Asma Jlizi

Identification of the CCR5-Δ32 HIV resistance allele and new mutations of the CCR5 gene in different Tunisian populations

Polymorphisms in some chemokine receptor genes are associated with susceptibility to and progression of human immunodeficiency virus-1 (HIV-1) infection. Most mutations detected in the CC-chemokine receptor 5 (CCR5) gene are specific to different populations. In this study, we focused on polymorphisms of the CCR5 coding region in three healthy populations from Tunisia, corresponding to a cosmopolitan population from Tunis, and two isolated Berber populations. In addition to the CCR5-Delta32 deletion, eleven single nucleotide polymorphisms were detected. Some of these point mutations were associated with the same genotype and even the same haplotype. The (L55Q-C101X), I124, V131F, T143N, A159V, I237, T239A and G301R alleles have not been described previously, whereas the CCR5-Delta32, L55Q, A335V and Y339F variants have already been reported in the literature. The distribution and frequency of these variants were different among the three groups studied, a result in agreement with the mosaic genetic structure of the Tunisian population. To determine whether these alleles affect HIV-1 transmission, we compared allele frequencies between healthy and HIV-1 infected individuals from Tunis. The frequency of the CCR5-Delta32 variant was significantly different between the two groups, leading us to conclude that this mutation might confer protection against HIV infection in Tunisian populations.

First report of molecular diagnosis of Tunisian hemophiliacs A: Identification of 8 novel causative mutations

AbstractIntroductionHemophilia A is an X linked recessive hemorrhagic disorder caused by mutations in the F8 gene that lead to qualitative and/or quantitative deficiencies of coagulation factor VIII (FVIII). Molecular diagnosis of hemophilia A is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. Molecular studies of these mutations are essential in order to reinforce our understanding of their pathogenic effect responsible for the disorder.AimIn this study we have performed molecular analysis of 28 Tunisian hemophilia A patients and analyzed the F8 mutation spectrum.MethodsWe screened the presence of intron 22 and intron 1 inversion in severe hemophilia A patients by southern blotting and polymerase chain reaction (PCR). Detection of point mutations was performed by dHPLC/sequencing of the coding F8 gene region. We predict the potential functional consequences of novel missense mutations with bioinformatics approaches and mapping of their spatial positions on the available FVIII 3D structure.ResultsWe identified 23 different mutations in 28 Tunisian hemophilia A patients belonging to 22 unrelated families. The identified mutations included 5 intron 22 inversions, 7 insertions, 4 deletions and 7 substitutions. In total 18 point mutations were identified, of which 9 are located in exon 14, the most mutated exonic sequence in the F8 gene. Among the 23 mutations, 8 are novel and not deposited in the HAMSTeRS database nor described in recently published articles.ConclusionThe mutation spectrum of Tunisian hemophilia A patients is heterogeneous with the presence of some characteristic features.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1693269827490715

HIV-1 drug resistance genotyping from antiretroviral therapy (ART) naïve and first-line treatment failures in Djiboutian patients

AbstractIn this study we report the prevalence of antiretroviral drug resistant HIV-1 genotypes of virus isolated from Djiboutian patients who failed first-line antiretroviral therapy (ART) and from ART naïve patients.Patients and methodsA total of 35 blood samples from 16 patients who showed first-line ART failure (>1000 viral genome copies/ml) and 19 ART-naïve patients were collected in Djibouti from October 2009 to December 2009. Both the protease (PR) and reverse transcriptase (RT) genes were amplified and sequenced using National Agency for AIDS Research (ANRS) protocols. The Stanford HIV database algorithm was used for interpretation of resistance data and genotyping.ResultsAmong the 16 patients with first-line ART failure, nine (56.2%) showed reverse transcriptase inhibitor-resistant HIV-1 strains: two (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. Analysis of the DNA sequencing data indicated that the most common mutations conferring drug resistance were M184V (38%) for NRTI and K103N (25%) for NNRTI. Only NRTI primary mutations K101Q, K103N and the PI minor mutation L10V were found in ART naïve individuals. No protease inhibitor resistant strains were detected. In our study, we found no detectable resistance in ∼ 44% of all patients who experienced therapeutic failure which was explained by low compliance, co-infection with tuberculosis and malnutrition. Genotyping revealed that 65.7% of samples were infected with subtype C, 20% with CRF02_AG, 8.5% with B, 2.9% with CRF02_AG/C and 2.9% with K/C.ConclusionThe results of this first study about drug resistance mutations in first-line ART failures show the importance of performing drug resistance mutation test which guides the choice of a second-line regimen. This will improve the management of HIV-infected Djiboutian patients.Virtual slidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/2051206212753973

Profile of drug resistance mutations among HIV-1-infected Tunisian subjects failing antiretroviral therapy

