New Born Screening of Hemoglobinopathies in a Center Tunisian Population.

Sickle cell diseases, ß-thalassemia, and other hemoglobinopathies are common in Africa. Their distribution differs from one region to another. There are higher frequencies in Western and Northern Africa. Their clinical complications presented a real public health problem in each country. For this, early treatment can improve the severity of these diseases. Hemoglobinopathies targeted by screening are associated with SCD, ß, and α thalassemia. Our study aim is to report our experience with newborn screening for hemoglobinopathy in Tunis. The 156 newborn's cord blood was collected at the time of childbirth in the center region (Farhat Hached Hôspital). We opted for hemoglobin exploration to achieve maximum efficiency and effectiveness in screening. After that, all patients suspected to have hemoglobinopathies are affected by molecular investigation. Our findings showed the presence of some hemoglobinopathies such as ß-thalassemia and α-thalassemia with the following frequencies: 12% and 0.33%. The molecular results show the presence of HBB: c.93-21G>A, IVS-I-110G>A, HBBc. -106G>A -56G>C, HBBc.404T>C, Hb Yaounde described for the first time in Tunisia and α 3,7 . In conclusion, newborn screening diagnoses neonates with different examples of hemoglobinopathies, which will be beneficial not only for the care of the child but also for genetic counseling of the potential risk's parents.

Emotional and behavioral attitudes of Tunisian youth towards childhood leukemia: health education and primary prevention in perspective.

BACKGROUND: Given the increasing blood cancer incidence in Tunisia and recent discoveries proving the involvement of environmental factors, this study examined the environmental health literacy (EHL) of Tunisian secondary school students concerning not only this disease, but also their emotional and behavioral attitudes towards leukemia risks. METHODS: A cross-sectional survey was conducted among Tunisian youths (N = 372, 16-20 years; 68% females, 32% males). Data collection took place in four representative public secondary schools in the North, Center, and South of Tunisia. Students completed a paper and pencil questionnaire and described their EHL level of blood cancer, as well as their attitudes and interests in this disease. The statistical software (SPSS, v.25.0) was used to analyze the data collected. RESULTS: The results indicated low EHL levels of leukemia. Most youths failed to identify all the leukemogenic (except tobacco and pollution) and non- leukemogenic risk factors. Pesticide use and exposure to low frequency electromagnetic radiation were not considered risk factors. Proximity to heavy-traffic roads and benzene exposure were not perceived by youth as risk factors. Despite these low levels, most participants were interested in having more information about leukemia and cancers in general. CONCLUSION: This investigation shows a lack of knowledge about leukemia. Low EHL levels will incite educational actors and curriculum designers to optimize content and innovate ICT adapted to this environmental health challenge.

Hematological relevance of JAK2 V617F and calreticulin mutations in Tunisian patients with essential thrombocythemia.

BACKGROUND: The genetic investigation of essential thrombocythemia(ET) has highlighted the presence of driver mutations in ET. Janus kinase JAK2V617F and calreticulin(CALR) mutations are the most frequent driver mutations and have significantly improved the molecular diagnosis of ET. The impact of genetic heterogeneity on clinical features has not been fully elucidated. This is the first study which aimed to determine the frequency of JAK2V617F and CALR exon9 mutations in Tunisian ET patients and to establish the correlation between hematological characteristics and mutational status. METHODS: This study included Tunisian patients suspected with ET and was conducted between September 2017 and March 2021. Genomic DNA of patients was isolated from peripheral blood samples. JAK2V617F was detected by AS-PCR and CALR mutations were detected by PCR/direct sequencing. Clinical and hematological characteristics were also analyzed. RESULTS: Two hundred and fifty ET patients were enrolled in this study. JAK2V617F mutation was found in 166/250 (66.4%) of patients, whereas CALR mutations were detected in 27/84 (32.1%) patients without JAK2V617F. Compared with JAK2V617F-positive patients, those with CALR mutations showed lower hemoglobin level and lower leucocytes count (p = 0.007 and p = 0.004,respectively). CALR type 2 was the most frequent mutation of CALR detected in 55.55% of CALR mutated. Six of seven patients with thrombotic events harbored JAK2V617F mutation. CONCLUSION: The prevalence of driver mutations JAK2V617F or CALR mutations was 77.2% in Tunisian ET patients. Moreover, patients with JAK2 V617F mutation had a higher risk of thrombosis. The mutational status is necessary to improve the diagnosis and contribute to the therapeutic decisions.

