Neji Tebib

Consanguinity, endogamy, and genetic disorders in Tunisia

Consanguinity refers to marriages between individuals who share at least one common ancestor. In clinical genetics, a consanguineous marriage is defined as a union between two individuals who are related as second cousins or closer, with the inbreeding coefficient (F) equal or higher than 0.0156 (Bittles2001). However, reports on consanguinity rates may sometimes include marriages between third cousins or more distantly related individuals (Hamamy2011). It is estimate that more than 690 million people in the world are consan- guineous (Bittles and Black 2010 ). Middle East, Northern Africa, and South Asia are regions that have historically and culturally had a high rate of consanguineous unions (Al- Awadi et al. 1985; Al-Gazali et al.1997; Jaber et al.1997;Bittles et al.2002; Bener and Alali2006). Recent studieshave shown that 20 % to 50 % of marriages in Arab countries are between relatives (Tadmouri et al. 2009;Bittles2011; Hamamy et al.2011). The rate was 68 % inEgypt (Mokhtar and Abdel-Fattah2001), 51-58 % in Jordan

Phenylketonuria is still a major cause of mental retardation in Tunisia despite the possibility of treatment

BACKGROUND AND OBJECTIVE Accumulation of phenylalanine following a deficiency of phenylalanine hydroxylase activity generates a brain damage with mental retardation: phenylketonuria (PKU). In the developing countries, where PKU systematic neonatal screening program is not established yet, the management of PKU handicap is not properly carried out. The aim of this study was to estimate the frequency of the PKU diagnosed following clinical features anomalies, to provide information about the untreated PKU patients profile in Tunisia not covered by neonatal screening. Also it is stressed that treated patients have a normal development. PATIENTS AND METHODS This is a retrospective study of 156 cases of PKU detected in Tunisia over 20 years following symptoms suggestive of inherited metabolic disease. Phenylalaninemia level was performed by fluorometric method. Among them 9 patients were treated. RESULTS The PKU estimated frequency was 1/7631. The diagnosis mean age was 4 years. The phenylalaninemia mean was 1680 μmol/L; the classical PKU form accounted for 85.3% of cases and the dominant clinical symptoms were: mental retardation (88.2%), motor delays (87.7%), speech difficulties (83.2%) and pigmentation anomalies (61.7%). The treated patients responded to treatment and showed a normal development. CONCLUSION The establishment of neonatal screening should be a priority to avoid cases of mentally retardation.

Severe respiratory complex III defect prevents liver adaptation to prolonged fasting

BACKGROUND & AIMS Next generation sequencing approaches have tremendously improved the diagnosis of rare genetic diseases. It may however be faced with difficult clinical interpretation of variants. Inherited enzymatic diseases provide an invaluable possibility to evaluate the function of the defective enzyme in human cell biology. This is the case for respiratory complex III, which has 11 structural subunits and requires several assembly factors. An important role of complex III in liver function is suggested by its frequent impairment in human cases of genetic complex III defects. METHODS We report the case of a child with complex III defect and acute liver dysfunction with lactic acidosis, hypoglycemia, and hyperammonemia. Mitochondrial activities were assessed in liver and fibroblasts using spectrophotometric assays. Genetic analysis was done by exome followed by Sanger sequencing. Functional complementation of defective fibroblasts was performed using lentiviral transduction followed by enzymatic analyses and expression assays. RESULTS Homozygous, truncating, mutations in LYRM7 and MTO1, two genes encoding essential mitochondrial proteins were found. Functional complementation of the complex III defect in fibroblasts demonstrated the causal role of LYRM7 mutations. Comparison of the patients clinical history to previously reported patients with complex III defect due to nuclear DNA mutations, some actually followed by us, showed striking similarities allowing us to propose common pathophysiology. CONCLUSIONS Profound complex III defect in liver does not induce actual liver failure but impedes liver adaptation to prolonged fasting leading to severe lactic acidosis, hypoglycemia, and hyperammonemia, potentially leading to irreversible brain damage. LAY SUMMARY The diagnosis of rare genetic disease has been tremendously accelerated by the development of high throughput sequencing technology. In this paper we report the investigations that have led to identify LYRM7 mutations causing severe hepatic defect of respiratory complex III. Based on the comparison of the patients phenotype with other cases of complex III defect, we propose that profound complex III defect in liver does not induce actual liver failure but impedes liver adaptation to prolonged fasting.