Three years after the introduction of antiretroviral therapy (ART) in Tunisia (North Africa), we aimed to determine the prevalence of drug resistance mutations in Tunisian HIV-1-infected patients failing ART. Plasma samples of 80 patients were tested for genotypic resistance using two distinct line probe assays, LiPA HIV-1 reverse transcriptase RT and LiPA HIV-1 protease assay. Of the 80 patients, 82.5% showed resistance to at least one antiretroviral molecule. In the RT gene, resistance to nucleoside RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) were recognized in 66.25 and 37.5%, respectively, with M184V, T215Y and K103N being the codons most frequently involved. Resistance to protease inhibitors (PIs) was found in 46.25% of cases. Despite the presence of different mutations, the viral variants were still susceptible to other RTIs and PIs that are currently not available in Tunisia. Thus, alternative therapeutic options exist but are not yet accessible.

Identification of genetic defects underlying FVII deficiency in 10 patients belonging to eight unrelated families of the North provinces from Tunisia

Inherited factor VII (FVII) deficiency is a rare disorder characterized by a bleeding phenotype varying from mild to severe. To date, more than 200 mutations have been described along the F7 gene encoding for FVII. The aim of this study was the identification of genetic defects underlying FVII deficiency in 10 patients belonging to eight unrelated families of the North provinces from Tunisia. Mutation detection was performed by sequencing the whole F7 gene coding region, exon-intron boundaries and about 400 bp of the promoter region. We identified 5 mutations in five unrelated families; the novel p.F328Y mutation and the reported mutations: p.R304Q, p.M298I, IVS1aG > A and p.G-39G. For the remaining 5 patients we didn’t identified any mutations using PCR/Sequencing protocol. In conclusion, this study represents the first comprehensive molecular series of FVII deficiency affected patients in Tunisia from the North. We will try in the future to continue the molecular study for Tunisian patients from Center and South provinces in order to have a complete idea about the FVII deficiency mutational profile in our country.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1288044089753085

Structural analysis of two novel mutations in MCFD2 gene causing combined coagulation factors V and VIII deficiency

Combined factor V and factor VIII deficiency (F5F8D) is a rare autosomal recessive bleeding disorder reported usually in the context of consanguinous marriage. F5F8D is characterized by mild-tomoderate bleeding and coordinate reduction in plasma FV and FVIII levels, as well as platelet FV level (OMIM 227300) [1]. The disease is caused by mutations in genes encoding lectin mannose binding protein (LMAN1) and multiple coagulation factor deficiency 2 (MCFD2), which are the components of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC-53) involved in the FV and FVIII intracellular transport [1, 2]. LMAN1 is a type-I integral membrane protein that was first described as a 53-kDa marker of the ERGIC [3], whereas MCFD2 is a soluble luminal protein

Structural insight into the binding complex: β-arrestin/CCR5 complex

The chemokine receptor 5 (CCR5) belongs to the superfamily of serpentine G protein-coupled receptors (GPCRs). The DRY motif (Asp, Arg, Tyr) of the intracellular loop 2 (ICL2), which is highly conserved in the GPCRs has been shown to be essential for the stability of folding of CCR5 and the interaction with β-arrestin. But the molecular mechanism by which it recognizes and interacts with β-arrestin has not been elucidated. In the present study, we described the active state of the β-arrestin structure using normal mode analysis and characterized the binding cleft of CCR5-ICL2 with β-arrestin using SABRE© docking tool and molecular dynamics simulation. Based on our computational results, we proposed a mode of binding between the ICL2 loop of CCR5 and β-arrestin structure, and modeled the energetically stable β-arrestin/CCR5 complex. In view of CCR5’s importance as a therapeutic target for the treatment of HIV, this observation provides novel insight into the β-arrestin/CCR5 pathway. As a result, the current computational study of the detailed β-arrestin/CCR5 binding complex could provide the rationale for the development of next generation of HIV peptide inhibitors as therapeutic agents.

Identification of novel and recurrent mutations in Tunisian haemophilia B patients

