Genetic Correlation of HBB, HFE and HAMP Genes to Endocrinal Complications in Egyptian Beta Thalassemia Major Patients.

Iron loading is regarded as the primary cause of endocrine abnormalities in thalassemia major patients. Thus, the purpose of the current research was to explore the impact of thalassemia genotypes, hepcidin antimicrobial peptide (HAMP) and hereditary hemochromatosis (HFE) gene variants, and hepcidin expression on serum ferritin and endocrinal complications in thalassemia patients. The study comprised fifty beta-thalassemia cases and fifty age- and sex-matched controls. Genotyping of the Beta-globin gene (HBB), HAMP, and exon 2 of the HFE gene was performed using Sanger sequencing. C282Y (c.845G > A) variant of the HFE gene was determined by PCR-RFLP. Hepcidin mRNA expression was assessed by qRT-PCR. Biochemical and hormonal studies were done for all patients. Hypogonadism and short stature were found in 56% and 20% of the investigated cases, respectively. Molecular studies reported a statistically higher frequency of the HAMP variant c.-582A > G in thalassemic patients than controls. Significant downregulation of hepcidin expression was found in cases compared to healthy subjects that was significantly associated with short stature. Considering the thalassemia alleles, the IVSI.1G > A (ß0) allele was statistically related to hypogonadism. Our results proposed that thalassemia genotypes and downregulated hepcidin expression were the potential risk factors for endocrinopathies in our cases. We also demonstrated an increased incidence of the HAMP promoter variant c.- 582A > G that might have a role in the pathogenesis of iron overload in thalassemic cases. Significant downregulation of hepcidin expression, that contributes to increased iron burden, could be used as a future therapeutic target in these patients.

Altered expression of miR-17 and miR-148b in pediatric familial mediterranean fever patients.

Familial Mediterranean fever (FMF) is a recessively inherited autoinflammatory disorder with wide phenotypic variation that has been observed among individuals who have the same genotype. Modifying genes, epigenetic factors, or environmental factors might all have an impact on genotype-phenotype correlation in FMF. The current research aims to determine the expression levels of microRNAs (miR-148b and miR-17) in Egyptian FMF participants. We also aimed to investigate Caspase -1 gene expression to make a correlation with disease severity. The study comprised 25 clinically diagnosed FMF cases and 25 healthy subjects matched for age and sex. The molecular diagnosis of FMF cases was assessed using real-time SNP genotyping assay. MiR-148b and miR-17 expression were profiled using TaqMan assay technology. The expression level of Caspase -1 gene was also verified using qRT-PCR. MiR-17 in the studied cases was significantly upregulated compared to healthy individuals (P = 0.006), whereas miR-148b was significantly downregulated in the examined patients (P = 0.030). Moreover, statistically significant upregulation of Caspase-1 expression was also elucidated in relation to normal subjects (P = 0.033). The results obtained indicated that miR-17 and miR-148b might be potential regulatory biomarkers in FMF cases. We further hypothesized that the upregulation of Caspase-1 could hint at its significance as a future therapeutic target to alleviate the inflammatory process in these patients. Key Points • The role of miRNAs in FMF and various mechanisms involved in FMF pathogenesis has received increasing attention. • Studying the expression profiles of miR-17 and miR-148b in FMF patients revealed their potential role as regulatory biomarkers in these patients. • Significant upregulation of Caspase-1 expression in FMF cases could hint at its significance as a future therapeutic target. • Future studies on larger cohorts are warranted to clarify and better understand the role of miRNAs in the pathogenesis and severity of FMF.

Studying the pathogenicity of 26 variants characterized in the first molecular analyses of Egyptian aplastic anemia patients.

