[COPD characteristics in subjects carrying the rare alpha-1 antitrypsin variant PIMMmalton]. / Aspects de la BPCO chez les porteurs de la mutation déficitaire rare de l'alpha-1 antitrypsine PIMMmalton.

INTRODUCTION: Several studies have investigated the clinical feature of COPD in subjects carrying the common alpha-1 antitrypsin deficiency mutations PIS and PIZ. However, there are few data on COPD due to rarer deficient variants. In this study, we aimed to explore the features of COPD in subjects carrying the PIMMmalton mutation, which is the most prevalent alpha-1 antitrypsin variant in Tunisia. MATERIAL AND METHODS: Five individuals, heterozygous for PIMMmalton were analyzed and compared to 97 non-deficient COPD patients. Demographic data as well as clinical and functional outcomes from subjects were collected. Blood gases and plasma alpha-1 antitrypsin levels were recorded. RESULTS: PIMMmalton subjects did not show any significant difference in terms of predicted FEV1 (35±13.2%), predicted forced vital capacity (34.2±9.6%) and FEV1 decline (148.6±114mL/year) compared to usual COPD patients (respectively 41.7±17.2%, P=0.500; 43.8±18.8%, P=0.300; 197.9±191mL/year, P=0.800). However, PaO2 was significantly reduced in PIMMmalton subjects (58.8±4.0mmHg) compared to usual COPD (69.9±10.6mmHg; P=0.029) and those patients with chronic bronchitis and centrolobular emphysema (71.0±10.9mmHg; P=0.038). CONCLUSION: PIMMmalton subjects were significantly hypoxic, similar to that observed in PiZZ homozygous rather than observed in heterozygous individuals.

A novel deletion in ZBTB24 in a Lebanese family with immunodeficiency, centromeric instability, and facial anomalies syndrome type 2.

The immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is a rare autosomal recessive disease characterized by targeted chromosome breakage, directly related to a genomic methylation defect. It manifests with phenotypic and clinical variability, with the most consistent features being developmental delay, facial anomalies, cytogenetic defects and immunodeficiency with a reduction in serum immunoglobulin levels. From the molecular point of view, ICF syndrome was always divided into ICF type I (ICF1) and ICF type 2 (ICF2). Mutations in DNMT3B gene are responsible for ICF1, while mutations in ZBTB24 have been reported to be responsible for ICF2. In this study, we describe a Lebanese family with three ICF2 affected brothers. Sanger sequencing of the coding sequence of ZBTB24 gene was conducted and revealed a novel deletion: c.396_397delTA (p.His132Glnfs*19), resulting in a loss-of-function of the corresponding protein. ZBTB24 belongs to a large family of transcriptional factors and may be involved in DNA methylation of juxtacentromeric DNA. Detailed molecular and functional studies of the ZBTB24 and DNMT3B genes are needed to understand the pathophysiology of ICF syndrome.

Relationship between glutathione S-transferase P1 polymorphisms and chronic obstructive pulmonary disease in a Tunisian population.

Chronic obstructive pulmonary disease (COPD) is a multifactorial disease with possible genetic predisposition and involvement of various environmental factors. Several candidate genes have been reported as potentially associated with this lung disease. The glutathione S-transferase P1 gene (GSTP1) was proposed to be involved in susceptibility to develop COPD. It belongs to the GST family, which is a group of phase II enzymes that catalyze the glutathione conjugation of many endogenous and exogenous electrophilic compounds, such as carcinogens, therapeutic drugs, environmental toxins, and oxidative stress products. We conducted a case-control study to investigate genetic polymorphisms of this enzyme [exon 5 (Ile105Val) and exon 6 (Ala114Val)] in 234 unrelated COPD cases and 182 healthy controls from a Tunisian population. Genotyping was carried out using polymerase chain reaction and restriction fragment length polymorphism methods. GSTP1 Ala114/Val114 and Val114/Val114 genotypes were not found in either patients or healthy controls. However, there were differences in the distribution of various exon 5 GSTP1 genotypes between COPD patients and healthy controls. GSTP1 Val105/Val105 was significantly more common in patients compared to controls (OR = 2.67; 95%CI = 1.45-4.92; P = 0.0013). Multivariate logistic regression analysis confirmed a significant relationship between the mutant genotype and COPD (OR = 2.58; 95%CI = 1.31-5.09; P = 0.026), after adjustment for classic risk factors. Analysis of variance showed no correlation between age, body-mass index, pack-years, percentage of predicted FEV1 values, and any of the GSTP1 genotypes. We conclude that subjects with GSTP1 Val105 allele are at higher risk of COPD.

