Ridha Mrad

Molecular investigation of distal renal tubular acidosis in Tunisia, evidence for founder mutations.

BACKGROUND Distal renal tubular acidosis (dRTA) is a rare genetic disease caused by mutations in different genes involved in the secretion of H+ ions in the intercalated cells of the collecting duct. Both autosomal dominant and recessive forms have been described; the latter is also associated with sensorineural hearing loss. METHODS Twenty-two Tunisian families were analyzed for mutations in the ATP6V1B1 and ATP6V0A4 genes by direct sequencing. Dating of the founder mutations was performed. RESULTS Two founder mutations in the ATP6V1B1 gene were found in 16/27 dRTA cases. The p.Ile386Hisfs*56 founder mutation was estimated to be older than 2400 years and no correlations were found with deafness. For the remaining patients, two mutations in the ATP6V0A4 gene, one of them being novel, were found in three Tunisian cases. The presence of a heterozygous missense mutation p.T30I, of the ATP6V1B1 gene, was identified in six patients, while no mutations of the second gene were detected. No deleterious mutations of either ATP6V1B1 or ATP6V0A were found for the two probands. CONCLUSION Our study gives evidence of phenotypic and genotypic heterogeneity of dRTA in the Tunisian population. Five different mutations were found, two of them were due to a founder effect, and screening of these mutations could provide a rapid and valuable tool for diagnosis of dRTA.

Exploring the 7p22.1 Chromosome as a Candidate Region for Autism

A high incidence of de novo chromosomal aberrations in a population of persons with autism suggests a causal relationship between certain chromosomal aberrations and the occurrence of autism. A previous study on a Tunisian boy carrying a t(7;16) translocation identified the 7p22.1 as a positional candidate region for autism on chromosome 7. The characterization of the chromosomal breakpoints helped us to identify new candidate regions on chromosome 16p11.2 which contain no known genes and the other one on 7p22.1 containing a portion of genes (NP 976327.1, RBAK, Q6NUR6 also called RNF216L and MMD2). We proposed Q6NUR6 (RNF216L) as a candidate gene for autism due to its vicinity to the translocation breakpoint on the chromosome derivative 7. Q6NUR6 is predicted to be an E3ubiquitin-ligase. Quantitative PCR demonstrates that Q6NUR6 gene has an ubiquitous expression and that it is strongly expressed in fetal and adult brain. The Q6NUR6 expression is increased in the patient blood cells in comparison to controls. This is the first report of Q6NUR6 gene (E3 ubiquitin ligase TRIAD3 EC 6.3.2) increasing blood levels in a patient with autism. Its probably caused by a position effect involving this gene and modifying its expression.

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.

Bathing suit ichthyosis caused by a TGM1 mutation in a Tunisian child

Bathing suit ichthyosis (BSI) is an uncommon phenotype classified as a minor variant of autosomal recessive congenital ichthyosis (ARCI).

GJB2 and GJB6 screening in Tunisian patients with autosomal recessive deafness

UNLABELLED Autosomal recessive nonsyndromic deafness (ARNSD or DFNB) is a very common genetically heterogenous disorder. Although DFNB1 mutations are known to be the most frequent cause of this disorder, they are largely dependent on ethnic groups. The aims of our study are to specify the prevalence and the spectrum of GJB2 mutations as well as the prevalence of GJB6 large deletion in Tunisian population. PATIENTS AND METHODS 95 unrelated patients with moderate to severe sensorineural hearing loss have been tested. The GJB2 coding region has been studied by PCR/Sequencing and the del(GJB6-D13S1830) mutation has been screened by fluorescent PCR multiplex. RESULTS 27.36% of patients present mutations on both alleles of GJB2 gene and no one has the del(GJB6-D13S1830) mutation. The c.35delG mutation represents 86.5% of GJB2 deafness alleles and is found in homozygous state in 22 patients and in heterozygous state in one patient. Four other mutations are detected in four probands: two are compound heterozygous for the p.V37I/p.E47X and the c.35delG/p.R184P mutations, and two are homozygous for the p.E47X and the c.333-334delAA mutations. CONCLUSION Our results showed that c.35delG is the most common but not the only GJB2 mutation and that the del(GJB6-del D13S1830) is absent in our cohort. Consequently, we propose a systematic sequencing of GJB2 coding region for ARNSD Tunisian patients and we suggest additional studies to specify the real prevalence of del(GJB6-D13S1830) in our population.

The experience of a Tunisian referral centre in prenatal diagnosis of Xeroderma pigmentosum.

Aims: Xeroderma pigmentosum (XP, OMIM 278700-278780) is one of the most severe genodermatoses and is relatively frequent in Tunisia. In the absence of any therapy and to better manage the disease, we aimed to develop a molecular tool for DNA-based prenatal diagnosis. Methods: Six consanguineous Tunisian XP families (4 XP-A and 2 XP-C) have benefited from a prenatal diagnosis. Screening for mutations was performed by direct sequencing, while maternal-foetal contamination was checked by genotyping. Results: Among the 7 prenatal diagnoses, 4 foetuses were heterozygous for the screened mutation. Exclusion of contamination by maternal cells was checked. Mutations were detected at a homozygous state in the remaining cases, and the parents decided to terminate pregnancy. Conclusion: Our study illustrates the implementation of prenatal diagnosis for better health support of XP in Tunisia.

