Evaluation of X chromosome inactivation in endemic Tunisian pemphigus foliaceus.

BACKGROUND: Almost 5% of the world's population develops an autoimmune disease (AID), it is considered the fourth leading cause of disability for women, who represent 78% of cases. The sex ratio when it comes to the most prevalent AID varies from 9:1 in systemic lupus erythematosus (SLE) to 13:1 in endemic Tunisian pemphigus foliaceus (PF). METHODS: To test the potential involvement of skewed x-inactivation in the pathogenesis of Tunisian PF, we analyzed the methylation status of a highly polymorphic CAG repeat in the androgen receptor gene and evaluated the x chromosome inactivation (XCI) patterns in peripheral blood-leukocyte-derived DNA samples of female patients with PF (n = 98) compared to healthy control (HC) subjects (n = 150), as well as female patients with SLE (n = 98) were enrolled as a reference group. RESULTS: XCI status was informative for 50 of the 98 PF patients (51%) and 70 of the 150 HC women (47%). Extremely skewed XCI patterns were more frequent in PF and SLEwomen than HC, but the difference was statistically significant only in women with SLE. No statistical difference was observed in XCI patterns between PF and SLE patients. PF phenotype-XCI correlation analysis revealed that (i) skewed XCI patterns may be involved in the disease's subtype and (ii) it was more pronounced in the endemic group than the sporadic one. Furthermore, preferential XCI showed an increase in heterozygote genotypes of PF's susceptibility polymorphisms in immunity-related X genes (FOXP3, AR, and TLR7) in PF patients compared to HC. CONCLUSION: Our results suggest that skewed XCI could lead to hemizygosity of X-linked alleles that might unmask X-linked deleterious alleles.

Role of innate immune receptors TLR4 and TLR2 polymorphisms in systemic lupus erythematosus susceptibility.

AIM: Through their recognition of various bacterial cell wall components, TLR2 and TLR4 participate in the innate response and modulate the activation of adaptive immunity. Therefore, the genetic background of these receptors might play a crucial role in autoimmune diseases such as systemic lupus erythematosus (SLE). In this study, we investigated the possible association between polymorphisms within TLR2 and TLR4 genes with SLE susceptibility. MATERIAL AND METHODS: A total of 100 SLE patients and 200 unrelated healthy controls of the Tunisian population were enrolled in the study.TLR4rs4986790, TLR4rs4986791, and TLR2rs5743708 genotyping were performed using a polymerase chain reaction-restriction fragment length polymorphism method. The number of guanine-thymine (GT) repeat microsatellite in the intron 2 of TLR2 gene was analyzed by sequencing. RESULTS: We reported a lack of allelic and genotypic association between SNPs of TLR4 and TLR2 genes and SLE pathogenesis. No correlation was found with any SLE features. However, SLE susceptibility was associated with the GT repeat microsatellite polymorphism in the human TLR2 gene. Further subclassification of alleles into three subclasses revealed a significant association between the long-sized repeats ((GT) >23) and SLE. CONCLUSION: Though the results showed the absence of genetic association of TLR4 and TLR2 SNPs with the risk of developing SLE, we have identified a protective association between the microsatellite polymorphism in intron 2 of the TLR2 gene and SLE. Functionally, these (GT)n repeats may confer modifying effects or susceptibility to certain inflammatory conditions.

Chromosome 2q33genetic polymorphisms in Tunisian endemic pemphigus foliaceus.