Coinheritance of HbO Arab/ß0-thalassemia with Severe Manifestation in Newborn.

OBJECTIVE: In this study, we report a Tunisian newborn boy referred for neonatal hemolytic anemia with yellowish skin and enlarged spleen due to coinheritance of hemoglobin O (HbO) Arab and ß-thalassemia. STUDY DESIGN: Hematological parameters were collected using an automated blood cell counter. The amounts of Hb fractions were measured by capillary electrophoresis of Hb. Amplification and sequencing of the HBB gene were performed by Sanger's method. RESULTS: Family study and genetic analysis revealed that the proband was a carrier of two hemoglobinopathies: HbO Arab and ß0-thalassemia. CONCLUSION: The coexistence of these two pathologies complicated the general state of the newborn boy and led to a severe anemia at birth. KEY POINTS: · Severe neonatal anemia can be caused by hemoglobinopathy.. · Coinheritance of HbO Arab/ß0-thalassemia complicated the general state of the newborn.. · Diagnosing hemoglobinopathy at an early age improves patient care..

Tunisian Newborn's Cord Blood: Reference Values of Complete Blood Count and Hemoglobin Fractions.

OBJECTIVE: This study was aimed to establish local reference values for hematological indices and hemoglobin (Hb) fractions in umbilical cord blood (UCB) for the northern population of Tunisia. STUDY DESIGN: Our study included full-term newborns by vaginal deliveries. Hematological parameters were collected using an automated blood cell counter. The amounts of Hb fractions were measured by capillary electrophoresis of Hb. Statistical analysis was performed using R software. RESULTS: A total of 328 cord blood samples were analyzed. Among them, 154 (male: 44.8%, female: 55.2%) were used to establish reference values. The normal reference values of complete blood count (CBC) and Hb fractions were calculated. Mean neonatal Hb was 14.75 ± 2.26 g/dL. Gestational age affects the expression of CBC values as red blood cell (RBC), Hb, hematocrit (Hct), mean corpuscular volume (MCV), white blood cell (WBC), and the Hb profile. Umbilical blood hemogram parameters and Hb profile are affected by the environment; higher in newborns from urban regions but not affected by gender ratio. CONCLUSION: Reference ranges of normal CBC indices and Hb fractions have been successfully established in Tunisian neonates' UCB. Our data suggest reference values that could be useful for neonatal patients' laboratory results and clinical interpretation. KEY POINTS: · Reference values for CBC and hemoglobin fractions have been established.. · Hematological reference for UCB is useful to identify hemolytic anemia cases early.. · UCB hematological values are influenced by gestational age and probably by environmental factors..

Tyrosol Derivatives, Bearing 3,5-Disubstituted Isoxazole and 1,4-Disubstituted Triazole, as Potential Antileukemia Agents by Promoting Apoptosis.

In the present study, we assess tyrosol derivatives bearing 3,5-disubstituted isoxazoles and 1,4-disubstituted triazoles for their ability to inhibit the proliferation of K562 cells derived from leukemia as well as primary chronic myeloid leukemia (CML) cells obtained from the peripheral blood of 15 CML patients including 10 patients with untreated chronic phase and 5 patients with resistance against imatinib or multiple TKI. Our results showed that most derivatives displayed significant anti-proliferative activity against K562 cells in a dose-dependent manner. Among them, compounds 3d and 4a exhibited greater potent anticancer activity with respective IC50 values of 16 and 18 µg/mL (45 µM and 61 µM). Interestingly, compound 3d inhibited CML cell proliferation not only in newly diagnosed but also in imatinib-resistant patients. We demonstrated that the anti-proliferative effect of this compound is mediated by a pro-apoptotic activity by promoting oxidative stress and modulating the activity of the Akt, p38 MAPK and Erk 1/2 pathways. In conclusion, our data highlight the potential of this class of derivative as a novel promising therapeutic agent for CML therapy.

Enhanced Eryptosis in Glucose-6-Phosphate Dehydrogenase Deficiency.