[Protective effect of diclofenac towards the oxidative stress induced by paracetamol toxicity in rats]. / Effet protecteur du diclofénac contre le stress oxydatif induit par la toxicité du paracétamol chez le rat.

Association of paracetamol (PARA) and diclofenac (DiCF) is the aim of our study. 60 male rats "Albinos wistar" were treated by oral gavage (per os) during seven days. A control group was treated by mineral water (0+0) mg/kg and a second group was treated with only a toxic dose of 100 mg/kg of PARA (100+0). Remaining lots were treated with a combination of different toxic doses of PARA and a therapeutic dose of DiCF (15+3, 100+3, 200+3 and 400+3) mg/kg. Plasma concentration of amiotransferases (ASAT, ALAT), alkalines phosphatase (ALP), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione (GSH), glucose, cholesterol, creatinin, direct and total bilirubin, significantly varied in the treated rats regarding to the witness's rats. The toxicity of PARA revealed by a dose dependant blood increases of ASAT, ALAT, ALP, GPx, GR, glucose, creatinin, bilirubin, and by decreases of cholesterol concentration and tissue GSH in comparisons to controls. The depletion of GSH and the increase of the oxidative stress enzymes (GPx and GR) suggest a detoxification function of the glutathione system. The association (PARA + DiCF) revealed a protective effect, resulting in the increase of the concentrations of ASAT, ALAT, ALP, GPx, GR, bilirubin and the increase of GSH. Regarding to all these results, it has been suggested that DiCF has a protective action towards the toxic effects of PARA.

[Biochemical and molecular diagnosis of alpha 1 antitrypsin deficiency in a Tunisian family]. / Diagnostic biochimique et moléculaire du déficit en alpha 1 antitrypsine dans une famille tunisienne.

Our study investigated alpha 1 antitrypsin deficiency (AATD) diagnosis in a family originated from central Tunisia and showing a familial history of asthma. Biochemical and genetic diagnosis for AATD was performed according to current diagnostic standards. AAT level quantification in affected individuals showed plasma AAT levels consistent with intermediate AATD (ranged from 0.91 to 1.04 g/L). The molecular analysis was assessed using the genotyping of the most prevalent PI*S and PI*Z SERPINA1 mutations and the sequencing of AAT coding exons for rare AATD variants detection. No PI*S or PI*Z deficient variants were seen in this family. Sequencing results showed the inheritance of the deficient rare variant PI*M(wurzburg) (P369S) at the heterozygous state in the mother and two affected siblings. However, AATD status remains unexplained in the third affected case, with no mutations detected in the AAT coding exons.

[Alpha 1 antitrypsin polymorphism associated to asthma and emphysema in a central Tunisian population]. / Polymorphisme de l'alpha 1 antitrypsine associé à l'asthme et l'emphysème pulmonaire dans une population du centre tunisien.

