute to the rapid diagnosis of BS. The genetic confirmation of the mutation in BLM gene provides crucial information for genetic counseling and prenatal diagnosis.

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.

Molecular Analysis of Libyan Families with Allgrove Syndrome: Geographic Expansion of the Ancestral Mutation c.1331+1G>A in North Africa.

Background/Aims: Allgrove syndrome is a rare autosomal recessive disorder characterized by alacrima, achalasia, and adrenal insufficiency. It is caused by mutations of the AAAS gene located on chromosome 12q13 encoding the WD-repeat protein ALADIN. The c.1331+1G>A mutation is one of the most common mutations described in the literature and was identified in Tunisian and Algerian populations. Herein, we describe the clinical and genetic profile of two families from Libya in North Africa associated with Allgrove syndrome. Methods: Two unrelated families clinically diagnosed with Allgrove syndrome were evaluated for sequence variations in the AAAS gene. Blood samples were collected, and isolated DNA derived from the subjects was amplified. The entire sequence of the AAAS gene was analyzed by PCR-RFLP and direct sequencing. Results: Molecular analysis revealed the major homozygous mutation (c.1331+1G>A) in all patients. The presence of a major mutation in Tunisia, Algeria and, as discovered in this report, in Libya in patients with Allgrove syndrome suggests the existence of an ancestral mutation and a founder effect in North Africa. Conclusions: The findings allow for a fast genetic counseling in North African families with Allgrove syndrome. To the best of our knowledge, this is the first report of Allgrove syndrome in Libya.

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.

DNA Repair Gene Polymorphisms at XRCC1 (Arg194Trp, Arg280His, and Arg399Gln) in a Healthy Tunisian Population: Interethnic Variation and Functional Prediction

The genetic polymorphisms in DNA repair genes might affect the repair activities of the enzymes, predisposing individuals to cancer risk. Due to these genetic variants, interethnic differences in DNA repair capacity were observed in various populations. Hence, our study aimed to determine the prevalence of three nonsynonymous single-nucleotide polymorphisms (SNPs) in an X-ray repair cross-complementation group 1 gene (XRCC1) (Arg194Trp, Arg280His, and Arg399Gln) in a healthy Tunisian population (TUN) and to compare that with HapMap ( www.hapmap.org ) populations. Also, we predicted their eventual functional effect based on the protein conservation analysis by Sorting Intolerant From Tolerant (SIFT; http://sift.jcvi.org/www/SIFT_dbSNP.html ) software. The genotypes of 154 healthy individuals were determined by the polymerase chain reaction-restriction fragment length polymorphism. Tunisians showed a relative relatedness with Caucasians (European ancestry) for Arg194Trp and Arg399Gln that may be explained by the strategic geographic location of Tunisia in the Mediterranean, allowing exchanges with European countries. However, a characteristic pattern was observed in Arg280His polymorphism, which could be explained by the high inbreeding rate in TUN. The analysis of protein conservation showed that the three amino acid substitutions were predicted as damaged. The results presented here provide the first report on XRCC1 polymorphisms about Tunisians and may establish baseline database for our future clinical and genetic studies.

Splicing Defects in the AAAS Gene Leading to both Exon Skipping and Partial Intron Retention in a Tunisian Patient with Allgrove Syndrome

Background/Aims: Allgrove syndrome is a rare autosomal recessive disorder characterized by the triad of adrenal insufficiency, achalasia, and alacrima. This syndrome is caused by mutations in the AAAS gene. A major splice site mutation c.1331+1G>A was found previously in North African families affected by Allgrove syndrome. In this study, we analyzed in vivo and in silico the effect of this mutation on the splicing process. Methods: Using reverse transcriptase-polymerase chain reaction, sequencing and bioinformatics tools, we analyzed all transcripts produced by the AAAS gene containing this splice site mutation. Results: The altered splicing of mRNA produces two aberrant transcripts: one with exon 14 skipping, the other with concurrent exon 14 skipping and retention of 99 bp of intron 14, both outcomes resulting in frameshifts with a new stop codon generation in the untranslated region of the last exon. Using in silico bioinformatics tools, we demonstrated that this mutation abolishes the splice donor site of exon 14 and activates a new intronic cryptic splice site in intron 14. Conclusion: This study demonstrated that a single splicing mutation affects the AAAS transcripts and consequently the ALADIN protein structure and 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.