G. Besbes

Update of the spectrum of GJB2 gene mutations in Tunisian families with autosomal recessive nonsyndromic hearing loss

Hearing loss is the most frequent sensory disorder. It affects 3 in 1000 newborns. It is genetically heterogeneous with 60 causally-related genes identified to date. Mutations in GJB2 gene account for half of all cases of non-syndromic deafness. The aim of this study was to determine the relative frequency of GJB2 allele variants in Tunisia. In this study, we screened 138 patients with congenital hearing loss belonging to 131 families originating from different parts of Tunisia for mutations in GJB2 gene. GJB2 mutations were found in 39% of families (51/131). The most common mutation was c.35delG accounting for 35% of all cases (46/131). The second most frequent mutation was p.E47X present in 3.8% of families. Four identified mutations in our cohort have not been reported in Tunisia; p.V37I, c.235delC, p.G130A and the splice site mutation IVS1+1G>A (0.76%). These previously described mutations were detected only in families originating from Northern and not from other geographical regions in Tunisia. In conclusion we have confirmed the high frequency of c.35delG in Tunisia which represents 85.4% of all GJB2 mutant alleles. We have also extended the mutational spectrum of GJB2 gene in Tunisia and revealed a more pronounced allelic heterogeneity in the North compared to the rest of the country.

Whole exome sequencing identifies new causative mutations in Tunisian families with non-syndromic deafness.

Identification of the causative mutations in patients affected by autosomal recessive non syndromic deafness (DFNB forms), is demanding due to genetic heterogeneity. After the exclusion of GJB2 mutations and other mutations previously reported in Tunisian deaf patients, we performed whole exome sequencing in patients affected with severe to profound deafness, from four unrelated consanguineous Tunisian families. Four biallelic non previously reported mutations were identified in three different genes: a nonsense mutation, c.208C>T (p.R70X), in LRTOMT, a missense mutation, c.5417T>C (p.L1806P), in MYO15A and two splice site mutations, c.7395+3G>A, and c.2260+2T>A, in MYO15A and TMC1 respectively. We thereby provide evidence that whole exome sequencing is a powerful, cost-effective screening tool to identify mutations causing recessive deafness in consanguineous families.

Whole Exome Sequencing Identifies Mutations in Usher Syndrome Genes in Profoundly Deaf Tunisian Patients

Usher syndrome (USH) is an autosomal recessive disorder characterized by combined deafness-blindness. It accounts for about 50% of all hereditary deafness blindness cases. Three clinical subtypes (USH1, USH2, and USH3) are described, of which USH1 is the most severe form, characterized by congenital profound deafness, constant vestibular dysfunction, and a prepubertal onset of retinitis pigmentosa. We performed whole exome sequencing in four unrelated Tunisian patients affected by apparently isolated, congenital profound deafness, with reportedly normal ocular fundus examination. Four biallelic mutations were identified in two USH1 genes: a splice acceptor site mutation, c.2283-1G>T, and a novel missense mutation, c.5434G>A (p.Glu1812Lys), in MYO7A, and two previously unreported mutations in USH1G, i.e. a frameshift mutation, c.1195_1196delAG (p.Leu399Alafs*24), and a nonsense mutation, c.52A>T (p.Lys18*). Another ophthalmological examination including optical coherence tomography actually showed the presence of retinitis pigmentosa in all the patients. Our findings provide evidence that USH is under-diagnosed in Tunisian deaf patients. Yet, early diagnosis of USH is of utmost importance because these patients should undergo cochlear implant surgery in early childhood, in anticipation of the visual loss.

Clinical and genetic analysis of two Tunisian otosclerosis families

Otosclerosis is caused by an abnormal bone homeostasis of the otic capsule resulting in a conductive hearing loss when the free motion of the stapes is compromised. An additional sensorineural hearing loss arises in some patients, most likely due to otosclerotic foci that invade the cochlear endosteum. Otosclerosis is a very common hearing impairment among Caucasians with a prevalence of about 0.3–0.4% among white adults. In the majority of cases, otosclerosis can be considered as a complex disease, caused by both genetic as environmental factors, but autosomal dominant forms of otosclerosis exist. However, families large enough for genetic analysis are very rare and often show reduced penetrance. To date five loci have been reported, but none of the genes have been cloned yet. In this study, we analyzed two new autosomal dominant otosclerosis families from Tunisia, and genotyped them with microsatellite markers for the known loci, the collagen genes COL1A1 and COL1A2, and NOG gene. In the family LK, linkage to all known loci was excluded. However, the family LS shows suggestive linkage to the OTSC3 region on chromosome 6p21.3–p22.3. This result points out that, besides the five reported loci, there must be at least one additional locus for autosomal dominant otosclerosis.