INTRODUCTION: more than 100 alleles have been described on the alpha 1 antitrypsin gene. Normal variants (PiM1, PiM2 and PiM3) encodes AAT molecules which are different but functional and normally secreted. The more frequent risk variants are PiS and PiZ. In this study, an AAT polymorphism analysis in correlation with pulmonary diseases was conducted. MATERIAL AND METHODS: analyses were performed on 96 asthmatics, 67 emphysema cases and 318 control subjects. Alpha 1 antitrypsin phenotypes were studied by quantitative determination of AAT concentration and isoelectrofocusing. Genotyping was performed by RFLP PCR. RESULTS: PiM1, PiM2, PiM3, PiS and PiZ allelic frequencies were calculated (0.7395, 0.2291, 0.0156, 0.0104, 0.0052 in asthmatics; 0.7547, 0.1716, 0.0298, 0.0298, 0.0149 in emphysema patients and 0.8030, 0.1525, 0.0408, 0.006, 0.0000 in controls, respectively). Results showed an increase in PiM2 allele frequencies in both patients' groups compared to controls. Allelic frequencies difference is significant only with the asthmatic group (p=0,0179). PiS and PiZ deficiency alleles are more prevalent in the emphysema (0.0298, 0.0149) than in the asthmatic subjects (0.0104, 0.0052). Meanwhile, no significant difference in PiS and PiZ allelic frequencies was observed between patients and controls. CONCLUSION: PiM2 allele can be considered as genetic risk factor for asthma. PiS and PiZ alleles are very rare in Tunisia in comparison with the European population, leading to a very small contribution in pulmonary diseases pathogenesis in Tunisia.

Alpha 1 antitrypsin polymorphism in the Tunisian population with special reference to pulmonary disease.

OBJECTIVES: The study investigated alpha 1 antitrypsin (AAT) gene polymorphism in the Tunisian population. We aimed to analyze the correlation between Pi polymorphism and the risk of developing chronic obstructive pulmonary disease (COPD). PATIENTS AND METHODS: We focused our study on two samples originating from the Tunisian centre: 318 healthy controls and 90 patients suffering from COPD. Data analysis was investigated by AAT level quantification, serum isoelectric focusing (IEF) and RFLP-PCR performed with PiS and PiZ allele specific primers. RESULTS: We calculated PiM1, PiM2, PiM3, PiS and PiZ allele frequencies in patients and controls. The difference in allele frequencies is significant only for the PiM2 allele (P=0.00378). In COPD patients, we note the presence of PiZ allele. This allele mainly observed in European populations, is rare in sub-Saharian populations and not described in North Africa. CONCLUSION: PiZ allele is found in COPD sample and never in Tunisian controls. However, no significant difference in PiZ allele frequency between patients and controls can be concluded. PiM2 allele, which is considered as "normal" variant can be associated with COPD risk.

[C3 complement polymorphism in patients suffering from obstructive chronic bronchopneumopathy in Tunisia]. / Polymorphisme du C3 du complément chez des patients atteints de bronchopneumopathie chronique obstructive en Tunisie.

We analysed the C3*S and C3*F polymorphism of the third component of the complement (C3), first at the protein level by the electrophoresis of the plasma on agarose gel and second on the gene level by the ARMS PCR technique. We determined the phenotypic and genotypic frequencies of the C3 on a sample of 90 patients suffering from the obstructive chronic bronchopneumopathy (OCBP) disease. Comparisons have been done with frequencies observed on a control sample of 437 healthy individuals from the Tunisian population in order to establish a putative correlation between the polymorphism studied and the disease. Frequencies of the C3*S and C3*F alleles in OCBP patients are 0,788 and 0,212 respectively. They are not significantly different from those observed in control sample (0,834 and 0,152 respectively). Therefore, no correlation is observed between the C3 polymorphism and the risk of developing the OCBP disease.

HLA-C molecular characterization of a Lebanese population and genetic structure of 39 populations from Europe to India-Pakistan.

