Mutation spectrum of primary hyperoxaluria type 1 in Tunisia: Implication for diagnosis in North Africa

Background

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive inherited metabolic disease, characterized by progressive kidney failure due to renal deposition of calcium oxalate. Mutations in the AGXT gene, encoding the liver-specific enzyme alanine glyoxylate aminotransferase, are responsible for the disease. We aimed to determine the mutational spectrum causing PH1 and to provide an accurate tool for diagnosis as well as for prenatal diagnosis in the affected families.

Methods

Direct sequencing was used to detect mutations in the AGXT gene in DNA samples from 13 patients belonging to 12 Tunisian families.

Results

Molecular analysis revealed five mutations causing PH1 in Tunisia. The mutations were identified along exons 1, 2, 4, 5 and 7. The most predominant mutations were the Maghrebian “p.I244T” and the Arabic “p.G190R”. Furthermore, three other mutations characteristic of different ethnic groups were found in our study population. These results confirm the mutational heterogeneity related to PH1 in Tunisian population. All the mutations are in a homozygous state, reflecting the high impact of endogamy in our population.

Conclusion

Mutation analysis through DNA sequencing can provide a useful first line investigation for PH1. This identification could provide an accurate tool for prenatal diagnosis, genetic counseling and screen for potential presymptomatic individuals.     

Varied tolerance to NaCl salinity is related to biochemical changes in two contrasting lettuce genotypes

Salt stress perturbs a multitude of physiological processes such as photosynthesis and growth. To understand the biochemical changes associated with physiological and cellular adaptations to salinity, two lettuce varieties (Verte and Romaine) were grown in a hydroponics culture system supplemented with 0, 100 or 200 mM NaCl. Verte displayed better growth under 100 mM NaCl compared to Romaine, but both genotypes registered relatively similar reductions in growth under 200 mM NaCl treatment. Both varieties showed differences in net photosynthetic activity in the absence of salt and 8 days after salt treatment. These differences diminished subsequently under prolonged salt stress (14 days). Verte showed enhanced leaf proline and restricted total cations especially Na⁺, lesser malondialdehyde (MDA) formation and lignification in the roots under 100 mM NaCl salinity. Membrane damage estimated by electrolyte leakage increased with elevated salt concentrations in roots of both varieties, but Verte had significantly lower electrolyte leakage relative to Romaine under 100 mM NaCl. Moreover, Verte also accumulated greater levels of carotenoids under increasing NaCl concentrations compared to Romaine. Taken together, these findings suggest that the greater tolerance of Verte to 100 mM NaCl is related to the more restricted accumulation of total cations and toxic Na⁺ in the roots and enhanced levels of antioxidative metabolites in root and leaf tissue.     

Expression and functionality of the Na+/myo-inositol cotransporter SMIT2 in rabbit kidney

Myo-inositol (MI) is involved in several important aspects of cell physiology including cell signaling and the control of intracellular osmolarity i.e. by serving as a "compatible osmolyte". Currently, three MI cotransporters have been identified: two are Na(+)-dependent (SMIT1 and SMIT2) and one is H(+)-dependent (HMIT) and predominantly expressed in the brain. The goal of this study was to characterize the expression of SMIT2 in rabbit kidney and to compare it to SMIT1. First, we quantified mRNA levels for both transporters using quantitative real-time PCR and found that SMIT1 was predominantly expressed in the medulla while SMIT2 was mainly in the cortex. This distribution of SMIT2 was confirmed on Western blots where an antibody raised against a SMIT2 epitope specifically detected a 75 kDa protein in both tissues. Characterization of MI transport in brush-border membrane vesicles (BBMV), in the presence of d-chiro-inositol and l-fucose to separately identify SMIT1 and SMIT2 activities, showed that only SMIT2 is expressed at the luminal side of proximal convoluted tubules. We thus conclude that, in the rabbit kidney, SMIT2 is predominantly expressed in the cortex where it is probably responsible for the apical transport of MI into the proximal tubule.     

Mutation in OsLMS, a gene encoding a protein with two double-stranded RNA binding motifs, causes lesion mimic phenotype and early senescence in rice (Oryza sativa L.)