BACKGROUND: Several studies have suggested that polymorphisms within genes encoding T-lymphocyte immune regulating molecules: CD28, CTLA-4, and ICOS, may alter the signaling process and subsequently could be involved in susceptibility to a broad spectrum of autoimmune diseases. METHODS: This study aimed to replicate associations between common polymorphisms in the 2q33.2 cluster and susceptibility to pemphigus foliaceus (PF) in the Tunisian population. We investigated seven polymorphisms: rs3116496 and rs1879877 (CD28), rs231775, rs3087243, and (AT)n repeat (CTLA4); rs11889031 and rs10932029 (ICOS) in a case-control study which enrolled 106 Tunisian PF patients and 205 matched healthy controls. RESULTS: We confirmed the associations with CTLA4((AT)13 , p = 0.00137, OR = 3.96 and (AT)20 , p = 0.008, OR = 5.22; respectively) and ICOS genes (rs10932029>CT, p = 0.034, OR = 2.12 and rs10932029>TT, p = 0.04 and OR = 0.41). CONCLUSION: Our results indicate that susceptibility to PF is located in the proximal and the distal 3' flanking region of the CTLA4/ICOS promoter. These findings may open avenues to the treatment of patients with biological drugs targeting CTLA4/ICOS molecules, in a personalized manner to achieve more effective treatment.

Mutations in GAA Gene in Tunisian Families with Infantile Onset Pompe Disease: Novel Mutation and Structural Modeling Investigations.

Pompe disease, a rare, autosomal, recessive, inherited, lysosomal storage disorder, is caused by mutations in the acid α-glucosidase (GAA) gene leading to a deficiency of the lysosomal GAA enzyme. Some GAA mutations eliminate all enzymatic activities, causing severe infantile Pompe disease; others allow residual GAA activity and lead to middle adulthood forms. Here, we report a cohort of 12 patients, belonging to 11 unrelated families, with infantile Pompe disease. The mutational analysis of GAA gene revealed a novel c.1494G > A (p.Trp498X) mutation in one patient and three known mutatio,ns including the c.1497G > A (p.Trp499X) mutation, in two patients, the c.1927G > A (p.Gly643Arg) mutation in one patient and the common c.236_246del (p.Pro79ArgfsX13) mutation in eight patients. The high prevalence of c.236_246del mutation in our cohort (58%) was supported by the existence of a common founder ancestor that was confirmed by its segregation of similar SNPs haplotype, including four intragenic SNPs of GAA gene. In addition, a 3D structure model and a docking were generated for the mutant p.Gly643Arg using the crystal structure of human GAA as template and the 4-methylumbelliferyl-α-D-glucopyranoside as substrate. The results showed that the arginine at position 643 caused electrostatic changes in neighboring regions, leading to the repulsion between the amino acids located in the catalytic cavity of the GAA enzyme, thus restricting access to its substrate. These structural defects could cause the impairment of the transport and maturation previously reported for p.Gly643Arg mutation.

Association of HLA Alleles with Primary Sjögren Syndrome in the South Tunisian Population.

OBJECTIVE: In order to investigate human leukocyte antigen (HLA) genes predisposing to primary Sjögren syndrome (pSS), we conducted an association study using HLA loci (A, B, and DRB1) and 9 polymorphic microsatellite markers spanning the HLA region in pSS patients as compared to healthy individuals. SUBJECTS AND METHODS: Forty-four patients fitting the European criteria of pSS and 123 healthy controls were analyzed for their HLA class I and class II alleles. HLA class I typing was performed using a standard microlymphocytotoxicity method followed by PCR-SSP. HLA-DRB1 genotyping was performed using PCR-SSP. We studied the polymorphism of 9 microsatellite markers for both groups. Microsatellite genotyping was performed using the PCR fluorescent technique. RESULTS: We observed a positive association between HLA-B15 and pSS in the Tunisian population (p = 0.004, OR 7.57). The comparison of the frequencies of DRB1 alleles in pSS patients and controls confirmed the association of the DRB1*03 allele with pSS (p = 0.02, OR 2.36). On the other hand, the association study of microsatellite markers showed that the a9 allele of D6S265 marker and the a20 of C1.2.C were found to be positively associated with pSS as compared to controls (p =0.0003, OR 10.29, and p =0.001, OR 4.79, respectively). Using the "Haplo.stats" software analysis, we found that the most associated region was located in the HLA class I region and limited by HLA-A and D6S265 loci (p = 0.00056). CONCLUSION: The results of this study support the hypothesis of the existence of a susceptibility gene for pSS located in the HLA class I and III regions.