BACKGROUND/AIMS: Defects in the Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme enhance cellular oxidative damage, thus impairing erythrocytes and radically shortening their lifespan. We aimed to study programmed erythrocyte cell death in G6PD-deficient patients, describe the molecular genetics basis of G6PD and investigate phenotype-genotype correlations. METHODS: We explored eryptosis using the annexin V-binding assay, taken as an indicator of PS exposure at the erythrocyte surface. We assessed reactive oxygen species (ROS) production, intracellular calcium concentrations and ceramide formation at the cell surface. Prior to and following treatments, cells were analyzed by flow cytometry. Finally, we explored G6PD gene mutations through PCR-Sanger sequencing. RESULTS: Before stimulation, PS-exposing erythrocytes were significantly higher in G6PD-deficient patients than in healthy volunteers. This was paralleled by a significant increase in reactive oxygen species production, suggesting that oxidative stress is the main trigger of PS exposure in G6PD-deficient erythrocytes. Five previously described mutations were detected in our patients. Two genotypes correlated with a significantly higher percentage of PS-exposing cells. CONCLUSION: Our study uncovers a novel effect detected in G6PD-deficient erythrocytes which is cell membrane scrambling with PS translocation to the erythrocyte surface. Our findings shed a light on the mechanisms of premature erythrocyte clearance in G6PD deficiency.

Biochemical, Cellular, and Proteomic Characterization of Hereditary Spherocytosis Among Tunisians.

BACKGROUND/AIMS: Hereditary Spherocytosis (HS) is the most common erythrocyte membrane disorder causing hemolytic anemia. The wide heterogeneity of both clinical and laboratory manifestations of HS contributes to difficulties associated with the diagnosis of this disorder. Although massive data previously reported worldwide, there is yet no data on HS among the Tunisian population. Here we aim to characterize HS in Tunisian patients at biochemical and cellular levels, identify the membrane protein deficiency, and compare the accuracy of the diagnostic tests to identify the most appropriate assay for HS diagnosis. METHODS: We investigated 81 patients with hemolytic anemia and 167 normal controls. The exploration of HS based on clinical and family history, physical examination, and the results of laboratory tests: blood smear, osmotic fragility test (OFT), cryohemolysis test (CT), pink test (PT), eosine-5'-maleimide (EMA) test, and erythrocyte membrane protein electrophoresis. RESULTS: We identified 21 patients with HS, classified as severe (6/21;28.5%), moderate (10/21;47.6%), and mild (5/21;23.8%). The most prevalent protein deficiency was the band 3 protein detected in ten Tunisian HS patients. The EMA test showed a high specificity (97.5%) and sensitivity (94.7%) for HS diagnosis compared to the other screening tests. Interestingly, fourteen among sixteen patients presenting with homozygous sickle cells HbSS showed an increase of EMA fluorescence intensity compared to other anemic patients. CONCLUSION: Our study highlights the efficiency of the EMA dye for the detection of HS whatever the nature of the involved protein deficiency. We report for the first time, the most prevalent protein deficiency among Tunisians with HS. Moreover, we found that the combination of the EMA-binding test with PT or incubated OFT improves the diagnosis sensitivity while maintaining a good specificity.

First Observation of HbM-Saskatoon at the Origin of Neonatal Cyanosis in a Tunisian Baby.

Several causes are known to be at the origin of neonatal cyanosis among them methemoglobinemia is by inheritance of an hemoglobin (Hb) M variant. This is a rare condition never been reported in Tunisia so far. Here, we report a Tunisian newborn with refractory cyanosis since birth. As cardiac and respiratory diseases were ruled out, methemoglobinemia was suspected. Hematological parameters, concentration of methemoglobin, capillary electrophoresis, and amplification sequencing of the HBB gene were performed. Computational analysis was achieved by different in silico tools to investigate the mutation effect. The diagnosis was established by a raised MetHb, confirmed by the presence HbM-Saskatoon [Beta63 (E7) His>Tyr] by capillary electrophoresis and molecular analysis. The identified mutation occurred as a de novo mutation. In silico analysis confirmed the pathogenicity of the mutation. To our knowledge, this is the first time that this mutation has been reported in the Tunisian population. In view of its low incidence rate, clinicians might misdiagnose cyanosis caused by HbM, which can lead to inappropriate treatment and clinical complications. An up-to-date literature review of HbM disease is presented in this study.

Chronic myeloid leukemia patients in Tunisia: epidemiology and outcome in the imatinib era (a multicentric experience).