The rice (Oryza sativa L.) lesion mimic and senescence (lms) EMS-mutant, identified in a japonica cultivar Hitomebore, is characterized by a spontaneous lesion mimic phenotype during its vegetative growth, an accelerated senescence after flowering, and enhanced resistance to rice blast (Magnaporthe oryzae). To isolate the OsLMS gene, we crossed the lms mutant to Kasalath (indica), and used mutant F(2) plants to initially map the candidate region to about 322-kb on the long arm of chromosome 2. Illumina whole-genome re-sequencing of the mutant and aligning the reads to Hitomebore reference sequence within the candidate region delineated by linkage analysis identified a G to A nucleotide substitution. The mutation corresponded to the exon-intron splicing junction of a novel gene that encodes a carboxyl-terminal domain (CTD) phosphatase domain and two double stranded RNA binding motifs (dsRBM) containing protein. By PCR amplification, we confirmed that the mutation causes splicing error that is predicted to introduce a premature stop codon. RNA interference (RNAi) transgenic lines with suppressed expression of LMS gene exhibited the lesion mimic phenotype, confirming that the mutation identified in LMS is responsible for the mutant phenotype. OsLMS shares a moderate amino-acid similarity to the Arabidopsis FIERY2/CPL1 gene, which is known to control many plant processes such as stress response and development. Consistence with this similarity, the lms mutant shows sensitivity to cold stress at the early growth stage, suggesting that LMS is a negative regulator of stress response in rice.     

Founder mutations in Tunisia: implications for diagnosis in North Africa and Middle East

ABSTRACT: Background Tunisia is a North African country of 10 million inhabitants. The native background population is Berber. However, throughout its history, Tunisia has been the site of invasions and migratory waves of allogenic populations and ethnic groups such as Phoenicians, Romans, Vandals, Arabs, Ottomans and French. Like neighbouring and Middle Eastern countries, the Tunisian population shows a relatively high rate of consanguinity and endogamy that favor expression of recessive genetic disorders at relatively high rates. Many factors could contribute to the recurrence of monogenic morbid trait expression. Among them, founder mutations that arise in one ancestral individual and diffuse through generations in isolated communities. Method We report here on founder mutations in the Tunisian population by a systematic review of all available data from PubMed, other sources of the scientific literature as well as unpublished data from our research laboratory. Results We identified two different classes of founder mutations. The first includes founder mutations so far reported only among Tunisians that are responsible for 30 genetic diseases. The second group represents founder haplotypes described in 51 inherited conditions that occur among Tunisians and are also shared with other North African and Middle Eastern countries. Several heavily disabilitating diseases are caused by recessive founder mutations. They include, among others, neuromuscular diseases such as congenital muscular dystrophy and spastic paraglegia and also severe genodermatoses such as dystrophic epidermolysis bullosa and xeroderma pigmentosa. Conclusion This report provides informations on founder mutations for 73 genetic diseases either specific to Tunisians or shared by other populations. Taking into account the relatively high number and frequency of genetic diseases in the region and the limited resources, screening for these founder mutations should provide a rapid and cost effective tool for molecular diagnosis. Indeed, our report should help designing appropriate measures for carrier screening, better evaluation of diseases burden and setting up of preventive measures at the regional level.     

Contribution of redox status to hepatitis C virus E2 envelope protein function and antigenicity