A Monoallelic Two-Hit Mechanism in PLCD1 Explains the Genetic Pathogenesis of Hereditary Trichilemmal Cyst Formation.

Trichilemmal cysts are common hair follicle-derived intradermal cysts. The trait shows an autosomal dominant mode of transmission with incomplete penetrance. Here, we describe the pathogenetic mechanism for the development of hereditary trichilemmal cysts. By whole-exome sequencing of DNA from the blood samples of 5 affected individuals and subsequent Sanger sequencing of a family cohort including 35 affected individuals, this study identified a combination of the Phospholipase C Delta 1 germline variants c.903A>G, p.(Pro301Pro) and c.1379C>T, p.(Ser460Leu) as a high-risk factor for trichilemmal cyst development. Allele-specific PCRs and cloning experiments showed that these two variants are present on the same allele. The analysis of tissue from several cysts revealed that an additional somatic Phospholipase C Delta 1 mutation on the same allele is required for cyst formation. In two different functional in vitro assays, this study showed that the protein function of the cyst-specific 1-phosphatidylinositol 4, 5-bisphosphate phosphodiesterase delta-1 protein variant is modified. This pathologic mechanism defines a monoallelic model of the two-hit mechanism proposed for tumor development and other hereditary cyst diseases.

Molecular Genotyping of Candida parapsilosis Species Complex.

BACKGROUND: The Candida parapsilosis complex species has emerged as an important cause of human disease. The molecular identification of C. parapsilosis isolates at the species level can be helpful for epidemiological studies and then for the establishment of appropriate therapies and prophylactic measures. METHODS: The present study was undertaken to analyze 13 short tandem repeat (STR) markers (7 minisatellites and 6 microsatellites) in a global set of 182 C. parapsilosis complex isolates from different origins including invasive and superficial clinical sites. RESULTS: Upon the analysis of 182 strains of C. parapsilosis complex species, 10-17 haplotypes were detected for each minisatellite marker. The combination of 7 minisatellite markers yielded 121 different genotypes with a 0.995 D value. Upon the analysis of 114 isolates (68 from invasive infections and 46 from superficial infections), 21-32 genotypes were detected for each microsatellite marker. The combination of all 13 markers yielded 96 different genotypes among 114 isolates with a high degree of discrimination (0.997 D value). The same multilocus genotype was shared by isolates recovered from some patients and from the hand of theirs correspondent healthcare worker. For another patient, the same multilocus genotype of C. metapsilosis was detected in blood and skin confirming that candidemia usually arises as an endogenous infection following prior colonization. CONCLUSIONS: These STR markers are a valuable tool for the differentiation of C. parapsilosis complex strains, to support epidemiological investigations especially studies of strain relatedness and pathways of transmission.

HLA Class III: A susceptibility region to systemic lupus erythematosus in Tunisian population.

BACKGROUND AND OBJECTIVES: Short tandem repeats (STR) are usually used as informative polymorphic markers for genetic mapping and for disease susceptibility analysis. The involvement of these microsatellite markers localized in the MHC region was reported in many auto-immune diseases. In this study we analyzed for the first time eight polymorphisms of microsatellite loci at the HLA region: D6S291, D6S273, TNFa, b and c, MICA, D6S265 and D6S276, in Tunisian systemic lupus erythematosus (SLE) patients. MATERIALS AND METHODS: We performed a case control study in which the microsatellite loci were amplified using specific primers labeled with NED, VIC, PET or 6-FAM and analyzed using GeneScan software 3.7. For the statistical analysis, we used SPSS software and we performed a sub-haplotype scoring test using the haplo.stats software developed in the R language. RESULTS: We found that two mean associated regions existed; the most statistically significant encompassed the 3 TNF markers (p = 0.0003, OR = 19.34); the latter covered the DR region. In fact, when scoring haplotypes in 3 marker- sliding windows, the p value increased as we moved away from the TNF region and decreased again when we approached the DRB1 locus. We also established for the first time the negative association between alleles of D6S291 and SLE. The majority of clinical and serological correlations were noted with TNF alleles. CONCLUSION: Our results confirm the association between TNF and DRB1 polymorphisms and SLE. The association between alleles of D6S291 and SLE needs however to be verified by the analysis of other markers beyond this region.

