H. Chaabouni

Targeted high-throughput sequencing for diagnosis of genetically heterogeneous diseases: efficient mutation detection in Bardet-Biedl and Alström Syndromes

Background Bardet-Biedl syndrome (BBS) is a pleiotropic recessive disorder that belongs to the rapidly growing family of ciliopathies. It shares phenotypic traits with other ciliopathies, such as Alström syndrome (ALMS), nephronophthisis (NPHP) or Joubert syndrome. BBS mutations have been detected in 16 different genes (BBS1-BBS16) without clear genotype-to-phenotype correlation. This extensive genetic heterogeneity is a major concern for molecular diagnosis and genetic counselling. While various strategies have been recently proposed to optimise mutation detection, they either fail to detect mutations in a majority of patients or are time consuming and costly. Method We tested a targeted exon-capture strategy coupled with multiplexing and high-throughput sequencing on 52 patients: 14 with known mutations as proof-of-principle and 38 with no previously detected mutation. Thirty genes were targeted in total including the 16 BBS genes, the 12 known NPHP genes, the single ALMS gene ALMS1 and the proposed modifier CCDC28B. Results This strategy allowed the reliable detection of causative mutations (including homozygous/heterozygous exon deletions) in 68% of BBS patients without previous molecular diagnosis and in all proof-of-principle samples. Three probands carried homozygous truncating mutations in ALMS1 confirming the major phenotypic overlap between both disorders. The efficiency of detecting mutations in patients was positively correlated with their compliance with the classical BBS phenotype (mutations were identified in 81% of ‘classical’ BBS patients) suggesting that only a few true BBS genes remain to be identified. We illustrate some interpretation problems encountered due to the multiplicity of identified variants. Conclusion This strategy is highly efficient and cost effective for diseases with high genetic heterogeneity, and guarantees a quality of coverage in coding sequences of target genes suited for diagnosis purposes.

Only two mutations detected in 15 Tunisian patients with 11β-hydroxylase deficiency: the p.Q356X and the novel p.G379V.

Kharrat M, Trabelsi S, Chaabouni M, Maazoul F, Kraoua L, Ben Jemaa L, Gandoura N, Barsaoui S, Morel Y, M’rad R, Chaabouni H. Only two mutations detected in 15 Tunisian patients with 11β‐hydroxylase deficiency: the p.Q356X and the novel p.G379V.

Clinical and genetic characterization of Bardet–Biedl syndrome in Tunisia: defining a strategy for molecular diagnosis

Bardet–Biedl syndrome (BBS, OMIM 209900) is a rare genetic disorder characterized by obesity, retinitis pigmentosa, post axial polydactyly, cognitive impairment, renal anomalies and hypogonadism. The aim of this study is to provide a comprehensive clinical and molecular analysis of a cohort of 11 Tunisian BBS consanguineous families in order to give insight into clinical and genetic spectrum and the genotype–phenotype correlations. Molecular analysis using combined sequence capture and high‐throughput sequencing of 30 ciliopathies genes revealed 11 mutations in 11 studied families. Five mutations were novel and six were previously described. Novel mutations included c.1110G>A and c.39delA (p.G13fs*41) in BBS1, c.115+5G>A in BBS2, c.1272+1G>A in BBS6, c.1181_1182insGCATTTATACC in BBS10 (p.S396Lfs*6). Described mutations included c.436C>T (p.R146*) and c.1473+4A>G in BBS1, c.565C> (p.R189*) in BBS2, deletion of exons 4–6 in BBS4, c.149T>G (p.L50R) in BBS5, and c.459+1G>A in BBS8; most frequent mutations were described in BBS1 (4/11, 37%) and BBS2 (2/11, 18%) genes. No phenotype–genotype correlation was evidenced. This data expands the mutations profile of BBS genes in Tunisia and suggests a divergence of the genetic spectrum comparing Tunisian and other populations.

Hexasomy of the Prader-Willi/Angelman critical region, including the OCA2 gene, in a patient with pigmentary dysplasia: case report.

Derivatives of chromosome 15, often referred to as inv dup(15), represent the most common supernumerary marker chromosome (SMC). SMC(15)s can be classified into two major groups according to their length: small SMC(15) and large ones. Depending on the amount of euchromatin, the carriers may either present with a normal phenotype or with a recognizable syndrome. Here we describe a patient with severe mental retardation, epilepsy, dysmorphic features and pigmentary dysplasia. His karyotype was 47,XY,+mar[41]/46,XY[9]. Chromosomal fluorescence in situ hybridization (FISH) showed the SMC to be originating from chromosome 15, dicentric and containing four copies of the Prader-Willi/Angelman Syndrome Critical Region (PWACR), including the OCA2 gene. Molecular studies indicated that it is maternally derived. This report supports the previous observations assuming that severity of phenotype in patients with SMC(15) depends on the dosage of the PWACR and that skin pigmentation is correlated to OCA2 gene copy number.

Prevalence and incidence estimation of large NPHP1 homozygous deletion in Tunisian population.

Nephronophthisis NPHP (MIM 256100) is a well-known and common hereditary renal disease especially in regions with a high rate of consanguineous marriages as in Tunisia. NPHP is an autosomal recessive cystic kidney disease. It accounts for 6 to 10% of end-stage renal disease in children. Ten genes (NPHP1-NPHP9 and NPHP11) responsible for nephronophthisis have been identified. Their encoded proteins products are located at the primary cilium, basal bodies and centrosomes thus supporting a theory that describes the renal cystic disease as ‘‘ciliopathy’’ [1]. NPHP1 mutations were found in 30 to 85% of juvenile NPHP. The major NPHP1 gene defect is a large homozygous deletion [2,3]. Little information is available concerning NPHP1 mutations in the Tunisian population. This study investigates Tunisian patients with clinical pictures of tubulo-interstitiel kidney disorder and chronic renal failure to estimate the large NPHP1 homozygous deletion prevalence and incidence in our population.

Syndrome of ocular, skeletal & abdominal abnormalities

We describe two sisters who had a unique association of facial, ocular & skeletal defects and abdominal muscle hypoplasia. The two affected sisters are the fourth & the fifth sibs, born to healthy first cousin parents originating from Tunisia, indicating autosomal recessive inheritance. The family history is unremarkable. Many of these features overlap those previously found in other malformation syndromes. However, the constellation of defects observed in these patients appears to represent a OSA syndrome previously reported in 1996 by Mingarelli et al. In the best of our knowledge, its the second report of this syndrome.

Genetic study of two Tunisian Ehlers Danlos syndrome type VI

Ehlers Danlos syndromes (EDS) represent a clinically and genetically heterogeneous category of connective tissue disorders characterized by hyperextensibility of the skin and hypermobility of the joints.Clinical genetic and biochemical grounds distinguish eight types. The type VI is characterized by specific biochemical defect of lysyl hydroxylase.We report 2 cases of EDS type VI, clinically and biochemically confirmed. We focused our interest on the range of clinical severity in these patients and the comparison between our patients clinical data and clinical findings reviewed in the literature.