Data are limited in developing countries regarding the clinicopathologic features and response to therapy of chronic myeloid leukemia (CML) in the era of imatinib (IM). The objective of this study is to report on the clinicoepidemiologic features of CML in Tunisia, to evaluate the long-term outcome of patients in chronic (CP) or accelerated phase (AP) treated with IM 400 mg daily as frontline therapy, and to determine imatinib's efficacy and safety. From October 2002 to December 2014, 410 CML patients were treated with IM in six Tunisian departments of hematology. Response (hematologic, cytogenetic, and molecular responses) and outcome-overall survival (OS), event-free survival (EFS), and progression-free survival (PFS)-were evaluated. The following prognostic factors were analyzed for their impact on the European leukemia net (ELN) response, OS, EFS, and PFS at 5 years: age, sex, leukocyte count, Sokal score, European Treatment and Outcome Study (EUTOS) score, CML phase, time to starting IM, and impact of adverse events. The median age was 45 years (3-85 years). Two hundred ten (51.2%) patients were male. Splenomegaly was present in 322 of the 410 (79%). Additional cytogenetic abnormalities were encountered in 25 (6.3%) patients. At diagnosis, 379 (92.4%) patients were in CP, 31 (7.6%) were in AP. The Sokal risk was low in 87 (22.5%), intermediate in 138 (35.7%), and high in 164 patients (41.9%). The EUTOS risk was low in 217 (74%), and high in 77 (26%) patients. The rates of cumulative complete cytogenetic response (CCyR), major molecular response (MMR), and molecular response 4/5 log (MR4.5) in CP/AP-CML patients were 72, 68.4, and 46.4%, respectively. The median time to reach CCyR, MMR, and MR4.5 was 6 months (3-51), 18 months (3-72), and 24 months (3-100), respectively. According to the ELN criteria, optimal, suboptimal response, and failure were noted in 206 (51.8%), 61 (15.3%), and 125 (31.4%) patients, respectively. Five-year event-free survival (EFS), progression-free survival (PFS), and overall survival (OS) were 81, 90, and 90%, respectively. By multivariate analysis, AP, high EUTOS risk, and baseline WBC ≥ 150G/l remained independent predictive factors of non-optimal response to IM. The adverse events (AE) of IM were moderate and tolerable. With the caveats that the monitoring of the disease was not optimal, response rates were similar to those reported in previous studies. It is clear to us that improvements should be made in treatment of AP-CML and high Sokal risk group of CP-CML. The frontline use of second-generation tyrosine kinase inhibitor (TKI) is expected to improve the results of the first-line treatment of these high-risk Tunisian patients, but cost and accessibility of this therapy remain the problems in developing countries.

Regulatory network analysis of microRNAs and genes in imatinib-resistant chronic myeloid leukemia.

Targeted therapy in the form of selective breakpoint cluster region-abelson (BCR/ABL) tyrosine kinase inhibitor (imatinib mesylate) has successfully been introduced in the treatment of the chronic myeloid leukemia (CML). However, acquired resistance against imatinib mesylate (IM) has been reported in nearly half of patients and has been recognized as major issue in clinical practice. Multiple resistance genes and microRNAs (miRNAs) are thought to be involved in the IM resistance process. These resistance genes and miRNAs tend to interact with each other through a regulatory network. Therefore, it is crucial to study the impact of these interactions in the IM resistance process. The present study focused on miRNA and gene network analysis in order to elucidate the role of interacting elements and to understand their functional contribution in therapeutic failure. Unlike previous studies which were centered only on genes or miRNAs, the prime focus of the present study was on relationships. To this end, three regulatory networks including differentially expressed, related, and global networks were constructed and analyzed in search of similarities and differences. Regulatory associations between miRNAs and their target genes, transcription factors and miRNAs, as well as miRNAs and their host genes were also macroscopically investigated. Certain key pathways in the three networks, especially in the differentially expressed network, were featured. The differentially expressed network emerged as a fault map of IM-resistant CML. Theoretically, the IM resistance process could be prevented by correcting the included errors. The present network-based approach to study resistance miRNAs and genes might help in understanding the molecular mechanisms of IM resistance in CML as well as in the improvement of CML therapy.

Molecular monitoring of Tunisian patients with chronic myeloid leukemia.