Human leukocyte antigens-DRB1*03 is associated with systemic lupus erythematosus and anti-SSB production in South Tunisia.

INTRODUCTION: Systemic lupus erythematosus (SLE) is an autoimmune disease with various presentations. This variation is due to the interaction of hormonal, environmental, and genetic factors. Associations between human leukocyte antigens and SLE have long been recognized in different ethnic populations and have been suggested to represent the most important association. OBJECTIVES: The objectives of this paper were to determine susceptibility and protection human leukocyte antigens (HLA) Class II markers for SLE and to highlight, for the first time, associations between HLA alleles and clinical and serological features in South Tunisia. METHODS: We conducted a case-control study on 75 SLE patients and 123 healthy controls. The HLA Class II DRB1/DQB1 of all patients and controls was genotyped using polymerase chain reaction-sequence specific primer technique. Statistical analysis was performed using SPSS software. RESULTS: HLA-DRB1*03 was the principal Class II allele associated with the genetic susceptibility to SLE (pc = 0.02; OR = 2.57; CI = [1.39-4.75]; this allele was also associated with anti-SSB production (P = 0.016; OR = 4.00; CI = [1.24-12.96]). HLA-DRB1*01 was significantly more expressed in SLE patients with neurologic disorders (P = 0.013; OR = 20.25; CI = [1.87-219.21]). No allele was found to be protective against SLE in our study group. CONCLUSION: Our results show that in South Tunisia SLE is associated with HLA-DRB1*03 and that some clinical features of SLE may be influenced by specific DRB1 and DQB1 alleles.

Measurement of absolute copy number variation of Glutathione S-Transferase M1 gene by digital droplet PCR and association analysis in Tunisian Rheumatoid Arthritis population.

BACKGROUND: The investigation of copy number variations (CNVs) analysis of candidate genes is currently an important research area in modulating human diseases. We aimed to quantify CNVs in glutathione S-transferase M1 (GSTM1) gene and determine its genetic contribution in Tunisian rheumatoid arthritis (RA) and its subsets through an innovative technique for quantification. METHODS: A total of 165 RA cases and 102 healthy controls were included in the study. Using a recently powerful approach of digital droplet PCR (ddPCR), we quantified GSTM1 gene to determine the presence of no, one, or multiple copy number (CN) at high levels of sensitivity and specificity. Odds ratio and Fisher exact test were performed to estimate the association risk for GSTM1CNVs in RA. RESULTS: Copy number identified by ddPCR was 0, 1, and 2 copies per diploid genome. A high frequency of '0' copy was revealed with 54% in RA patients. The deletion ('0' copy) of GSTM1 was found to be a significant risk factor for anti-cyclic citrullinated peptide (anti-CCP) positive RA (OR=4.16, CI95% =[1.17-14.7]). In addition, a lack of association was found when comparing between the CNVs of RA patients and those of controls. CONCLUSION: This study highlights the powerful accuracy of ddPCR for the quantification of CNVs and suggests that the variation in the CN of GSTM1 is associated with anti-CCP positivity in RA. However, it does not indicate a specific role in the susceptibility to the disease in our Tunisian sample.

Beyond Human Leukocyte Antigen Class I Antigens: Hereditary Hemochromatosis Gene Mutations in Recurrent Aphthous Oral Ulcers and Behçet Disease in the South of Tunisia.