Lebanon is located at a continental crossroad between Europe, Africa, and Asia. This region has been the center of wide-scale movements of populations as well as the theater of genetic and cultural trade off among neighboring populations. In this study, HLA-C alleles were characterized by a PCR-SSOP (sequence-specific oligonucleotide probes) hybridization protocol in a sample of 97 Lebanese. A total of 23 alleles were identified with four predominant, Cw*0401, Cw*0602, Cw*0701/06, and Cw*1203, accounting for almost 60% of HLA-C allele frequencies. We included the Lebanese data into a broad analysis of the HLA-C genetic structure of a large set of populations located in Europe, the Middle East, and the Indian subcontinent. Our results indicate that Lebanese exhibit an intermediate genetic profile among the populations from the Middle East, which constitute a rather homogeneous genetic group. In Europe, a high correlation coefficient is found between genetic and geographic distances. In this continent, we also identified a significant genetic frontier following a north-east to south-west axis. This frontier cuts through the Alps and the Pyrenees, thus separating the north-western European populations from those located in the eastern and Mediterranean areas. Finally, the populations from India - Pakistan are very heterogeneous, particularly the Dravidians. Their differentiation has probably been caused by rapid genetic drift under complex influences of cultural, linguistic, and/or religious barriers. Overall, the results show that the HLA-C genetic patterns of these three geographic regions, i.e., the Middle East, Europe, and India-Pakistan, have been shaped by very different genetic histories.

Population structure in the Mediterranean basin: a Y chromosome perspective.

The Mediterranean region has been characterised by a number of pre-historical and historical demographic events whose legacy on the current genetic landscape is still a matter of debate. In order to investigate the degree of population structure across the Mediterranean, we have investigated Y chromosome variation in a large dataset of Mediterranean populations, 11 of which are first described here. Our analyses identify four main clusters in the Mediterranean that can be labelled as North Africa, Arab, Central-East and West Mediterranean. In particular, Near Eastern samples tend to separate according to the presence of Arab Y chromosome lineages, suggesting that the Arab expansion played a major role in shaping the current genetic structuring within the Fertile Crescent.

Local application of IGF1 on dental pulp mechanically exposed; in vivo study on rabbit.

IGF1 (Insulin Growth Factor, 1) was intentionally applied onto pulp tissues, aiming to provoque a dentine regeneration process through the stimulation of the dentinoblasts' potententials. 72 cavities were hence performed on rabbit molars, intentionally exposing the dental pulp. Different concentrations of IGF1 were then applied; The histo and anatomo-pathological observations showed persistent vitality of the pulp without any sign of necrosis, even 6 weeks after the IGF1 application. Dentinoblasts layers (as an indication of the regeneration activity) were counted, according to a pre-established protocol, at days 7, 14, 22, 28 and 42. The type of the applied IGF1, was carefully selected to be "Binding Protein Resistant" (IGF-BPR), so to avoid any inhibition of the IGF1 action by the endogenous binding proteins (Hochscheid and coll). The results were conclusive in indicating the IGF1 as an efficient dental pulp capping product.

Joining the pillars of Hercules: mtDNA sequences show multidirectional gene flow in the western Mediterranean.

Phylogenetic analysis of mitochondrial DNA (mtDNA) performed in Western Mediterranean populations has shown that both shores share a common set of mtDNA haplogroups already found in Europe and the Middle East. Principal co-ordinates of genetic distances and principal components analyses based on the haplotype frequencies show that the main genetic difference is attributed to the higher frequency of sub-Saharan L haplogroups in NW Africa, showing some gene flow across the Sahara desert, with a major impact in the southern populations of NW Africa. The AMOVA demonstrates that SW European populations are highly homogeneous whereas NW African populations display a more heterogeneous genetic pattern, due to an east-west differentiation as a result of gene flow coming from the East. Despite the shared haplogroups found in both areas, the European V and the NW African U6 haplogroups reveal the traces of the Mediterranean Sea permeability to female migrations, and allowed for determination and quantification of the genetic contribution of both shores to the genetic landscape of the geographic area. Comparison of mtDNA data with autosomal markers and Y-chromosome lineages, analysed in the same populations, shows a congruent pattern, although female-mediated gene flow seems to have been more intense than male-mediated gene flow.

