Mohamed Samir Boubaker

Heminecrolysin, the first hemolytic dermonecrotic toxin purified from scorpion venom

Envenomation caused by Hemiscorpius (H.) lepturus from Liochlidae family presents clinical features that have not been previously described for the Buthidae family scorpions. The most significant manifestations of H. lepturus envenomation are hemolysis and dermonecrosis which could lead in severe cases to renal, cardio-respiratory failure, and death. In this study, we aimed to identify and characterize the protein(s) causing these effects. We have purified a 33 kDa protein from the venom of H. lepturus and named it Heminecrolysin. Tryptic digestion and MS/MS analysis of obtained peptides showed homology with previously described brown spider sphingomyelinases D. Functional characterization of Heminecrolysin indicated a sphingomyelinase D, a complement-dependent hemolysis properties and a dermonecrosis activity. Heminecrolysin displayed higher hemolytic activity to human erythrocytes (ED50 of 0.025 μg/ml), a stronger inflammatory and dermonecrotic effects when injected intra-dermally to rabbit skins, while its efficiency to hydrolyze sphingomyelin seems weaker than other known spider dermonecrotic SMasesD (149 ± 32.5 nmol/mg). Step of sensitization of human erythrocytes by Heminecrolysin was shown to be Mg²⁺ and Ca²⁺-independent while hemolysis step in the presence of complement required both bivalent ions. Heminecrolysin is the first hemolytic dermonecrotic toxin identified in venom other than spiders. Except in spider Loxosceles genus and some pathogenic strains of Corynebacteria, sphingomyelinase D activity is unknown in the animal kingdom.

Particular Mal de Meleda Phenotypes in Tunisia and Mutations Founder Effect in the Mediterranean Region

Mal de Meleda (MDM) is a rare, autosomal recessive form of palmoplantar keratoderma. It is characterized by erythema and hyperkeratosis of the palms and soles that progressively extend to the dorsal surface of the hands and feet. It is caused by mutations in SLURP-1 gene encoding for secreted mammalian Ly-6/uPAR-related protein 1 (SLURP-1). We performed mutational analysis by direct sequencing of SLURP-1 gene in order to identify the genetic defect in three unrelated families (families MDM-12, MDM-13, and MDM-14) variably affected with transgressive palmoplantar keratoderma. A spectrum of clinical presentations with variable features has been observed from the pronounced to the transparent hyperkeratosis. We identified the 82delT frame shift mutation in the SLURP-1 gene in both families MDM-12 and MDM-13 and the missense variation p.Cys99Tyr in family MDM-14. To date, the 82delT variation is the most frequent cause of MDM in the world which is in favour of a recurrent molecular defect. The p.Cys99Tyr variation is only described in Tunisian families making evidence of founder effect mutation of likely Tunisian origin. Our patients presented with very severe to relatively mild phenotypes, including multiple keratolytic pits observed for one patient in the hyperkeratotic area which was not previously reported. The phenotypic variability may reflect the influence of additional factors on disease characteristics. This report further expands the spectrum of clinical phenotypes associated with mutations in SLURP1 in the Mediterranean population.

A founder large deletion mutation in Xeroderma pigmentosum-Variant form in Tunisia: implication for molecular diagnosis and therapy.

Xeroderma pigmentosum Variant (XP-V) form is characterized by a late onset of skin symptoms. Our aim is the clinical and genetic investigations of XP-V Tunisian patients in order to develop a simple tool for early diagnosis. We investigated 16 suspected XP patients belonging to ten consanguineous families. Analysis of the POLH gene was performed by linkage analysis, long range PCR, and sequencing. Genetic analysis showed linkage to the POLH gene with a founder haplotype in all affected patients. Long range PCR of exon 9 to exon 11 showed a 3926 bp deletion compared to control individuals. Sequence analysis demonstrates that this deletion has occurred between two Alu-Sq2 repetitive sequences in the same orientation, respectively, in introns 9 and 10. We suggest that this mutation POLH NG_009252.1: g.36847_40771del3925 is caused by an equal crossover event that occurred between two homologous chromosomes at meiosis. These results allowed us to develop a simple test based on a simple PCR in order to screen suspected XP-V patients. In Tunisia, the prevalence of XP-V group seems to be underestimated and clinical diagnosis is usually later. Cascade screening of this founder mutation by PCR in regions with high frequency of XP provides a rapid and cost-effective tool for early diagnosis of XP-V in Tunisia and North Africa.