Compound heterozygosity for dominant and recessive GJB2 mutations in a Tunisian family and association with successful cochlear implant outcome.

OBJECTIVES Mutations of GJB2 encoding connexin 26 are the most common cause of hearing loss. They are responsible for up to 50% of ARNSHL. The pathogenic mutations in this gene are generally inherited recessively. Dominant mutations in GJB2 also cause hearing loss, either in isolated non-syndromic form or as part of a syndrome associated with various skin disorders. METHODS We screened a Tunisian child affected by congenital, bilateral, profound, sensorineural hearing loss for mutations in GJB2 gene using PCR and direct sequencing. RESULTS The proband was found to be compound heterozygous for recessive and dominant GJB2 mutations respectively p.V37I (c.109G > A) and p.R143Q (c.428G > A). Surprisingly the hearing mother is a carrier for this dominant GJB2 mutation. This proband underwent a cochlear implant at four years old. The evaluation using APCEI and IT-MAIS tests at six months post implantation indicates a successful cochlear implant outcome since the deaf child began to acquire language abilities and auditory sensation. CONCLUSIONS The p.R143Q mutation was described for the first time in Tunisia. We confirm the low penetrance of this mutation since the proband mother is a carrier despite her normal hearing. We show the effectiveness of cochlear implant to restore the communication abilities and auditory sensation for our patient.

Estimation of Recent and Ancient Inbreeding in a Small Endogamous Tunisian Community Through Genomic Runs of Homozygosity.

Runs of homozygosity (ROHs) are extended genomic regions of homozygous genotypes that record populations’ mating patterns in the past. We performed microarray genotyping on 15 individuals from a small isolated Tunisian community. We estimated the individual and population genome‐wide level of homozygosity from data on ROH above 0.5 Mb in length. We found a high average number of ROH per individual (48.2). The smallest ROH category (0.5–1.49 Mb) represents 0.93% of the whole genome, while medium‐size (1.5‐4.99 Mb) and long‐size ROH (≥5 Mb) cover 1.18% and 0.95%, respectively. We found that genealogical individual inbreeding coefficients (Fped) based on three‐ to four‐generation pedigrees are not reliable indicators of the current proportion of genome‐wide homozygosity inferred from ROH (FROH) either for 0.5 or 1.5 Mb ROH length thresholds, while identity‐by‐descent sharing is a function of shared coancestry. This study emphasizes the effect of reproductive isolation and a prolonged practice of consanguinity that limits the genetic heterogeneity. It also provides evidence of both recent and ancient parental relatedness contribution to the current level of genome‐wide homozygosity in the studied population. These findings may be useful for evaluation of long‐term effects of inbreeding on human health and for future applications of ROHs in identifying recessive susceptibility genes.

Paradoxical reaction associated with cervical lymph node tuberculosis: predictive factors and therapeutic management

OBJECTIVES The aims of this study were to determine predictive factors of paradoxical reaction in patients with cervical lymph node tuberculosis (TB) and to discuss the therapeutic management of this condition. MATERIALS AND METHODS A retrospective study was performed of 501 patients managed for cervical lymph node TB over a period of 12 years (from January 2000 to December 2011). Statistical data were analyzed using IBM SPSS Statistics version 20.0. RESULTS Paradoxical reaction occurred in 67 patients (13.4%), with a median delay to onset after starting TB treatment of 7 months. Lymph node size ≥3cm and associated extra-lymph node TB were independently associated with paradoxical reaction. Treatment consisted of surgical excision (71.6%), restarting quadruple therapy (10.4%), reintroduction of ethambutol (23.8%), and addition of ciprofloxacin (20.8%); steroids were given in two cases . All patients recovered after an average treatment duration of 14.91±7.03 months. CONCLUSION The occurrence of paradoxical reaction in cervical lymph node TB seems to be predicted by associated extra-lymph node TB and a swelling size ≥3cm. The treatment of paradoxical reaction remains unclear and more randomized trials are necessary to improve its management.