Disulfide bonding contributes to the function and antigenicity of many viral envelope glycoproteins. We assessed here its significance for the hepatitis C virus E2 envelope protein and a counterpart deleted for hypervariable region-1 (HVR1). All 18 cysteine residues of the antigens were involved in disulfides. Chemical reduction of up to half of these disulfides was compatible with anti-E2 monoclonal antibody reaction, CD81 receptor binding, and viral entry, whereas complete reduction abrogated these properties. The addition of 5,5'-dithiobis-2-nitrobenzoic acid had no effect on viral entry. Thus, E2 function is only weakly dependent on its redox status, and cell entry does not require redox catalysts, in contrast to a number of enveloped viruses. Because E2 is a major neutralizing antibody target, we examined the effect of disulfide bonding on E2 antigenicity. We show that reduction of three disulfides, as well as deletion of HVR1, improved antibody binding for half of the patient sera tested, whereas it had no effect on the remainder. Small scale immunization of mice with reduced E2 antigens greatly improved serum reactivity with reduced forms of E2 when compared with immunization using native E2, whereas deletion of HVR1 only marginally affected the ability of the serum to bind the redox intermediates. Immunization with reduced E2 also showed an improved neutralizing antibody response, suggesting that potential epitopes are masked on the disulfide-bonded antigen and that mild reduction may increase the breadth of the antibody response. Although E2 function is surprisingly independent of its redox status, its disulfide bonds mask antigenic domains. E2 redox manipulation may contribute to improved vaccine design.     

Antifungal mechanism of the combination of Cinnamomum verum and Pelargonium graveolens essential oils with fluconazole against pathogenic Candida strains

The present study aimed to investigate the anti-Candida activity of ten essential oils (EOs) and to evaluate their potential synergism with conventional drugs. The effect on secreted aspartic protease (SAP) activity and the mechanism of action were also explored. The antifungal properties of essential oils were investigated using standard micro-broth dilution assay. Only Cinnamomum verum, Thymus capitatus, Syzygium aromaticum, and Pelargonium graveolens exhibited a broad spectrum of activity against a variety of pathogenic Candida strains. Chemical composition of active essential oils was performed by gas chromatography-mass spectrometry (GC-MS). Synergistic effect was observed with the combinations C. verum/fluconazole and P. graveolens/fluconazole, with FIC value 0.37. Investigation of the mechanism of action revealed that C. verum EO reduced the quantity of ergosterol to 83%. A total inhibition was observed for the combination C. verum/fluconazole. However, P. graveolens EO may disturb the permeability barrier of the fungal cell wall. An increase of MIC values of P. graveolens EO and the combination with fluconazole was observed with osmoprotectants (sorbitol and PEG6000). Furthermore, the combination with fluconazole may affect ergosterol biosynthesis and disturb fatty acid homeostasis in C. albicans cells as the quantity of ergosterol and oleic acid was reduced to 52.33 and 72%, respectively. The combination of P. graveolens and C. verum EOs with fluconazole inhibited 78.31 and 64.72% SAP activity, respectively. To our knowledge, this is the first report underlying the mechanism of action and the inhibitory effect of SAP activity of essential oils in synergy with fluconazole. Naturally occurring phytochemicals C. verum and P. graveolens could be effective candidate to enhance the efficacy of fluconazole-based therapy of C. albicans infections.     

Apoptose et ségrégation méiotique dans les spermatozoïdes d'hommes porteurs de translocations

Men with a chromosomal translocation produce a significant percentage of unbalanced spermatozoa. In order to determine a correlation between chromosomal anomalies and apoptosis in human sperm, we analysed DNA fragmentation and meiotic segregation in sperm from men with a (13;14) Robertsonian translocation. We studied sperm from 12 (13;14) translocation carriers and 9 proven fertile men with a normal karyotype. Meiotic segregation of chromosomes 13 and 14 was analysed using dual-colour fluorescencein situ hybridization with locus-specific probes for chromosomes 13 and 14. Apoptosis in spermatozoa was measured byin situ TUNEL assay. The meiotic segregation study showed a significantly increased frequency of unbalanced spermatozoa for chromosomes 13 and 14 in (13;14) carriers (15.9%) compared to the control population (1.3%) (p=0.00016). The study of apoptosis showed an increase of DNA fragmentation in (13;14) carriers (34.9%) compared to the control population (13.8%) (p=0.0036). This increased apoptosis was observed in spermatozoa presenting an increase of unbalanced chromosomal anomalies concerning chromosomes 13 and 14, but with a predominance of balanced spermatozoa compared to the theoretical risk of meiotic segregation. These results suggest that apoptosis could be involved as a regulatory mechanism to eliminate unbalanced chromosomal spermatozoa in men with a (13;14) Robertsonian translocation.