The first Mal de Meleda case in Libya: identification of a SLURP1 mutation

Laboratoire de Génomique Biomédicale et Oncogénétique (LR11IPT05), Institut Pasteur de Tunis, Université de Tunis El Manar, Tunis, Tunisia, Département de Dermatologie,Hôpital de Ben Ghazi, Ben Ghazi, Libya Département de Dermatologie, Hôpital La Rabta, Tunis, Tunisia, Unité de recherche “Troubles héréditaires de la kératinisation, UR24/04”, Hôpital La Rabta de Tunis, Tunis, Tunisia, and Laboratoire d’anatomie pathologique humaine et expérimentale, Institut Pasteur de Tunis, Tunis, Tunisia

Further Evidence of Mutational Heterogeneity of the XPC Gene in Tunisian Families: A Spectrum of Private and Ethnic Specific Mutations

Xeroderma Pigmentosum (XP) is a rare recessive autosomal cancer prone disease, characterized by UV hypersensitivity and early appearance of cutaneous and ocular malignancies. We investigated four unrelated patients suspected to be XP-C. To confirm linkage to XPC gene, genotyping and direct sequencing of XPC gene were performed. Pathogenic effect of novel mutations was confirmed by reverse Transciptase PCR. Mutation screening revealed the presence of two novel mutations g.18246G>A and g.18810G>T in the XPC gene (NG_011763.1). The first is present in one patient XP50NEF, but the second is present in three unrelated patients (XP16KEB, XP28SFA, and XP45GB). These 3 patients are from three different cities of Southern Tunisia and bear the same haplotype, suggesting a founder effect. Reverse Transciptase PCR revealed the absence of the XPC mRNA. In Tunisia, as observed in an other severe genodermatosis, the mutational spectrum of XP-C group seems to be homogeneous with some clusters of heterogeneity that should be taken into account to improve molecular diagnosis of this disease.

Genetic basis of dominant dystrophic epidermolysis bullosa in tunisian families and co-occurrence of dominant and recessive mutations

epithelial–myoepithelial structures within a chondromyxoid and fibrous stroma (Fig. 1b,c). Decapitation secretion was occasionally seen in the space (Fig. 1d). No cellular atypia was seen, and only occasional mitoses were found. From these findings, the diagnosis of MTS was made. Retrospectively, the elastic moderate lesion (visualized green in Fig. 2a) demonstrated in elastography may pathologically correspond to closely aggregated myoepithelial cells around the sweat gland. The elastic soft lesion (visualized in yellow to red in Fig. 2a) that was revealed in elastography may be histologically comparable to a broadly mucinous region, empty space and the chondroid portion. To compare the present case with ordinary epidermal cyst, we presented five images of cases that had already been histologically confirmed as epidermal cyst. Ultrasonography (B-mode) of epidermal cyst revealed the tumour to be well circumscribed, the inside of the tumour to be a heterogeneous high-echoic region, posterior echo enhancement to be evident (Fig. 2b–f; blue arrowhead) and lateral shadow to be seen (Fig. 2b–f; red arrowhead). Almost of all these findings of epidermal cysts were extremely similar to the B-mode image of MTS. On the other hand, elastography of epidermal cysts uniformly visualized the inside of the tumour as blue or green (Fig. 2b–f; yellow arrowhead), finding that were completely different from the MTS image of our own case (Fig. 2a). In our case, the combination of B-mode and elastographic findings helped us to keep cystic lesion in mind as a differential diagnosis. When similar elastographic findings forMTS of the scalp are accumulated, it may become more easily diagnosable. We should be encouraged to examine skin tumours on the scalp in conjunction with elastography tomake better clinical diagnoses.

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

Mutational founder effect in recessive dystrophic epidermolysis bullosa families from Southern Tunisia

Abstract Dystrophic epidermolysis bullosa (DEB) is a group of heritable bullous skin disorders caused by mutations in the COL7A1 gene. One of the most severe forms of DEB is the severe generalized [recessive dystrophic epidermolysis bullosa (RDEB-SG)] subtype, which is inherited in an autosomal recessive manner. This subtype is most often due to COL7A1 mutations resulting in a premature termination codon on both alleles. We report here, the molecular investigation of 15 patients belonging to 14 nuclear families from the city of Sfax in Southern Tunisia, with clinical features of RDEB-SG complicated by squamous cell carcinoma in 3 patients. We identified two novel mutations, p.Val769LeufsX1 and p.Ala2297SerfsX91, in addition to one previously reported mutation (p.Arg2063Trp). The p.Val769LeufsX1 mutation was shared by 11 families and haplotype analysis indicated that it is a founder mutation. The p.Ala2297SerfsX91 mutation was a private mutation found in only one family. Together with the previously described recurrent mutations in Tunisia, screening for the founder p.Val769LeufsX1 mutation should provide a rapid molecular diagnosis tool for mutation screening in RDEB patients from Southern Tunisia and possibly from other Mediterranean populations sharing the same genetic background.

Clinical and molecular investigation of Buschke-Fischer-Brauer in consanguineous Tunisian families.

Punctate palmoplantar keratoderma type I (PPPK‐BFB), also called Buschke‐Fischer‐Brauer disease (MIM 148600) is a rare autosomal dominant disorder of keratinization, characterized by multiple hyperkeratotic lesions on the palms and soles. Recently, PPPK‐BFB has been shown to be associated with mutations in the AAGAB gene in several families of European, African, Canadian and Asian origins.