A gain-of-function mutation in zinc cluster transcription factor Rob1 drives Candida albicans adaptive growth in the cystic fibrosis lung environment.

Candida albicans chronically colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens (e.g. Pseudomonas aeruginosa) and contributes to disease severity. We hypothesize that C. albicans undergoes specific adaptation mechanisms that explain its persistence in the CF lung environment. To identify the underlying genetic and phenotypic determinants, we serially recovered 146 C. albicans clinical isolates over a period of 30 months from the sputum of 25 antifungal-naive CF patients. Multilocus sequence typing analyses revealed that most patients were individually colonized with genetically close strains, facilitating comparative analyses between serial isolates. We strikingly observed differential ability to filament and form monospecies and dual-species biofilms with P. aeruginosa among 18 serial isolates sharing the same diploid sequence type, recovered within one year from a pediatric patient. Whole genome sequencing revealed that their genomes were highly heterozygous and similar to each other, displaying a highly clonal subpopulation structure. Data mining identified 34 non-synonymous heterozygous SNPs in 19 open reading frames differentiating the hyperfilamentous and strong biofilm-former strains from the remaining isolates. Among these, we detected a glycine-to-glutamate substitution at position 299 (G299E) in the deduced amino acid sequence of the zinc cluster transcription factor ROB1 (ROB1G299E), encoding a major regulator of filamentous growth and biofilm formation. Introduction of the G299E heterozygous mutation in a co-isolated weak biofilm-former CF strain was sufficient to confer hyperfilamentous growth, increased expression of hyphal-specific genes, increased monospecies biofilm formation and increased survival in dual-species biofilms formed with P. aeruginosa, indicating that ROB1G299E is a gain-of-function mutation. Disruption of ROB1 in a hyperfilamentous isolate carrying the ROB1G299E allele abolished hyperfilamentation and biofilm formation. Our study links a single heterozygous mutation to the ability of C. albicans to better survive during the interaction with other CF-associated microbes and illuminates how adaptive traits emerge in microbial pathogens to persistently colonize and/or infect the CF-patient airways.

Catalyzing precision: unraveling the diagnostic conundrum of tunisian familial hypophosphatasia case through integrative clinical and molecular approaches.

Familial Hypophosphatasia presents a complex diagnostic challenge due to its wide-ranging clinical manifestations and genetic heterogeneity. This study aims to elucidate the molecular underpinnings of familial Hypophosphatasia within a Tunisian family harboring a rare c.896 T > C mutation in the ALPL gene, offering insights into genotype-phenotype correlations and potential therapeutic avenues. The study employs a comprehensive approach, integrating biochemical examination, genetic analysis, structural modeling, and functional insights to unravel the impact of this rare mutation. Genetic investigation revealed the presence of the p.Leu299Pro mutation within the ALPL gene in affected family members. This mutation is strategically positioned in proximity to both the catalytic site and the metal-binding domain, suggesting potential functional consequences. Homology modeling techniques were employed to predict the 3D structure of TNSALP, providing insights into the structural context of the mutation. Our findings suggest that the mutation may induce conformational changes in the vicinity of the catalytic site and metal-binding domain, potentially affecting substrate recognition and catalytic efficiency. Molecular dynamics simulations were instrumental in elucidating the dynamic behavior of the tissue-nonspecific alkaline phosphatase isozyme (TNSALP) in the presence of the p.Leu299Pro mutation. The simulations indicated alterations in structural flexibility near the mutation site, with potential ramifications for the enzyme's overall stability and function. These dynamic changes may influence the catalytic efficiency of TNSALP, shedding light on the molecular underpinnings of the observed clinical manifestations within the Tunisian family. The clinical presentation of affected individuals highlighted significant phenotypic heterogeneity, underscoring the complex genotype-phenotype correlations in familial Hypophosphatasia. Variability in age of onset, severity of symptoms, and radiographic features was observed, emphasizing the need for a nuanced understanding of the clinical spectrum associated with the p.Leu299Pro mutation. This study advances our understanding of familial Hypophosphatasia by delineating the molecular consequences of the p.Leu299Pro mutation in the ALPL gene. By integrating genetic, structural, and clinical analyses, we provide insights into disease pathogenesis and lay the groundwork for personalized therapeutic strategies tailored to specific genetic profiles. Our findings underscore the importance of comprehensive genetic and clinical evaluation in guiding precision medicine approaches for familial Hypophosphatasia.