OBJECTIVE: The aim of this work was to establish human leukocyte antigen (HLA) class I and hereditary hemochromatosis gene (HFE) mutation associations with recurrent aphthous oral ulcers (RAOU) and Behçet disease (BD) in a cohort of Southern Tunisian patients. SUBJECTS AND METHODS: A total of 232 patients with RAOU and 123 healthy controls (HCs) were enrolled in this study. The patients were divided into 2 groups based on the presence (BD+: n = 62) or absence of BD (BD-, n = 170). In the BD+ group, 28 patients had severe manifestations of BD. In the BD- group, RAOU was isolated in 81 patients, associated with mucocutaneous manifestations in 58 and with joint symptoms in 25. Complement-dependent microlymphocytotoxicity assay and polymerase chain reaction-restriction fragment length polymorphism were used to study HLA class I polymorphism and HFE mutations, respectively. RESULTS: HLA-B51 was positively associated with BD, particularly in those with severe manifestations. No association was detected with HLA class I polymorphism among the BD group. Based on stratification to clinical manifestations, the isolated RAOU was negatively associated with HLA-A1 with a difference close to significance (12 [14.81%] vs. 32 [26.02%] in HCs; p = 0.06). Furthermore, patients with mucocutaneous features had a higher frequency of HLA-B51 (14, 24.14%) than patients without mucocutaneous involvement (11, 11.37%). Considering HFE mutations, patients with isolated RAOU had a higher frequency of H63D when compared with other subgroups, especially after limiting the comparison to 27 patients of at least 5 years of follow-up. CONCLUSION: This study showed that, unlike BD, RAOU were not associated with HLA-B51. Moreover, we suggest that H63D mutation was positively associated with isolated RAOU.

Novel cases of Tunisian patients with mutations in the gene encoding 17ß-hydroxysteroid dehydrogenase type 3 and a founder effect.

17ß-Hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is expressed almost exclusively in the testis and converts Δ4-androstene-3,17-dione to testosterone. Mutations in the HSD17B3 gene causing 17ß-HSD3 deficiency are responsible for a rare recessive form of 46, XY Disorders of Sex Development (46, XY DSD). We report novel cases of Tunisian patients with 17ß-HSD3 deficiency due to previously reported mutations, i.e. p.C206X and p.G133R, as well as a case with the novel compound heterozygous mutations p.C206X and p.Q176P. Moreover, the previously reported polymorphism p.G289S was identified in a heterozygous state in combination with a novel non-coding variant c.54G>T, also in a heterozygous state, in a male patient presenting with micropenis and low testosterone levels. The identification of four different mutations in a cohort of eight patients confirms the generally observed genetic heterogeneity of 17ß-HSD3 deficiency. Nevertheless, analysis of DNA from 272 randomly selected healthy controls from the same geographic area (region of Sfax) revealed a high carrier frequency for the p.C206X mutation of approximately 1 in 40. Genotype reconstruction of the affected pedigree members revealed that all p.C206X mutation carriers harbored the same haplotype, indicating inheritance of the mutation from a common ancestor. Thus, the identification of a founder effect and the elevated carrier frequency of the p.C206X mutation emphasize the importance to consider this mutation in the diagnosis and genetic counseling of affected 17ß-HSD3 deficiency pedigrees in Tunisia.

Analysis of two susceptibility SNPs in HLA region and evidence of interaction between rs6457617 in HLA-DQB1 and HLA-DRB1*04 locus on Tunisian rheumatoid arthritis.

Previous genomewide association studies (GWAS) and meta-analyses have enumerated several genes/loci in major histocompatibility complex region, which are consistently associated with rheumatoid arthritis (RA) in different ethnic populations. Given the genetic heterogeneity of the disease, it is necessary to replicate these susceptibility loci in other populations. In this case, we investigate the analysis of two SNPs, rs13192471 and rs6457617, from the human leukocyte antigen (HLA) region with the risk of RA in Tunisian population. These SNPs were previously identified to have a strong RA association signal in several GWAS studies. A case-control sample composed of 142 RA patients and 123 healthy controls was analysed. Genotyping of rs13192471 and rs6457617 was carried out using real-time PCR methods by TaqMan allelic discrimination assay. A trend of significant association was found in rs6457617 TT genotype with susceptibility to RA (P = 0.04, pc = 0.08, OR = 1.73). Moreover, using multivariable analysis, the combination of rs6457617*TT-HLA-DRB1*04+ increased risk of RA (OR = 2.38), which suggest a gene-gene interaction event between rs6457617 located within the HLA-DQB1 and HLA-DRB1. Additionally, haplotypic analysis highlighted a significant association of rs6457617*T-HLA-DRB1*04+ haplotype with susceptibility to RA (P = 0.018, pc = 0.036, OR = 1.72). An evidence of association was shown subsequently in antiCCP+ subgroup with rs6457617 both in T allele and TT genotype (P = 0.01, pc = 0.03, OR = 1.66 and P = 0.008, pc = 0.024, OR = 1.28, respectively). However, no association was shown for rs13192471 polymorphism with susceptibility and severity to RA. This study suggests the involvement of rs6457617 locus as risk variant for susceptibility/severity to RA in Tunisian population. Secondly, it highlights the gene-gene interaction between HLA-DQB1 and HLA-DRB1.