Haemophilia B disease is a recessively inherited X-linked bleeding disorder which results from deficiency of factor IX (F9). Haemophilia B has a frequency of approximately 1 in 25 000 men worldwide [1]. Haemophilia B results from heterogeneous mutations spread throughout the F9 gene [2]. According to the World Federation of Hemophilia Report on the annual global survey 2007, 51 haemophilia B in Tunisia have been reported [3]. In this first study on Tunisian haemophilia B, we report the molecular analysis of 16 unrelated haemophilia B families. Patients involved in this study were from the Hemophilia Treatment Center, Aziza Othmana hospital, Tunisia. Informed consent was obtained from all patients. Molecular analysis was performed using the following strategy: polymerase chain reactions for the entire coding sequence of the F9 gene were prepared as described previously [4]. The mutation detection protocol was performed by dHPLC on a WAVE DNA Fragment Analysis System (Transgenomics, San Jose, USA). Altered profiles detected by dHPLC were sequenced using ABI Dye Terminator Cycle Sequencing (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) and analysed using a capillary sequencer Genetic Analyser ABI PRISM310 (Perkin-Elmer Applied Biosystems, Foster City, CA, USA [4]. Results were analysed using BLAST (http://www.ncbi.nlm. nih.gov/blast) program against the normal F9 gene sequence (GenBank Accession No. K02402) and the mutations were compared with the haemophilia B mutation database (http://www.umds.ac.uk/molgen). To evaluate the nature of missense mutations, we used PolyPhen (Polymorphism Phenotyping) (http://genetics.bwh.harvard.edu/pph). Our cohort is composed of 30 patients belonging to 16 unrelated families who represent 60% of total haemophilia B Tunisian population. A total of 15 different mutations were detected (Table 1), except for one family that did not show any mutations. In addition, the polymorphism g.20421A>G in exon 6 was also identified in five families. Five novel mutations were identified in five patients including 2 missense mutations, 1 nonsense mutation, 1 splice site mutation and one small deletion. For patient Hb 2, a deletion of CAG sequence from the 17795 to 17797 position inducing the loss of the last acid Ala173 in exon 5 (The numbering of the amino acids is according to the Swiss-Prot PZES (P00740)). For patient Hb10, a T to A substitution at nucleotide position 113 which changes a Cys acid in a codon stop (Cys27X) in exon 1, which will result in nonsense-mediated RNA decay and produce a severe phenotype as no protein will be translated, has been revealed. Patient Hb12 shows an acceptor splice substitution at the position 10507 in intron 4 (+2T > C). For Patient Hb14, a substitution of T to A at the position 31286 in exon 8, change the Cys 435 to Ser. Patient Hb16 present a substitution of G to A at the position 30932 in exon 8, which change Ala 317 to Thr. Replacement of a non-polar amino acid residue by a polar one is likely to affect the function, secretion or stability of the protein. Using PolyPhen these two mutations are predicted to be probably damaging with a score of 1.000 and 0.995 respectively. The question of whether these two candidate mutations Cys435Ser and Ala317Thr are pathogenic and alter the three-dimensional structure and function of F9 protein needs further investigation. However, as the latter mutations along with, Ala173Del, Ala317Thr and Cys435Ser occurred at amino acid residues highly conserved among different species, they may be involved in the F9 destabilization. Compared with previously published reports [5], we found that the two deletions identified in our patients bearing a severe disease. However, in our patient cohort the two nonsense mutations were associated with different phenotypes, severe and moderate disease respectively in patient Hb17(FIX:C level of 4) which is at variance with the majority of entries on the haemophilia B database for this mutation (which cite FIX:C and antigen levels of <1 for most of the 56 examples). Our observation in Hb17 is actually the exception for this particular mutation and for most nonsense mutations in general. In patient Hb3, we could not detect any mutations in the F9 gene using first dHPLC (no altered profile was observed) then sequencing. It is possible that this family might have pathological translocation, duplication or inversion in the factor IX gene leading to the disease. Further investigation is needed. To our knowledge this study is the first comprehensive molecular analysis of haemophilia B patients in Tunisia. Five novel mutations were identified and our data are globally in agreement with other reports in the international database. When requested, the data obtained from this study will be used for carrier testing and prenatal diagnosis. The identification of the mutations can also be used to estimate the risk of inhibitor development. It can also be valuable when planning future studies including gene therapy.

Structural insight into a novel human CCR5-V130I variant associated with resistance to HIV-1 infection.

We report the identification of a novel CC chemokine receptor 5 (CCR5) variant that seems associated with resistance to HIV-1 infection. The V130I mutation of the CCR5 receptor is located in the intracellular loop ICL2 known as DRY box and described in the literature as a nonsynonymous mutation present in nonhuman primates group. Extensive molecular modeling and dynamics simulations were performed to elucidate the mechanism by which the V130I mutation may induce conformational change of the CCR5 folding protein and prevent the interaction with the β-arrestin protein. Our study provides new mechanistic insight into how a specific mutation in the regulatory domain of CCR5 might alter the structural folding of the DRY box and the possible ICL2 loop binding with the β-arrestin protein, as described in our previous computational study. The results from our large-scale simulations complement recent experimental results and clinical features and offer useful insights into the mechanism behind CCR5 protein folding and signal transduction. In order for HIV, the entry of the virus to the cells must fuse with the CCR5 receptor that sits on the surface of T-helper immune cells. The described V130I mutation in the gene encoding the CCR5 protein may results in a defective CCR5-Arrestin binding complex that blocks entry of the virus.