Vitamin D receptor gene BsmI (rs1544410) polymorphism: role in multiple sclerosis and genotype-phenotype correlations.

BACKGROUND: Multiple sclerosis (MS) has a complex pathophysiology which depends on many endogenous and exogenous factors. Vitamin D involvement has been largely studied in MS. The large distribution of the vitamin D receptor (VDR) in different immune cells is suggestive of an immunomodulatory role. The VDR gene polymorphisms have been proposed as potential risk factors for MS development or evolution with non-conclusive results. METHODS AND RESULTS: We conducted a cross-sectional study including patients ≥ 18 years, with a diagnosis of relapsing remitting MS according to the McDonald Criteria and having a minimum follow-up period of one year after starting a disease modifying therapy. Two study groups were compared based on the Multiple Sclerosis Severity Scale or MSSS: "a slow progressor" group for an MSSS ≤ 5, and a "fast progressor" group for an MSSS > 5. The rs1544410 VDR gene polymorphism was studied for all patients. Eighty patients were included. The fast progressor groups had a higher EDSS at onset, a higher total number of relapses, more frequent and shorter time to secondary progression. The progression profile was not statistically different between genotypes and alleles of the VDR gene polymorphism rs1544410. The CC genotype and wild-type allele exhibited a more aggressive disease phenotype with a higher number of relapses the first year, shorter time to secondary progression and cerebral atrophy on assessment. CONCLUSIONS: Our results suggest potential genotype-phenotype correlations for the rs1544410 VDR gene polymorphism in the disease course of MS. Future research on a larger scale is needed to confirm these findings.

Association analysis of the surfactant protein-C gene to childhood asthma.

OBJECTIVES: This study aims to describe the molecular variability in the SFTPC gene in a childhood chronic respiratory disease, asthma, in the Tunisian population and to identify the implications based on a case-control study of p.Thr138Asn (T138N) and p.Ser186Asn (S186N) variants. METHODS: We used direct sequencing for the genotyping of the SFTPC gene within 101 asthmatic children. The study of T138N and S186N variants in 110 controls is conducted by the PCR-RFLP technique. RESULTS: The molecular study revealed 26 variants including 24 intronic variations and 2 exonic variations (T138N and S186N) with respective frequencies of 16.8% and 18.3%. We conducted a case-control study of the two identified exonic variations. A different genotypic and allelic distribution between the two groups was noted. Only the T138N polymorphism showed a significant association with asthma disease (p < 1 0 -3). Statistical analysis elaborated four haplotypes with the following frequencies in patients vs controls: 138Thr-186Ser (79.5% vs 57.6%), 138Thr-186Asn (3.7% vs 7.8%), 138Asn-186Thr (2.2% vs 20.2%) and 138Asn-186Asn (14.6% vs 14.4%). A significant difference (p < 1 0 -3) was highlighted in haplotype distribution. The 138Asn-186Ser (OR [95%CI] = 0.14[0.04-0.54], p = 0.004, R2=0.93) and 138Thr-186Asn (OR [95%CI] = 0.35[0.12-0.54], p = 0.047, R2=0.88) haplotypes showed a negative association with asthma which may constitute a protective factor against the disease. CONCLUSION: In Tunisia, this work constitutes the first report interested in the SFTPC gene and highlights the genetic variability of the SFTPC gene in asthma. Therefore, the case-controls analysis may be useful in the study of surfactant proteins dysfunction in chronic respiratory disease at an early age.

Analysis of δ-globin gene alleles in Tunisians: description of three new delta-thalassemia mutations.