Genetic diversity and haplotype structure of 21 Y-STRs, including nine noncore loci, in South Tunisian Population: Forensic relevance.

Y chromosome STRs (Y-STRs) are being used frequently in forensic laboratories. Previous studies of Y-STR polymorphisms in different groups of the Tunisian population identified low levels of diversity and discrimination capacity (DC) using various commercial marker sets. This definitely limits the use of such systems for Y-STRs genotyping in Tunisia. In our investigation on South Tunisia, 200 unrelated males were typed for the 12 conventional Y-STRs included in the PowerPlex® Y System. Additional set of nine noncore Y-STRs including DYS446, DYS456, DYS458, DYS388, DYS444, DYS445, DYS449, DYS710, and DYS464 markers were genotyped and evaluated for their potential in improving DC. Allele frequency, gene diversity, haplotype diversity (HD), and DC calculation revealed that DYS464 was the most diverse marker followed by DYS710 and DYS449 markers. The standard panel of 12 Y-STRs (DC = 80.5%) and the nine markers were combined to obtain DC of 99%. Among the 198 different haplotypes observed, 196 haplotypes were unique (HD = 99.999). Out of the nine noncore set, six Y-STRs (DYS458, DYS456, DYS449, DYS710, DYS444, and DYS464) had the greatest impact on enhancing DC. Our data provided putative Y-STRs combination to be used for genetic and forensic applications.

Association study of MICA-TM polymorphism with inflammatory bowel disease in the South Tunisian population.

BACKGROUND: Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory disorders of the gastro-intestinal tract with unknown etiology. Both environmental and genetic factors are involved in the pathogenesis of these inflammatory bowel diseases (IBD). AIM: The purpose of the present study was to determine the association between the polymorphism of the transmembrane region of MICA (MICA-TM), and the genetic susceptibility in Tunisian patients with IBD. PATIENTS AND METHODS: A total of 102 Tunisian patients (66 with UC, 36 with CD) and 123 healthy controls were enrolled in our study. MICA-TM was genotyped by a semiautomatic fluorescent-labelled PCR method, amplicons were analysed on an ABI Prism 310 genotyper. Comparisons of allele frequencies between patients and controls, and between patients' subgroups were performed using SPSS 13.0. RESULTS: No MICA allele was significantly increased in both groups of IBD compared to controls. The MICA-A5.1 allele was significantly decreased in CD patients (p=0.006, pc=0.03). In UC, MICA-A6 was associated with the presence of extraintestinal manifestations (p=0.04, pc=0.2), whereas MICA-A5 was associated with late age of onset (p=0.04). In CD, MICA-A6 was significantly increased in active disease patients when compared to moderately active or inactive disease (p=0.03, pc=0.15). CONCLUSION: Some clinical features of CD and UC may be influenced by specific MICA-TM alleles. In our South Tunisian population, MICA plays a disease modifying role, rather than being an important gene in the susceptibility for developing IBD.

Analysis of HLA-A, -B, -C, -DR, -DQ polymorphisms in the South Tunisian population and a comparison with other populations.