Asymptomatic deficiency in the peptide transporter associated to antigen processing (TAP).

Human HLA class I deficiency is a rare disease which, in most of the patients described to date, results from a defect in subunit 1 or 2 of the peptide transporter associated with antigen processing (TAP). The clinical features of TAP deficiency include a chronic inflammation of the respiratory tract and/or granulomatous skin lesions. In this report, we describe two adult siblings with an HLA class I deficiency. One individual had only spontaneously-healing skin granulomatous lesions, while the second did not display any of the symptoms associated with HLA class I deficiency and could be considered to be healthy. We show that the patients display a homozygous TAP2 mutation which blocks the maturation of HLA class I molecules. Cell surface expression of these molecules is strongly reduced, but three times higher than on cells from other previously described TAP-deficient individuals. This higher expression results, at least in part, from the presence of HLA-B7 molecules which are probably empty of peptide. The numbers of CD8+ alphabeta T cells are almost normal in these patients. The anti-EBV T-cell response of one patient is mediated by HLA-B7 restricted CD8+ alphabeta T lymphocytes recognizing the BMRF1 nuclear EBV antigen, demonstrating that CD8+ alphabeta T cells can participate in anti-viral responses. This study shows that TAP deficiency can remain totally asymptomatic for several decades, and suggests that in some cases, TAP-independent immune responses provide efficient protection from most of the common intracellular pathogens.

Pathophysiology of limb girdle muscular dystrophy type 2A: hypothesis and new insights into the IkappaBalpha/NF-kappaB survival pathway in skeletal muscle.

Limb girdle muscular dystrophies (LGMDs) are a group of clinically heterogeneous genetic diseases characterized by progressive weakness and atrophy of scapular and pelvic muscles, with either a dominant or recessive autosomic mode of inheritance. The first symptoms of the disorder appear during the first 20 years of life and progresses gradually, and a walking disability develops 10-20 years later. The gene responsible for LGMD2A has been identified and encodes calpain 3, a protease expressed mainly in skeletal muscle. Apoptotic myonuclei were recently detected in muscular biopsy specimens of LGMD2A patients, and apoptosis was found to be correlated with altered subcellular distribution of inhibitory protein kappaBalpha (IkappaBalpha) and nuclear factor kappaB (NF-kappaB), resulting in sarcoplasmic sequestration of NF-kappaB. Calpain 3 dependent IkappaBalpha degradation was reconstituted in vitro, supporting a possible in vivo sequence of events leading from calpain 3 deficiency to IkappaBkappa accumulation, prevention of nuclear translocation of NF-kappaB, and ultimately apoptosis. Therefore calpain 3, present in healthy muscle as sarcoplasmic and nuclear forms, may control IkappaBalpha turnover and indirectly regulate NF-kappaB dependent expression of survival genes. Recent data reported from a new model of LGMD2A in mice and from other muscular disorders strengthen understanding of the molecular links between calpain 3 and the Ikappaalpha/NF-kappaB pathway. Finally, in light of the lack of apoptosis observed in inflammatory myopathies, a unifying model for the control of cell survival in muscle is proposed and discussed

Haplotype diversity and linkage disequilibrium at human G6PD: recent origin of alleles that confer malarial resistance.

The frequencies of low-activity alleles of glucose-6-phosphate dehydrogenase in humans are highly correlated with the prevalence of malaria. These "deficiency" alleles are thought to provide reduced risk from infection by the Plasmodium parasite and are maintained at high frequency despite the hemopathologies that they cause. Haplotype analysis of "A-" and "Med" mutations at this locus indicates that they have evolved independently and have increased in frequency at a rate that is too rapid to be explained by random genetic drift. Statistical modeling indicates that the A- allele arose within the past 3840 to 11,760 years and the Med allele arose within the past 1600 to 6640 years. These results support the hypothesis that malaria has had a major impact on humans only since the introduction of agriculture within the past 10,000 years and provide a striking example of the signature of selection on the human genome.