BACKGROUND: Thalassemia is one of the most prevalent worldwide autosomal recessive disorders characterized by a great molecular and clinical expression heterogeneity. Alpha and beta-thalassemia are the main two types observed in case of mutations affecting alpha and beta-globin genes respectively. Delta-thalassemia is noted when mutations occur on the delta-globin gene. In Tunisia, ß-thalassemia prevalence is estimated at 2.21% of carriers. However, few reports investigated the delta-globin gene. OBJECTIVES: In this work, we aimed to perform a molecular study to help define the molecular spectrum of δ-thalassemia mutations in Tunisia. PATIENTS AND METHODS: The study involved 7558 patients among whom we selected 179 individuals with abnormal HbA2 values or fractions. Hemoglobin analysis was performed using Capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). DNA sequencing was performed on ABI prism 310 Genetic Analyzer Applied Biosystems. CUPSAT (Cologne University Protein Stability Analysis Tool) was used for the prediction of protein stability changes upon missense mutations and mutants were modeled via DeepView-SwissPdbViewer and POV-Ray softwares for molecular dynamics simulation studies. RESULTS: We identified four mutations: HbA2-Yialousa described for the first time in Tunisia ( in 72.72% of cases) and 3 mutations reported for the first time in the world: (i) c.442 T > C Stop147Arg ext 15aa-stop observed in 18.18% of cases, (ii) c.187 G > C (Ala62Pro) noted in 4.54% of cases and (iii) c.93-1G > C found in 4.54% of cases. CONCLUSION: Our data provide genetic basis that would be especially useful in screening for beta-thalassemia trait during delta-beta thalassemia associations.

Refractory epilepsy in PSEN 1 mutation (I83T).

Mutations in the presenilin-1 gene (PSEN1) on chromosome 14 are the most common cause of autosomal dominant Alzheimer's disease (ADAD), which has a broad clinical phenotype, encompassing not only dementia but a variety of other neurological features. We report the case of a 32 years old man with a family history of  early onset AD associated with a PSEN1 mutation in the exon 4 (I83T). The proband's, carrying the mutation,  present a refractory epilepsy predating cognitive decline. We discuss the physiopathological mechanisms of epilepsy during AD associated with PSEN 1 mutation, the possibility of linking this epilepsy to the mutation?.

Association of M470V polymorphism of CFTR gene with variability of clinical expression of asthma: Case-report study.

INTRODUCTION AND OBJECTIVES: Asthma is a complex genetic disorder. Several genes have been found associated with asthma. The cystic fibrosis transmembrane conductance regulator (CFTR) gene is one of them. The aim of this study was to perform a comparative analysis of the genotype and allele frequency distributions of the biallelic marker M470V within the CFTR gene on mutant and wide chromosomes. PATIENTS AND METHODS: The molecular approach consists in the genotyping of the M470V marker by the PCR-RFLP technique in 105 asthmatic patients, aged between four months and 17 years, and 105 healthy subjects. RESULTS: We found a significant difference in the genotype frequencies between the two studied groups (χ2=9.855, P=0.007). The V/V genotype was over represented in the asthmatic group as compared to the controls (32.38% vs. 16.19%). Whereas, the M/V genotype is more frequent in healthy subjects (40.95% vs. 28.71%). We also noted a significant difference in allelic distribution of M470V with associated diseases (χ2=9.610, P=0.022). CONCLUSIONS: The present study is the first report on the distribution of the M470V polymorphism in asthmatic Tunisian patients. We noticed that the M470V variant could modulate the clinical phenotype of asthmatic patients. This preliminary study will establish the molecular basis of this disease in Tunisia.

Clopidogrel utilization in patients with coronary artery disease and diabetes mellitus: should we determine CYP2C19*2 genotype?

PURPOSE: Clopidogrel non-responsiveness is multifactorial; several genetic and non-genetic factors may contribute to impaired platelet inhibition. The goal of this study is to determine the effect of the cytochrome P450 CYP2C19*2 polymorphism on the platelet response to clopidogrel in patients with and without diabetes mellitus (DM). METHODS: We conducted an observational study in patients with coronary artery disease and consequent exposure to clopidogrel therapy (75 mg/day for at least 7 consecutive days). We have analyzed two groups of patients: group I (DM patients) and group II (non-diabetes mellitus patients). Platelet reactivity was assessed by the VerifyNow P2Y12 assay and high on clopidogrel platelet reactivity (HPR) was defined as P2Y12 reaction units (PRU) ≥ 208. Genotyping for CYP2C19*2 polymorphism was performed by PCR-RFLP. RESULTS: We have included 150 subjects (76 DM and 74 non-diabetes mellitus patients). The carriage of CYP2C19*2 allele, in DM patients, was significantly associated to HPR (odds ratio (OR) 4.437, 95% confidence interval (CI) 1.134 to 17.359; p = 0.032). Furthermore, 8.4% of the variability in percent inhibition by clopidogrel could be attributed to CYP2C19*2 carrier status. However, in non-diabetes mellitus patients, there was no significant difference in platelet response to clopidogrel according to the presence or absence of CYP2C19*2 allele carriage (OR 1.260, 95% CI 0.288 to 5.522; p = 0.759). CONCLUSIONS: Our study suggests that the carriage of CYP2C19*2 polymorphism, in DM patients, might be a potential predictor of persisting HPR in these high-risk individuals. TRIAL REGISTRATION: Clinical Trials.gov NCT03373552 (Registered 13 December 2017).