BACKGROUND: The Human Leucocyte Antigen (HLA) system is often used as a genetic marker for analysing populations. HLA antigen distribution among the Tunisian population is not well defined because of the lack of a general population study. AIM: The aim of the present study was to investigate the polymorphism of HLA-A, -B, -C, -DR and -DQ loci in the South Tunisian population. SUBJECTS AND METHODS: This study has investigated HLA-A, -B, -C, -DR and -DQ polymorphisms in 123 unrelated healthy individuals originating from the south of Tunisia. HLA class I was studied by serology and completed by polymerase chain reaction-sequence specific primer (PCR-SSP). HLA class II was performed using PCR-SSP. RESULTS: The most common alleles were A-2 (0.2154), B-44 (0.1179), C7 (0.2114), DR4 (0.1626) and DQ2 (0.313). A1-B-8-C7-DR3-DQ2 (2.84%) was the predominant haplotype in this population. Comparisons with data of other worldwide populations based on phylogenetic tree and multidimensional scaling analysis were done. This study suggests that both HLA class I and class II polymorphism specificities demonstrate a high diversity in this South Tunisian population, which reflects ancient and recent admixture with neighbouring populations. CONCLUSION: The results provide useful information for further studies of Tunisian population evolution, anthropology and for resolving HLA frequencies when searching for HLA-compatible donors in transplantation and for the analysis of disease associations.

Microsatellite analysis of Candida isolates from recurrent vulvovaginal candidiasis.

Candida albicans and Candida glabrata are the most common causative agents of both vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC). Studying the population structure and genotype differentiation of Candida species that cause RVVC may lead to a significant improvement in clinical management. A total of 106 isolates were collected from 55 patients who were subdivided into three groups. Group I comprised 15 patients with RVVC (n=50 isolates); group II comprised 16 patients, who had a history of at least two episodes of VVC in the last year (n=32 isolates, two from each patient); and group III comprised 24 patients (n=24 isolates) who had experienced a single episode of VVC in the previous 1 year period. C. albicans microsatellite markers CAI, CAIII and CAIV and C. glabrata RPM2, MTI and ERG3 microsatellites were amplified in a multiplex PCR. All isolates were subjected to population genetic analysis, which provided evidence that there is a predominantly clonal population structure of C. albicans in each group. However, recombination was detected to some degree in C. albicans isolates in group III. A genetic homogeneity between the different C. albicans groups was observed. Although, C. glabrata isolates showed an important genetic differentiation between group I and group III (F(ST)=0.207). Genotype analysis revealed that the dominant genotypes of C. glabrata and C. albicans strains were more prevalent in patients with RVVC. The frequent scenario for cases of recurrent infection in our study was strain replacement (53.3%). In conclusion, the identification of recurrence-associated genotypes and a specific C. glabrata population structure in the RVVC group could be a significant marker for further investigations of virulence factors and RVVC management.

Association study of human leukocyte antigen-DRB1 alleles with rheumatoid arthritis in south Tunisian patients.

The aim of this study is to explore relationship between HLA-DRB1 alleles and the susceptibility and clinical features of rheumatoid arthritis (RA) in the south Tunisian population. We studied 142 RA patients and 123 controls matched for age, sex, and ethnicity. HLA-DRB1 genotyping and HLA-DRB1*04 subtypes were performed using polymerase chain reaction/sequence-specific primers. Association was assessed based on the χ (2) test and odds ratio with 95% confidence interval. For multiple comparisons, p value was corrected (p (c)) with Bonferroni test. Two alleles, HLA-DRB1*04 (p=0.045, p(c)=NS) and HLA-DRB1*10 (p=0.021, p(c)=NS), were found to have increased frequencies in RA patients compared to controls. In contrast HLA-DRB1*08 allele was found to have a decreased frequency in patients compared to controls (p=0.044, p(c)=NS). Molecular subtyping of the most prevalent allele (DRB1*04) revealed increased frequencies of HLA-DRB1*04:05 in patients compared to controls (p=0.013, p(c)=NS) whereas HLA-DRB1*04:02 showed a protective effect (p=0.005, p(c)=0.04). Moreover, stratified analyses indicated statistically significant associations between HLA-DRB1*04 allele and anti-cyclic peptides antibodies positivity (ACPA(+)) and rheumatoid factor positivity (RF(+); p(c)=0.03, for both subgroups), HLA-DRBI*10 and ACPA(+) and the presence of another autoimmune disease (p(c)=0.05 and p(c)=0.007, respectively), and HLA-DRB1*04:05 and RF(+) and erosion (p(c)=0.005 and p(c)=0.049; respectively). A significant decrease in the frequency of the DRB1*04:02 allele was observed in patients with ACPA(+) and RF(+) subgroups (p(c)=0.04 and p(c)=0.02, respectively). Our results showed that there was a trend of positive association of HLA-DRB1*04 and HLA-DRB1*10 with RA as such and significant associations with the disease severity in the south Tunisian population.