A novel UBE3A truncating mutation in large Tunisian Angelman syndrome pedigree.

We identified in a large Tunisian pedigree a novel UBE3A frameshift mutation in exon 16 coding region, and we expect that the resulting UBE3A truncated protein in our patients is non‐functional since the mutation implies the catalytic region of the enzyme. The family includes 14 affected patients born from four sisters. This mutation was found in all surviving affected individuals and their mothers pointing out the importance of genetic counseling possibility in Angelman syndrome (AS). All patients had severe mental retardation with epilepsy and microcephaly. Minor clinical expression variation was observed among the investigated patients. The severity of clinical expression is related to the detected molecular variation: deletion of 15 bp and insertion of 7 bp. These results are concordant with the gene expression observed in previously reported individuals with AS and truncated UBE3A protein.

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.

Novel and recurrent mutations in the TAT gene in Tunisian families affected with Richner-Hanhart Syndrome

Tyrosinemia type II, also designated as oculocutaneous tyrosinemia or Richner-Hanhart syndrome (RHS), is a very rare autosomal recessive disorder. In the present study, we report clinical features and molecular genetic investigation of the tyrosine aminotransferase (TAT) gene in two young patients, both born to consanguineous unions between first-degree cousins. These two unrelated families originated from Northern and Southern Tunisia. The clinical diagnosis was based on the observation of several complications related to Richner-Hanhart syndrome: recurrent eye redness, tearing and burning pain, photophobia, bilateral pseudodendritic keratitis, an erythematous and painful focal palmo-plantar hyperkeratosis and a mild delay of mental development. The diagnosis was confirmed by biochemical analysis. Sequencing of the TAT gene revealed the presence of a previously reported missense mutation (c.452G>A, p.Cys151Tyr) in a Tunisian family, and a novel G duplication (c.869dupG, p.Trp291Leufs 6). Early diagnosis of RHS and protein-restricted diet are crucial to reduce the risk and the severity of long-term complications of hypertyrosinemia such as intellectual disability.

High frequency of W1327X mutation in glycogen storage disease type III patients from central Tunisia

Glycogen storage disease type III (GSD III) is an autosomal recessive disorder caused by the deficiency of glycogen debranching enzyme (AGL). It is characterized by hepatomegaly, progressive myopathy, cardiomyopathy and fasting hypoglycemia. Several mutations in AGL gene have been described in different populations. The W1327X mutation was reported in one Tunisian patient resident in Italy. We looked in this report to determine the frequency of W1327X mutation among Tunisian patients. The W1327X mutation was screening in 26 GSD III patients originated from various geographic locations in Tunisia. The sequence analysis revealed that among nine patients carried the W1327X mutation. Eight of them were from six unrelated families and they were originated from Mahdia (centre of Tunisia) suggesting the existence of a founder effect in this region. Taking into account historical migratory waves, screening for this mutation should be performed in priority for molecular diagnosis confirmation of GSD III in North African populations.

Phenylketonuria in Tunisian institutions for the mentally handicapped

Phenylketonuria (PKU) is the most frequent inherited metabolic disorder and results in progressive mental retardation. The incidence of PKU varies widely in neonates: it is high in Turkey (1:2600) and Ireland (1:4500) and low in Japan (1:125 000).1 The incidence of the disease is unknown in Tunisia and North Africa. In the absence of a newborn screening program for PKU and considering the high rate of consanguineous marriages in Tunisia, this study aimed to assess the incidence of PKU among institutionalised patients with a mental handicap. A …

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.

Gaucher disease in Tunisia: High frequency of the most common mutations

a Molecular Investigation of Genetic Orphan Diseases Research Unit, Institut Pasteur de Tunis, Tunisia b Inherited Metabolic Diseases Unit, Pediatric Department, La Rabta Hospital, Tunis, Tunisia c Child Neurological Diseases Unit; Faculty of Medicine, Tunis, Tunisia d Pediatric Department, Sahloul Hospital, Sousse, Tunisia e Neurobiology Genetic Department, Biology Molecular and Cellular Institute, Porto University, Portugal f Metabolic Genetic Laboratory, Medicine Faculty, Cochin, France