Factor V Leiden G1691A, Prothrombin G20210A, and MTHFR C677T and A1298C Mutations in Patients With Sickle Cell Disease in Tunisia.

Sickle cell disease is a genetic disorder characterized by a hypercoagulable state. Several complications in this hemoglobinopathy are increased by thrombosis. Factor V Leiden (FVL), prothrombin (PRT) G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C mutations are major inherited risk factors of thrombotic complications. In this study, our aim was to compare the frequencies of these mutations in sickle cell patients with healthy controls. The study population comprised 35 homozygous Hb S (HBB: c.20A>T) patients, 29 compound heterozygous patients [16 Hb S/ß0-thalassemia (ß0-thal), four Hb S/ß+-thal, seven Hb S/Hb C (HBB: c.19G>A) and two Hb S/Hb O-Arab (HBB: c.364G>A)] and 100 healthy subjects. All patients and controls were subjected to laboratory investigations as well as mutation genotyping. Our findings showed a severe anemia with the lowest values of protein S (PS), protein C (PC) and antithrombin (AT) in the homozygous Hb S group compared to Hb S/Hb C and Hb S/ß-thal subjects. No significant difference in FVL genotype between patients and controls was observed, whereas high frequencies of PRT G20210A, MTHFR C677T and MTHFR A1298C mutations in the Hb S patients and a significant association between the MTHFR C677T mutation and Hb S/ß0-thal were found.

Capillarys 2 Flex Piercing: Analytical performance assessment according to CLSI protocols for HbA1c quantification.

HbA1c is used for monitoring diabetic balance. In this paper we report an assessment of the analytical performances of Capillarys 2 Flex Piercing (C2FP) for HbA1c measurement using CE (Capillary Electrophoresis). CLSI (Clinical and Laboratory Standard Institute) protocols are used for the evaluation of apparatus performances: precision, linearity, method comparison, trueness and common interferences. HbA1c CVs average in intra-assay was 1.6% between run imprecision CV ranged from 0.1 to 1.8%. The linearity was demonstrated between 4.7 and 15.0%. The comparison study revealed that Bland Altman plot mean difference was equal to -0.03 (CI 95% (-0.05 to -0.0003)) and Passing-Bablok regression intercept was -0.05, CI95%(-0.13 -  -0.05); slope: 1.00, CI95%[1.00-1.01]. A strong correlation (r > 0.99) was proved. No significant effects of hemoglobin variants were seen with CE on HbA1c measurement. No problem related to sample-to-sample carry over was noted. No interferences of LA1c and cHb were observed. CE allowed quantification of HbA1c even at low level of total hemoglobin (40 g/L) in contrast to HPLC. Furthermore, this analyzer offered the opportunity of quantifying the HbA2 simultaneously with HbA1c . This evaluation showed that C2FP is a convenient system for the control of diabetes and the detection of hemoglobinopathies.

Early psychiatrics symptoms in familial Alzheimer's disease with presenilin 1 mutation (I83T).

Several clinical phenotypes were associated with presenilin 1 (PSEN1) mutation in early-onset familial Alzheimer's disease (EOFAD). We report the clinical phenotype of two members of a familial dementia kindred who presented with EOFAD and early psychiatric syndrome as behavioral abnormalities. Sequence analysis of the index patient and his brother's PSEN1 transcript revealed a novel T > C transition in exon 4 which was determined as a missense substitution at position 248 of the coding sequence (cDNA. 248T > C).