Novel PAX3 mutations causing Waardenburg syndrome type 1 in Tunisian patients

Waardenburg syndrome (WS) is an auditory-pigmentary disease characterized by a clinical and genetic variability. WS is classified into four types depending on the presence or absence of additional symptoms: WS1, WS2, WS3 and WS4. Type 1 and 3 are mostly caused by PAX3 mutations, while type 2 and type 4 are genetically heterogeneous. The aims of this study are to confirm the diagnostic of WS1 by the sequencing of PAX3 gene and to evaluate the genotype phenotype correlation. A clinical classification was established for 14 patients WS, as proposed by the Waardenburg Consortium, and noted a predominance of type 1 and type 2 with 6 patients WS1, 7 patients WS2 and 1 patient WS3. A significant inter and intra-familial clinical heterogeneity was also observed. A sequencing of PAX3 gene in the 6 patients WS1 confirmed the diagnosis in 4 of them by revealing three novel mutations that modify two functional domains of the protein: the c.942delC; the c.933_936dupTTAC and the c.164delTCCGCCACA. These three variations are most likely responsible for the phenotype, however their pathogenic effects need to be confirmed by functional studies. The MLPA analysis of the 2 patients who were sequence negative for PAX3 gene revealed, in one of them, a heterozygous deletion of exons 5 to 9 confirming the WS1 diagnosis. Both clinical and molecular approaches led to the conclusion that there is a lack of genotype-phenotype correlation in WS1, an element that must be taken into account in genetic counseling. The absence of PAX3 mutation in one patient WS1 highlights the fact that the clinical classification is sometimes insufficient to distinguish WS1 from other types WS hence the interest of sequencing the other WS genes in this patient.

Paralysie faciale périphérique suite à une otite moyenne aiguë

OBJECTS To discuss clinical presentation and therapeutic approaches of facial paralysis in acute otitis media. METHODS We present five cases of facial palsy in children with acute otitis media managed in our ENT department during a period of 12 years (2001-2012). RESULTS The mean age was 14.2 years; sex ratio was 0.66. All patients presented with a facial asymmetry, but only 3 of them had otalgia before the onset of facial asymmetry. The facial palsy delay was 3.3 days. The ear examination showed that the tympanic membrane was congestive in 4 patients, associated with a bulging in 2 patients, and a small perforation in one patient. Our patients presented grade III to IV initial facial palsy according to House and Brackmann staging. Computed tomography scan revealed a dehiscence of the bony facial canal in one patient. Antibiotic therapy associated with intravenous corticosteroids was administered in all patients. All patients underwent a facial kinesis therapy. A progressive improvement of facial palsy was observed in 4 patients and complete recovery of facial function in one case. DISCUSSION Conservative treatment associating intravenous antibiotic and corticosteroids with or without myringotomy is the standard approach.

A novel frameshift mutation (c.405delC) in the GJB2 gene associated with autosomal recessive hearing loss in two Tunisian families

OBJECTIVES Mutations in GJB2 are found to be responsible for 50% of congenital autosomal recessive non-syndromic hearing loss, one of the most important mutations in this gene is the c.35delG, which is responsible for the majority of GJB2 related deafness in the Tunisian population. The aim of this study was to determine the molecular etiology of hearing loss in two Tunisian individuals. METHODS We screened two Tunisian individuals affected by congenital, bilateral, profound, sensorineural hearing loss for mutations in GJB2 gene using PCR and direct sequencing. RESULTS We identified a novel frameshift mutation in the GJB2 gene, the c.405delC resulting in a truncated protein (p.Tyr136Thrfs*32). It was found in compound heterozygosity with the c.35delG in two non-consanguineous unrelated families from Tunisia. One patient underwent a cochlear implant at 4 years. Initial evaluations post-implantation indicate a successful cochlear implant outcome since the patient began to acquire language abilities and auditory sensation. CONCLUSIONS With this novel GJB2 mutation, the mutational spectrum of this gene continues to broaden in our population. The occurrence of biallelic GJB2 mutations for the other deaf girl, despite the neonatal pain and hypotension due to complicated delivery, led us to confirm the importance of GJB2 screening for cochlear implant candidates regardless of the etiology of deafness in populations with a relatively high frequency of GJB2 mutation carriers.