BACKGROUND: bcr-abl fusion gene is the hallmark of chronic myeloid leukemia (CML). RQ-PCR provides an accurate measure of the total leukemia cell mass and the degree to which bcr-abl transcripts are reduced by therapy correlates with progression free survival. AIM: We report molecular assessment of residual disease in CML Tunisian patients. METHODS: Between June 2003 and December 2014 we measured bcr-abl mRNA levels in peripheral blood from all Tunisian patients by quantitative real time polymerase chain reaction (RQ-PCR). RESULTS: A total of 708 patients with a mean age of 42 years were included in this study. Based on European Leukemia Net 2013, 80% of the patients achieved an optimal response 20% were in treatment failure. 38% of the patients achieved RM4 which corresponds to a bcr-abl/abl ratio <0.01%, 13% of the patients achieved RM4.5corresponding to bcr-abl/abl ratio of 0.0032%. CONCLUSION: CML patients had a good response to tyrosine kinase inhibitors treatment. RQ-PCR is helpful in detecting any residual disease and determining the depth of the treatment response.

Association of genetic variation in IKZF1, ARID5B, CDKN2A, and CEBPE with the risk of acute lymphoblastic leukemia in Tunisian children and their contribution to racial differences in leukemia incidence.

Recent genome-wide association studies (GWAS) focusing on pediatric acute lymphoblastic leukemia (ALL), the most common malignancy in children younger than 15 years old, have found evidence that single-nucleotide polymorphisms (SNPs) in IKZF1 (7p12.2), ARID5B (10q21.2), CDKN2A (9p21.3), and CEBPE (14q11.2) are strongly associated to the risk of developing pediatric ALL. These studies have been conducted in European and Thai populations, and it is unclear whether these observations generalize to other populations with a lower incidence of pediatric ALL. In order to explore the impact of these variants on pediatric ALL risk in the Tunisian population, we genotyped 58 cases of pediatric ALL and 150 controls for SNPs rs4132601 (7p12.2), rs7089424 (10q21.2), rs3731217 (9p21.3), and rs2239633 (14q11.2). Our results, which are consistent with findings in European populations, show that 3 SNPs, i.e., rs4132601 (P = .00116, odds ratio [OR] = 2.78, 95% confidence interval [CI] = [1.42, 5.87]), rs7089424 (P = .0022, OR = 0.49, 95% CI = [0.31, 0.79]), and rs2239633 (P = .0010, OR = 0.47, 95% CI = [0.29, 0.75]) are significantly associated with a higher risk of developing pediatric ALL (P < .05). Furthermore, we show differences in allele frequencies in SNPs between Tunisian and Caucasian and/or Thai populations (e.g., CEBPE, rs2239633; population attributable risk [PAR] ∼15-fold the PAR of Thai population). These differences, combined with differences in linkage disequilibrium structure between populations and differences in size between populations, may contribute to racial differences in pediatric ALL incidence.

Immunity in the Progeroid Model of Cockayne Syndrome: Biomarkers of Pathological Aging.

Cockayne syndrome (CS) is a rare autosomal recessive disorder that affects the DNA repair process. It is a progeroid syndrome predisposing patients to accelerated aging and to increased susceptibility to respiratory infections. Here, we studied the immune status of CS patients to determine potential biomarkers associated with pathological aging. CS patients, as well as elderly and young, healthy donors, were enrolled in this study. Complete blood counts for patients and donors were assessed, immune cell subsets were analyzed using flow cytometry, and candidate cytokines were analyzed via multi-analyte ELISArray kits. In CS patients, we noticed a high percentage of lymphocytes, an increased rate of intermediate and non-classical monocytes, and a high level of pro-inflammatory cytokine IL-8. In addition, we identified an increased rate of particular subtypes of T Lymphocyte CD8+ CD28- CD27-, which are senescent T cells. Thus, an inflammatory state was found in CS patients that is similar to that observed in the elderly donors and is associated with an immunosenescence status in both groups. This could explain the CS patients' increased susceptibility to infections, which is partly due to an aging-associated inflammation process.

Genetic polymorphisms of NQO1, CYP1A1 and TPMT and susceptibility to acute lymphoblastic leukemia in a Tunisian population.