Familial Mediterranean fever in Lebanon: mutation spectrum, evidence for cases in Maronites, Greek orthodoxes, Greek catholics, Syriacs and Chiites and for an association between amyloidosis and M694V and M694I mutations.

Seventy-nine unrelated Lebanese patients were tested for 15 mutations in the MEFV gene: A761H, A744S, V726A, K695R, M694V, M694I, M694del, M6801 (G --> C), M680I (G --> A) in exon 10, F479L in exon 5, P369S in exon 3, T267I, E167D and E148Q in exon 2, using PCR digestion, ARMS, DGGE and/or sequencing. Mutations were detected in patients belonging to all communities, most interestingly the Maronite, Greek orthodox, Greek catholic, Syriac and Chiite communities. The most frequent mutations are M694V and V726A (27% and 20% of the total alleles respectively). M694I, E148Q and M680I mutations account respectively for 9%, 8% and 5%. Each of the K695R, E167D and F479L mutations was observed once and all the remaining mutations were not encountered. Of the alleles 33% do not carry any of the studied mutations. The mutation spectra, clinical features and severity of the disease differed among the Lebanese communities. The genotype-phenotype analysis showed a significant association (P < 0.001) between amyloidosis and the presence of mutations at codon 694 in exon 10 (both M694V and M694I). None of the patients carrying other mutations developed amyloidosis.

A worldwide analysis of AG molecular diversity inferred from serology.

Ten population samples from different geographic origins were tested serologically for the AG polymorphism of human beta-lipoproteins. Their haplotype frequencies were used with previously published data to perform a wide analysis of AG genetic differentiations throughout the world. Coancestry coefficients were computed from weighted F(ST)s among populations by using a matrix of molecular distances among AG haplotypes, which is here determined on the basis of DNA studies. Coancestry coefficients derived from unweighted F(ST)s and more classical Prevosti distances were computed on the same data and used for a comparison. In all cases a highly significant correlation was found between genetics and geography on a worldwide scale, while the significance of the correlation with linguistics differed. A test of significance of the pairwise F(ST)s among populations also gave different results depending on whether the molecular distance matrix among AG haplotypes was included. Globally, this study shows that in spite of being highly significantly correlated to each other, different genetic distance measures can lead to different interpretations of the same data set. Moreover, the elucidation of the molecular models related to the presently known serological polymorphisms may represent an additional tool for analyzing such polymorphisms in human population genetics studies.

Familial Mediterranean Fever: association of elevated IgD plasma levels with specific MEFV mutations.

Familial Mediterranean Fever (FMF) is a recessively inherited disorder, characterized by episodic fever, abdominal and arthritic pain, as well as other forms of inflammation. Some FMF patients present higher IgD serum levels, and it is not yet known whether such an elevation is related to specific genotypes or correlated with a specific phenotype. In order to evaluate the association between known FMF-related mutations and IgD levels in confirmed patients, as well as the correlation between those levels and the presence of specific clinical signs, genotypic analysis and IgD plasma measurements were performed for 148 Lebanese and Jordanian FMF patients. Most common mutational patterns were M694V heterozygotes (19%) and homozygotes (17%), and V726A heterozygotes (18%) and homozygotes (5%), with an additional 11% combining both mutations. Twenty-one patients had higher IgD levels (superior to 100 microg/ml). The risk for higher IgD levels was significantly associated with M694V homozygote status (OR = 6.25) but not with heterozygotic one (OR = 1). Similarly, the risk for higher IgD was also found with V726A homozygotes (OR = 2.2) but not with heterozygotes (OR = 1.05). The use of colchicine was not statistically associated with IgD levels. Clinically, hyper IgD was also found significantly associated with arthritis (OR = 18). Thus, homozygotic status for M694V, and to a lesser extent V726A, is associated with increased risk for higher IgD plasma levels, regardless of colchicine use. Elevated IgD plasma levels are also correlated with the severity of FMF manifestations, and especially with arthritis.