Inflammatory bowel disease: susceptibility and disease heterogeneity revealed by human leukocyte antigen genotyping.

This study aimed to investigate the association between HLA DR/DQ and inflammatory bowel diseases (IBD) in Tunisian patients and to determine the relationship between HLA DR/DQ alleles with the clinical disease patterns. DNA typing of human leukocyte antigen (HLA) genes was performed in 70 ulcerative colitis (UC) patients, 40 Crohn's disease (CD) patients, and 123 healthy controls (HC) using a polymerase chain reaction sequence specific primer technique. Data were analyzed using Cochran-Mantel-Haenszel test and binary logistic regression. Compared with HC, IBD patients showed an increased frequency of the homozygous DRB1*07 genotype. This positive association was maintained when UC and CD were separately compared to HC. In UC patients, DQB1*03:02 was predictive of colonic extension whereas DRB1*13 and DQB1*03:01 were associated limited disease localization (left-sided colitis and proctitis). The DRB1*15 allele increased in patients with extraintestinal manifestations. In CD, female patients showed an increased frequency of DRB1*13, DRB1*15, and DQB1*06 alleles and DRB1*13-DQB1*06 haplotype, whereas a significant increase of DRB1*07, DQB1*02 alleles, and DRB1*07-DQB1*02 haplotype was noted in male patients. These results show a significant association of the homozygous HLA-DRB1*07 genotype with UC and CD and of several HLA DR/DQ alleles and haplotypes with the clinical phenotypes of these diseases in Tunisian patients. Because of limited statistical power, our study findings are subject to further investigation.

Interleukin-36-receptor antagonist deficiency and generalized pustular psoriasis.

BACKGROUND: Generalized pustular psoriasis is a life-threatening disease of unknown cause. It is characterized by sudden, repeated episodes of high-grade fever, generalized rash, and disseminated pustules, with hyperleukocytosis and elevated serum levels of C-reactive protein, which may be associated with plaque-type psoriasis. METHODS: We performed homozygosity mapping and direct sequencing in nine Tunisian multiplex families with autosomal recessive generalized pustular psoriasis. We assessed the effect of mutations on protein expression and conformation, stability, and function. RESULTS: We identified significant linkage to an interval of 1.2 megabases on chromosome 2q13-q14.1 and a homozygous missense mutation in IL36RN, encoding an interleukin-36-receptor antagonist (interleukin-36Ra), an antiinflammatory cytokine. This mutation predicts the substitution of a proline residue for leucine at amino acid position 27 (L27P). Homology-based structural modeling of human interleukin-36Ra suggests that the proline at position 27 affects both the stability of interleukin-36Ra and its interaction with its receptor, interleukin-1 receptor-like 2 (interleukin-1 receptor-related protein 2). Biochemical analyses showed that the L27P variant was poorly expressed and less potent than the nonvariant interleukin-36Ra in inhibiting a cytokine-induced response in an interleukin-8 reporter assay, leading to enhanced production of inflammatory cytokines (interleukin-8 in particular) by keratinocytes from the patients. CONCLUSIONS: Aberrant interleukin-36Ra structure and function lead to unregulated secretion of inflammatory cytokines and generalized pustular psoriasis. (Funded by Agence Nationale de la Recherche and Société Française de Dermatologie.).