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.

Congenital analbuminemia associated with compound heterozygous novel nucleotide variations in a young adult with coronary thrombosis.

Congenital analbuminemia (CAA) is a very rare genetic disorder characterized by a significant reduced or even complete absence of human serum albumin. Our data describe the clinical features and laboratory results of a case confirmed by mutation analysis of the albumin gene in a 35-year-old man presenting recurrent acute coronary syndrome. To the best of our knowledge, only two cases of coronary artery disease have been reported worldwide without recurrence. Our findings contribute to shed light on the clinical characteristics and biochemical parameters of this disease and confirm that cardiovascular complications must be taken seriously in this pathology. Mutational screening disclosed two novel compound heterozygous nucleotide variations located in intron 12 and in 3'UTR. The prediction of the functional and structural impact of the reported variations using different bioinformatics tools demonstrates that these genetics variations affect RNA transcription and mRNA folding.

Epidemiological and clinical characteristics of 66 Tunisian Sickle cell syndrome patients.

Introduction: Sickle cell syndrome (SCS) represent a real health problem. In this work, we propose to study the epidemiological and clinical features of 66 patients with SCS. Methods: This is a retrospective descriptive cross-sectional study carried out on a population of 66 patients with SCS, (36 S/S, 18 S/ß-thalassemia, seven S/C and five S/OArab), over a period of two years. Results: The average age of our population is 15.5 years ± 8.4. 36 patients (55%) were born to a consanguineous marriage and 35 (53%) had siblings with SCS. The average baseline hemoglobin in our patients is 9.1g/dL±1.51. S/C patients have significantly higher baseline hemoglobin than S/S, S/ß-thalassemia and S/OArab with p <0.05. Jaundice, mucosal skin pallor and hepatomegaly have been observed only in S/S, S/ß-thalassemia and S/OArab patients. The persistence of splenomegaly is more frequent in S/C than in S/S, and in S/-thalassemia than in S/S. The most common acute complications were vaso-occlusive attacks (69.7%) and worsening of anemia (54.54%). The most common chronic complication was cholelithiasis (36.36%). Conclusion: S/C patients present the best tolerated form and were the least affected by chronic complications and therefore can lead an almost normal life.

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.

Contribution of common CFTR variants (M470V, T854, and Q1463) to cystic fibrosis in Tunisia: haplotype analysis.

BACKGROUND & OBJECTIVES: Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane regulator (CFTR) protein, a chloride channel located in the epithelial cell membrane. Over than 2,000 CFTR mutations have been identified, which contribute to the variety of clinical phenotypes of CF. We performed a case-control study to determine p.Met470Val (M470V), p.Thr854= (T854) and p.Gln1463= (Q1463) polymorphisms frequencies in CF patients and healthy controls and to elaborate haplotype based on these SNPs. METHODS: The genotyping of M470V (exon 10), T854 (exon 14a), and Q1463 (exon 24) variants were identified using polymorphism restriction fragment length polymorphism (RFLP). RESULTS & CONCLUSION: Statistical difference was noted in the genotype distribution of two markers, M470V and T854, between CF and control groups. However, the Q1463 polymorphism is not identified in two studied groups. Three haplotypes were found in CF patients and controls. An exclusive association between the ancestral haplotype 1-1-2 and p.Phe508del (F508del) mutation was shown. In Tunisia, this is the first work to be interested in the analysis of M470V, T854 and Q1463 polymorphisms and haplotypes associated with the most common mutation, F508del, in the Tunisian population and worldwide.

Glucose-6-phosphate dehydrogenase deficiency in Tunisian jaundiced neonates.

BACKGROUND AND OBJECTIVES: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy worldwide associated with hemolysis as well as neonatal jaundice, kernicterus, and even death. The goal of this study is to determinate the prevalence of G6PD deficiency in icteric neonates and to investigate its biochemical, hematological and molecular characteristics. PATIENTS AND METHODS: This cross sectional study was carried out on 154 icteric newborns admitted to the Bechir Hamza Children's Hospital in Tunisia. Laboratory evaluations included complete blood count, total serum bilirubin level (TSB), and erythrocyte G6PD activity. The G6PD activity was determined using a quantitative assay, which allowed us to divide the total population into two groups: normal and deficient population. The common G6PD Tunisian mutations (GdA - and GdMed) were determined using the amplification refractory mutation system (ARMS-PCR) method. RESULTS: The prevalence of G6PD deficiency among total population (154 icteric newborns) was 18.83%. Male neonates showed a higher incidence of G6PD deficiency of 11.03% compared to females (7.79%). There was no statistical difference between the two groups (normal and deficient), in relation to the sex, peak TSB level, age at peak TSB, hemoglobin level, and hematocrit. However, there was a significant difference in gestational age. In the deficient group, 48.28% neonates presented the peak TSB level between 3 to 7 days and 55% of the cases show a peak TSB level greater than 250 µmol/L. The G6PD G202A variant was found in 41.37% of cases. CONCLUSION: This study shows a higher prevalence of G6PD deficiency in icteric newborns of Tunisia (18.83%). This emphasizes the necessity of neonatal screening for G6PD deficiency to prevent the exposure of these newborns to known hemolytic agents and, subsequently, to prevent kernicterus or other serious complications.