Association between ACE polymorphism, cognitive phenotype and APOE E4 allele in a Tunisian population with Alzheimer disease.

Angiotensin-converting enzyme (ACE) has shown altered activity in patients with neurological diseases. An insertion/deletion (I/D) polymorphism of the ACE gene encoding angiotensin-converting enzyme has been reported to be associated with the risk for Alzheimer's disease (AD), and is generally considered to be a disorder primarily affecting memory. We conducted a case-control study in a sample composed of 85 sporadic AD patients and 90 age- and sex-matched controls to investigate the possible effect of the polymorphism and cognitive profile. Our data revealed an association between the ACE polymorphism and AD risk. There was a significant difference in the ACE allele or genotype frequencies between cases and controls. The D/D genotype showed an increased risk for AD and in the amnestic group and the effect was independent on ApoE genotypes.

Association of MP6d9 Polymorphism with Clinical Variability in Cystic Fibrosis Patients.

BACKGROUND: In this work, we are interested to study for the first time the extragenic polymorphic marker MP6d9 in cystic fibrosis and healthy cohort in Tunisia to establish the contribution of MP6d9 polymorphism in the phenotypic variability of CF patients. METHODS: Our study enrolled 112 CF patients and 100 healthy controls. The analysis of the polymorphic marker MP6d9 was performed using the PCR-RFLP technique. RESULTS: Statistical difference was found in the genotype and allelic distribution between CF patients and control groups. We found that the 2/2 genotype was higher in CF patients than in controls (58.9% vs 23%). We noted that the 2/2 genotype is associated with severe clinical manifestations. CONCLUSION: Based on the above data, it seems that this genotype has led to the deterioration of our patient's clinical manifestation. This study enabled us to understanding the involvement of the MP6d9 marker in the CF clinical expression in the Tunisian population.

Setup of a Protocol of Molecular Diagnosis of ß-Thalassemia Mutations in Tunisia using Denaturing High-Performance Liquid Chromatography (DHPLC).

BACKGROUNDS: ß-Thalassemia is one of the most prevalent worldwide autosomal recessive disorders. It presents a great molecular heterogeneity resulting from more than 200 causative mutations in the ß-globin gene. In Tunisia, ß-thalassemia represents the most prevalent monogenic hemoglobin disorder with 2.21% of carriers. Efficient and reliable mutation-screening methods are essential in order to establish appropriate prevention programs for at risk couples. The aim of the present study is to develop an efficient method based on the denaturing high-performance liquid chromatography (DHPLC) in which the whole ß-globin gene (HBB) is screened for mutations covering about 90% of the spectrum. METHODS: We have performed the validation of a DHPLC assay for direct genotyping of 11 known ß-thalassemia mutations in the Tunisian population. RESULTS: DHPLC assay was established based on the analysis of 62 archival ß-thalassemia samples previously genotyped then validated with full concordance on 50 tests with blind randomized samples previously genotyped with DNA sequencing and with 96% of consistency on 40 samples as a prospective study. CONCLUSION: Compared to other genotyping techniques, the DHPLC method can meet the requirements of direct genotyping of known ß-thalassemia mutations in Tunisia and to be applied as a powerful tool for the genetic screening of prenatal and postnatal individuals.

First description of the rs45496295 polymorphism of the C/EBPE gene in ß-thalassemia intermedia patients.

The C/EBPE gene, located in 14q11.2, encodes for a B/zip-type transcription factor. The C/EBPɛ is involved in terminal differentiation and functional maturity of granulocyte progenitor cells and in cell apoptosis during myeloid differentiation. A C/EBPE gene has recently been described as a candidate gene involved in clinical variability of ß-thalassemia (ß-thal). In this study, the C/EBPE gene was sequenced in 146 subjects divided into the severe type of ß-thal major (ß-TM) and moderate type of ß-thal intermedia (ß-TI), and a control group. The analysis identified the rs45496295 (C > T) polymorphism in the heterozygous state in 73.9% ß-TI patients, which was not the case in the ß-TM patients or in the control group. Thus, the T allele is consequently associated with the ß-TI group (p = 10-3). According to the Human Splicing Finder (version 3.0, Marseille, France), the presence of the rs45496295 polymorphism leads the creation of a new intronic exotic splicing enhancer (ESE) site. Moreover, the T allele of rs45496295 is associated with a lower transfusion regimen (p = 10-3) and a higher pretransfusion hemoglobin (Hb) rate (p = .006). The comparison of several factors concerning T allele carriers and non-carriers showed that the T allele does not act on the Hb F rate. The T allele of rs45496295, associated with moderate type of ß-thal, seems to modify the C/EBPɛ action, thereby preventing the hemolysis.