Acute lymphoblastic leukemia (ALL) is the major pediatric cancer in developed countries. The etiology of ALL remains poorly understood, with few established environmental risk factors. These risks were influenced by co-inheritance of multiple low-risk genetic polymorphisms such as variants within cytochrome P450A1 (CYP1A1), NADPH: quinone oxidoreductase (NQO1) and Thiopurine methyltransferase (TPMT) genes. In this work, we conduct a case-control study to assess the impact of CYP1A1*2A (CYP1A1 T6235C); NQO1*2 (NQO1 C609T); TPMT*2 (TPMT G238C) and TPMT A719G polymorphisms on the risk of developing ALL. The frequencies of TPMT*2, TPMT A719G, NQO1*2 and CYP1A1*2 variants were examined in 100 patients with ALL and 106 healthy controls by allele specific PCR and/or PCR-RFLP methods using blood samples. We have found that NQO1 609CT genotype was overrepresented in patients and was associated with an aggravating effect compared to the reference group with NQO1 609CC genotype (p = 0.028, OR = 1.41; CI 95 %: 1.04-1.93). However, TPMT*2, TPMT 719*G and CYP1A1*2 variants did not appear to influence ALL susceptibility (p > 0.05). Moreover we have not found a significant correlation between the studied variants and Bcr-Abl transcript. In conclusion we retain that leukemogenesis of ALL is associated with carcinogens metabolism and consequently related to environmental exposures.

The prevalence and prognostic significance of KRAS mutation in bladder cancer, chronic myeloid leukemia and colorectal cancer.

Mutations in the KRAS gene have been shown to play a key role in the pathogenesis of a variety of human tumours. However the mutational spectrum of KRAS gene differs by organ site. In this study, we have analysed the mutational spectrum of KRAS exon 1 in bladder tumours, colorectal cancer (CRC) and chronic myeloid leukemia (CML). A total of 366 patients were included in the present study (234 bladder tumours, 48 CRC and 84 CML). The KRAS mutations are absent in BCR/ABL1 positive CML. This result suggests that BCR/ABL1 fusion gene and KRAS mutations were mutually exclusive. The frequency of KRAS mutations in bladder cancer was estimated at 4.27 %. All of mutations were found in codon 12 and 90 % of them were detected in advanced bladder tumours. However the correlation between KRAS mutations and tumour stage and grade does not report a statistical significant association. The KRAS mutations occur in 35.41 % of patients with CRC. The most frequent mutations were G12C, G12D and G13D. These mutations were significantly correlated with histological differentiation of CRC (p = 0.024). Although the high frequency of KRAS in CRC in comparison to bladder cancer, these two cancers appear to have the same mutational spectrum (p > 0.05).

[Does multiple myeloma response to induction therapy depend on plasma cell IL6 receptor gene expression?]. / L'expression de l'IL6-r par les plasmocytes influe-t-elle la réponse au traitement d'induction au cours du myélome multiple ?

BACKGROUND: Interleukine 6 (IL-6) is the most important cytokine involved in malignant plasma cells growth and survival. AIM: To analyse bone marrow plasma cells IL6 receptor gene expression in both multiple myeloma patients at diagnosis and healthy bone marrow donors. METHODS: Clinical and biological patients' features and responses to Dexamethasone-Thalidomide induction therapy were gathered. 47 patients and 16 case controls were analyzed: Bone marrow plasma cells were isolated; and IL6 receptor gene expression was quantified using Taqman quantitative PCr technology and 2-ΔCT formula. RESULTS: Quantitative and qualitative IL6 receptor gene expression were negatively correlated with the degree of response to therapy (p= 0.02). In this study, plasma cells IL6 receptor gene expression seems to be decisive in predicting the response to treatment. CONCLUSION: Understanding the mechanisms involved in plasma cells IL6 receptor gene expression may offer a better appreciation of the physiopathologic and anti-oncogenic ways of drug resistance in multiple myeloma and consequently the discovery of new specific drugs.

Identification of High-Risk Single Nucleotide Polymorphisms in the Human CYB5R3 Gene Responsible for Recessive Congenital Methemoglobinemia: A Computational Approach.