A new case of congenital atransferrinemia with a novel splice site mutation: c.293-63del.

Congenital atransferrinemia is an extremely rare autosomal recessive disorder resulting in the complete absence or extremely reduced amount of transferrin. In this study, we describe the first case of congenital atransferrinemia in Tunisia and the 18th patient in the reported data. The patient was referred to our hospital to explore a severe hypochromic and microcytic anemia. The laboratory evaluation including hematological and biochemical examination was performed in the proband and her parents. All exons of the transferrin gene were PCR amplified. The products were screened for mutations by direct sequencing. Based on laboratory and clinical findings, diagnosis of congenital atransferrinemia was confirmed. DNA sequencing revealed the presence of a novel homozygous deletion (c.293-63del) in the intron 13. This mutation is predicted to generate a higher score cryptic branch point leading to the production of an altered mRNA molecule. The second previously reported missense mutation p.Arg609Trp. Crystallographic structure analyzes demonstrate that the mutation would probably lead to significant conformational change not allowing the expression of transferrin protein. Current molecular characterization of this novel transferrin abnormality puts to the proof the variability in onset, first blood transfusion, and phenotypic expression in atransferrinemic patients.

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 (chi2 = 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 (chi2 = 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

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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).

Hypodysfibrinogenemia: A novel abnormal fibrinogen associated with bleeding and thrombotic complications.

BACKGROUND: Congenital disorders of fibrinogen are rare diseases resulting in the complete absence (afibrinogenemia), reduced concentration (hypofibrinogenemia) or altered function of circulating fibrinogen (dysfibrinogenemia). A combination of two different fibrinogen abnormalities with a significant functional and secretion defect (hypodysfibrinogenemia) reported in Tunisian family members, was investigated in this study. METHODS: The coagulation-related tests, kinetics of fibrin polymerization and lysis and fibrinogen analysis using gel electrophoresis were performed in the family members to characterize fibrinogen abnormalities. All exons including exon-intron boundaries of fibrinogen genes were screened by direct sequencing. RESULTS: Mutational screening of the fibrinogen genes disclosed novel missense mutations, BßCys197Arg, in exon 4 of the fibrinogen Bß-chain gene. After the loose of its partner in Bß-chain, the γCys135 was probably disulfide-bridged to its corresponding Cys residue of another abnormal fibrinogen molecule, forming dimmer with an abnormal electrophoretic profile. Homozygous form carried by the proband found to be directly involved in the bleeding phenotype by affecting fibrin polymerization. In contrast, affected family members bearing the heterozygous mutation showed an impaired fibrin polymerization and fibrinolysis leading to thrombosis. CONCLUSION: These results suggest that this mutation could alter the extremely conserved conformations of fibrinogen D domain and D-D lateral regions on fibrin assembly.

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.

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.

Fortuitous description of haemoglobin A2' [δ16 (A13) Gly→Arg (GGC→CGC)] in a Tunisian family: study of the molecular defect and its origin.

The most common inherited haemoglobin disorders encountered in Tunisia are ß-thalassemia and sickle cell disease, which result from mutations in the ß-globin gene. Few studies focused on δ-globin gene variations responsible for δ-thalassemia or HbA2 variants. HbA2' [δ16 (A13) Gly→Arg (GGC→CGC)] is a δ-chain variant that has been identified in several populations of African origin. We report herein for the first time the description of HbA2' in the Tunisian population. Identification of HbA2' in the studied family was carried out by high-performance liquid chromatography and confirmed by sequencing analyses of the whole δ-globin gene. Haplotypes of the ß-globin gene cluster were constructed by mapping the restriction sites using polymerase chain reaction followed by enzymatic digestion. Compound heterozygosity of HbA2' with HbO-Arab was identified in the proband. The mother and two other siblings showed heterozygous HbA2' whereas the father showed heterozygous HbO-Arab. The sum of HbA2 and HbA2' in all cases was less than 4%, thus excluding ß-thalassemia. ß-cluster haplotype analysis revealed that this mutation was associated with the F haplotype (-+--+++). The unique origin of this mutation in Africa is likely since the linked ß-cluster haplotype is one of the major haplotypes found in African populations.