1471 delTTCT a Common Mutation of Tunisian Patients with Lysinuric Protein Intolerance.

BACKGROUND: Lysinuric protein intolerance is an inherited aminoaciduria caused by defective cationic amino acid transport. It is an autosomal recessive disease caused by mutations in the SLC7A 7 gene. The objective of this study was to identify the mutations of Tunisians patients in order to offer the genetic counseling and the prenatal diagnosis to families. METHODS: Five affected Tunisian children (4 girls and 1 boy) belonging to four consanguineous families were considered. The diagnosis was made based on the plasma for amino acids quantification by Ion Exchange chromatography, the DNA for mutational analysis by DHPLC and sequencing, and the amniotic fluid for prenatal diagnosis. RESULTS: For the 5 patients, clinical features were dominated by failure to thrive, bone marrow abnormalities, hepatosplenomegaly, and mental retardation. The diagnosis for all patients was confirmed by biochemical analysis with hyperammonemia, hyperexcretion of urinary dibasic amino acids, and a high amount of orotic acid in the urine. The 1471 delTTCT mutation was identified in exon 9 in the homozygous state for all Tunisian patients. Genetic counseling was performed for three out of four families, four heterozygous and two homozygous healthy siblings were identified. The result of prenatal diagnosis showed the presence of the 1471 de1TTCT mutation in the homozygous state for the third pregnancy and heterozygous state for the fourth. CONCLUSIONS: The 1471 deITTCT mutation seems to be a common mutation of Tunisian population. The identification of this specific mutation provides a tool for confirmatory diagnosis, genetic counseling, and prenatal diagnosis.

Clinical and Biochemical Profile of Tyrosinemia Type 1 in Tunisia.

BACKGROUND: Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by a defect of fumarylacetoacetate hydrolase. This study aimed to estimate the prevalence of HT1 in Tunisia and report its clinical, biochemical and genetic features. METHODS: During the last 25 years, 69 patients were diagnosed with HT1 based on clinical features and increased succinylacetone (SA) in blood and urine. SA was detected by GC-MS after oximation and quantified by a spectrophotometric method. Nine prenatal diagnoses for HT1 have been done and nine unrelated patients were screened for the hotspot IVS6-1(G-T) mutation using PCR. RESULTS: Using the Hardy-Weinberg formula, the incidence of HT1 was estimated at 1/14804 births in Tunisia. According to clinical form, 21 patients (30%) had the acute form and 48 patients (70%) had the chronic form. Mean plasma and urine SA were higher in the acute form (24 and 193 µmol/L vs. 9 and 90 µmol/L, respectively). Diagnosis of HT1 was done for 4 fetuses. The hotspot IVS6-1(G-T) mutation was found in six of nine explored patients. CONCLUSIONS: The incidence of HT1 is relatively high in Tunisia with a predominance of the chronic form. It is important to diagnose the disease as early as possible to prevent unfavorable issues. Prenatal diagnosis should be recommended to minimize the recurrence of the disease.

Association between Alzheimer disease and the -491T allele of regulatory region polymorphism of Apolipoprotein E in a Tunisian population.

The apolipoprotein E (APOE) is a well-established risk factor for late-onset Alzheimer's disease (AD). Several studies have attempted to confirm the association between the polymorphism located at position -491 in the transcriptional regulatory region of the APOE gene and AD. We examined in 85 AD patients and 90 control subjects of a Tunisian population the potential involvement of this polymorphism as a risk factor for AD, either through an independent effect or through interaction with the existing APOE ε4 allele risk. The T allele frequency was significantly higher in the AD patients group (45.3 %) than in the controls group (32.78 %) and may possibly constitute a significant risk factor for AD. The APOE ε4 allele did not influence the distribution of the -491 polymorphism after stratification.