Distinctive findings in a boy with Simpson–Golabi–Behmel syndrome

Simpson–Golabi–Behmel syndrome (SGBS) is an X‐linked condition characterized by pre and post natal overgrowth, facial malformations, and visceral, skeletal, and neurological anomalies. The physical characteristics of SGBS have been well documented; however there is a lack of description regarding the behavioral phenotype. We report the case of a 6‐year‐old boy, with confirmed deletion of 6–8 exons of the glypican‐3 gene (GPC3) who presents three distinctive findings: the persistence of the craniopharyngeal canal, an immune‐allergic specificity, and a scarcely behavioral phenotype consisting in the association of Austim Spectrum Disorder with accompanying mild intellectual disability and language impairments. He also fulfilled the criteria of Attention Deficit Hyperactivity Disorder and Oppositional Defiant Disorder according to DSM 5 criteria. The specificities of the case are discussed in the light of recent pathophysiological data.

Clinical, genealogical and molecular investigation of the xeroderma pigmentosum type C complementation group in Tunisia

DEAR EDITOR, Xeroderma pigmentosum (XP) is an ultraviolet sensitivity syndrome characterized by skin hyperpigmentation, premature photoageing and early onset of skin cancers. XP is a rare disease. In the U.S.A. and Europe the estimated incidence is 1 per 1 million inhabitants, while it is much higher in Japan (1 in 22 000) and both North Africa and the Middle East (estimated 1 in 50 000). In Tunisia the frequency is about 1 in 10 000. There are seven complementation groups with defects in the nucleotide excision repair pathway (XP-A to XP-G). Patients with XP-C develop predominantly skin damage and early malignancies without neurological abnormalities. Nevertheless, several cases with neurological disorders have been reported. Here we report on the clinical, genealogical and molecular investigation of 44 families including a total of 64 patients with XP-C who were followed at three university hospitals (Charles Nicolle, Habib Thameur and La Rabta hospitals) during the period from 2006 to 2013 (Table 1). Patients were suspected to have XP-C on the basis of their clinical features (age at onset of erythema, age at appearance of skin cancers and absence of neurological abnormalities). Written informed consent was obtained from all participants or their legal guardians. All families were interviewed using a specific questionnaire to collect information about genealogical data, familial history and associated diseases. We performed molecular and histological analysis at Institut Pasteur de Tunis. The study was evaluated and approved by the institutional ethical committee and conducted according to the Declaration of Helsinki principles. Blood samples were obtained from all available family members and DNA was extracted using a salting-out procedure or Qiagen FlexiGene DNA kit (51206; Qiagen, Venlo, the Netherlands). Genomic DNA was amplified using XPC gene primers specific to exons 4–16. Polymerase chain reaction products were sequenced in an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, U.S.A.). Mutations were annotated using NG 011763 1 according to the Human Genome Variation Society version 2.0 (Mutalyzer 2.0.beta-26; https://www.mutalyzer.nl/). In the case of skin tumours, histopathological analyses were performed on biopsies using routine haematoxylin and eosin staining to define the histological type of the tumours. Acquired data were statistically analysed using SPSS 20.0 for Windows (IBM, Armonk, NY, U.S.A.). Descriptive statistical methods (frequency, mean, median, SD) were performed with their corresponding graphs. The association between protection levels and skin cancer development was calculated using a Pearson correlation test. A P-value ≤ 0 05 was considered statistically significant. The observed number of nonmelanoma skin cancers (NMSCs) was compared with the expected number from regional cancer registries for the 1995–2006 period after adjustment for age and sex. The exact Fisher test was used to predict the relative risk factor odds ratio (Table 2). Genealogical pedigrees were drawn using the R package Kinship2 (http://www.r-project.org/). Mutational analysis showed that 60 patients (94%) belonging to 43 families not known to be related shared the founder mutation XPC c.1643_1644delTG (p.Val548Alafs*25). Among them, 15 patients shared the same haplotype. This founder mutation c.1643_1644delTG has been identified in patients from various regions around the world, with higher frequencies in the Mediterranean region. It has been estimated that the common ancestor mutation occurred 1250 years ago. In addition, three unrelated patients (5%) had the second founder mutation c.850G>T and one patient (2%) had the c.779+1G>A mutation (Fig. 1). It is noteworthy that all of the patients were homozygous for the deleterious mutations, and no compound heterozygous mutation was identified. The mutations were confirmed in the parents. In addition, all available related individuals were screened. Among the latter, 24 were healthy heterozygous carriers and 18 were homozygous for the wild-type allele (Fig. 1). Presymptomatic diagnosis was performed for two patients under the age of 2 years (XP66 2MED and XP30 2MO). Four families (XP13KA, XP26B, XP46MED and XP76MED) asked for prenatal diagnosis (PND). The XP46MED family benefited from two consecutive PNDs. The results showed that one fetus was homozygous for the wild-type allele, three fetuses were heterozygous and one was affected homozygous for the screened mutation in the proband. The parents decided to terminate the pregnancy when the fetus was homozygous for the deleterious allele. Analysis of the geographical distribution of XPC mutations showed that southeast Tunisia and the coasts have the highest

Perinatal-lethal Gaucher disease presenting as hydrops fetalis

Perinatal-lethal Gaucher disease is very rare and is considered a variant of type 2 Gaucher disease that occurs in the neonatal period. The most distinct features of perinatal-lethal Gaucher disease are non-immune hydrops fetalis. Less common signs of the disease are hepatosplenomegaly, ichthyosis and arthrogryposis. We report a case of Gauchers disease (type 2) diagnosed in a newborn who presented with Hydrops Fetalis.