Introduction: NADH-cytochrome b5 reductase deficiency due to pathogenic variants in the CYB5R3 gene causes recessive congenital methemoglobinemia (RCM) type I or type II. In type I, cyanosis from birth is the only major symptom, and the enzyme deficiency is restricted only to erythrocytes. Whereas in type II, cyanosis is associated with severe neurological manifestations, and the enzyme deficiency is generalized to all tissues. Methods: In this study, several computational methods (SIFT, Polyphen-2, PROVEAN, Mutation Assessor, Panther, Phd-SNP, SNPs&GO, SNAP2, Align, GVGD, MutPred2, I-Mutant 2.0, MUpro, Duet, ConSurf and Netsurf-2.0 tools) were used to find the most deleterious nsSNPs in the CYB5R3 gene. Furthermore, structural analysis by Swiss-PDB viewer, protein-ligand docking using FTSite, and protein-protein interaction using STRING were carried out to evaluate the impact of these nsSNPs on the protein structure and function. Results: Our in silico analysis suggested that out of 339 nsSNPs of the CYB5R3 gene, 17 (L47H, L47P, R61P, L73R G76D, G76C, P96H, G104C, S128P, G144D, P145S, L149P, Y151H, M177T, I178T, I216N, and G251V), are the most deleterious. Among them, two (P96H and S128P) were reported to be associated with the severe form RCM type II, six are related to RCM type I (G104C, G144D, P145S, L149P, M177T, and I178T), and the remaining nine high-risk nsSNPs have not yet been reported in RCM patients. Discussion: This study highlighted the potential pathogenic nsSNPs of the CYB5R3 gene. To comprehend how these most harmful nsSNPs contribute to disease, it is crucial to experimentally validate their functional effects.

Molecular characterization of a novel homozygous deletion in ß-globin cluster causing (δß)0-Thalassemia among Tunisian family.

BACKGROUND: Deletions in the ß-globin cluster are uncommon and cause thalassemia (thal) with hereditary persistence of fetal hemoglobin. They constitute a heterogenous group of disorders characterized by absent or reduced synthesis of adult hemoglobin (Hb A) and increased synthesis of fetal hemoglobin (Hb F). Although the clinical severity of these disorders are asymptomatic owing to the increased Hb F levels, the molecular basis is very heterogenous due to the large deletions in the ß-globin cluster spanning both HBD and HBB genes. Here, we describe a Tunisian family carrying a novel deletion mutation causing (δß)°-thalassemia. METHODS: The amounts of hemoglobin fractions were measured by capillary electrophoresis of hemoglobin. Amplification and sequencing of different regions on the ß-gene cluster were performed by Sanger method. RESULTS: Family study and genetic analysis revealed a large deletion mutation in the ß-globin cluster of 14.5 kb (NG_000,007.3:g. 58,253 to g.72837del14584) at the homozygous state in the patient and at heterozygous state at the other members of the family. This deletion removes the HBD and HBB genes. CONCLUSIONS: In our knowledge, this new large deletion is described for the first time in the Tunisian population and in the world, designed Tunisian(δß)0 in Ithanet database (IthaID: 3971). Therefore, it is important to identify the deletion leading to δß-thalassemia carriers at the molecular level, to highlight the importance of recognizing the clinical features and implementing appropriate testing to clarify the diagnosis and manage the condition.

TP53 Gene 72 Arg/Pro (rs1042522) single nucleotide polymorphism increases the risk and the severity of chronic lymphocytic leukemia.

Background: Genetic variations in TP53 gene are known to be important in chronic lymphocytic leukemia (CLL) and may cause its inactivation which is associated with an aggressive form of the disease. Single nucleotide polymorphism (rs1042522:G>C) in TP53 gene at codon 72 encodes for arginine (Arg) or proline (Pro) variant which results in amino acid substitution affecting the apoptotic potential of TP53 protein. The aim of this study was to assess the correlation between TP53 codon 72 polymorphism and risk susceptibility as well as severity of CLL among Tunisian patients. Materials and methods: A case-control study was conducted in Tunisia from February 2019 to November 2021, 160 de novo CLL patients and 160 healthy volunteers matched in age and gender were involved. DNA was extracted from peripheral blood mononuclear cells and the rs1042522 was analyzed using PCR-RFLP. Results: Pro variant was associated with higher susceptibility to CLL than Arg variant (p= 0.023). A significant association was found between Pro variant and prognostic classification of Binet stage C (p= 0.001), low hemoglobin level (p= 0.003) and low platelet count (p= 0.016). Conclusion: We suggest that Pro variant may increase the risk of developing CLL in our population and could be associated with the severity of the disease.