BACKGROUND: Aplastic anemia (AA) is a bone marrow disorder characterized by peripheral pancytopenia and marrow hypoplasia which can lead to life-threatening complications. Our objective was to study the telomerase genes (TERT and TERC) variants, explore their relationship to telomere shortening and TERT gene expression, and to identify variants in the MPL gene within Egyptian AA patients. METHODS: Forty AA patients and 40 sex- and age-matched healthy individuals as the control group were studied through sequencing of TERT, TERC, and MPL genes. Quantitative real-time PCR (qRT-PCR) was used for measuring TERT gene expression. Telomere length (TL) was measured using the Quantitative Fluorescence In Situ Hybridization (Q-FISH) technique. In silico analysis was performed for the prediction of the pathogenicity of resultant variants. RESULTS: Sequencing of MPL, TERT, and TERC genes identified 26 variants. Eleven variants were identified in the MPL gene. Three of them are pathogenic: two missense [c.305 G>A, c.1589 C>T] and one splice site [g.9130T>G]. TERT gene sequencing showed thirteen variants, among them, four novel [c.484G>A, c.499G>A, c.512G>A, c.3164C>G] and two previously reported [c.835G>A, c.2031C>T] were predicted to be pathogenic. Two variants were characterized within the TERC gene; n.514A>G and n.463 C>T. TERT gene expression was downregulated in 70% of studied patients and the Q-FISH technique detected telomere shortening in 82.5% of patients. CONCLUSIONS: Twenty-six pathogenic and benign variants within the TERC, TERT, and MPL genes were identified among the studied AA patients that were in several cases associated with shortened telomeres and/or lower TERT gene expression. Genotype/phenotype correlation in AA patients is of great importance in explaining the disease severity and guiding therapeutic decisions.

MBL2 gene variants and susceptibility to meningitis in Egyptian patients.

BACKGROUND: Meningitis is inflammation of the membranes enclosing the brain and spinal cord. It is a fatal disease with severe morbidity and mortality. Mannose binding lectin (MBL) encoded by MBL2 gene activates complement system through lectin pathway in innate immunity to defense against the infections. OBJECTIVE: The current study aimed to investigate the promoter and exon 1 variants of MBL2 gene among Egyptian patients having meningitis to explore their role in disease susceptibility. PATIENTS AND METHODS: This case-control study, included 53 patients and 50 sex and age matched controls. MBL2 genotyping was done using Sanger sequencing. RESULTS: The frequency of one promoter (c.-290C > G) and four in exon 1 (c.161G > A, c.170G > A, c.154C > T and c.132C > T) as well as another one located in its 5'utranslated part (c.-66C > T) variants were estimated. The incidence of the four individual exonic variants was not significantly different between cases and healthy individuals (all P > 0.05). The promoter variant, c.-290C > G was found in all examined patients (84.9% of the patients in homozygote state and 15.1% of patients in heterozygous state) with a highly significant variance in the prevalence of this variant between cases and control group (p = 0.0001). Additionally, UTR variant (c.-66C > T) was also significantly higher in patients than controls (P = 0.033).In comparison with clinical outcome, it was found that c.170G > A variant named C allele was associated with favorable outcome in the studied patients (P = 0.025). CONCLUSION: The results obtained showed that the Promoter (c.-290C > G) and UTR (c.-66C > T) variants of MBL2 gene may be potential risk factors for disease susceptibility in Egyptian cases with meningitis. Our results also proposed that c.170G > A (C allele and CC genotype) could affect the severity and play a protective role in these patients. The other genetic variants of MBL2 gene, including c.132C > T, c.161G > A (A > B), and c.154C > T (A > D) that were investigated, did not show any association with susceptibility or severity of meningitis.

Dynamic disequilibrium-based pathogenicity model in mutated pyrin's B30.2 domain-Casp1/p20 complex.

BACKGROUND: The B30.2 variants lead to most relevant severity forms of familial Mediterranean fever (FMF) manifestations. The B30.2 domain plays a key role in protein-protein interaction (PPI) of pyrin with other apoptosis proteins and in regulation the cascade of inflammatory reactions. Pyrin-casp1 interaction is mainly responsible for the dysregulation of the inflammatory responses in FMF. Lower binding affinity was observed between the mutant B30.2 pyrin and casp1 without the release of the complete pathogenicity mechanism. The aim of this study was to identify the possible effects of the interface pocked residues in B30.2/SPRY-Casp1/p20 complex using molecular mechanics simulation and in silico analysis. RESULTS: It was found that Lys671Met, Ser703Ile, and Ala744Ser variants led mainly to shift of the binding affinity (∆G), dissociation constant (Kd), and root mean square deviation (RMSD) in B30.2/SPRY-Casp1/p20 complex leading to dynamic disequilibrium of the p20-B30.2/SPRY complex toward its complex form. The current pathogenicity model and its predicted implementation in the relevant colchicine dosage were delineated. CONCLUSION: The molecular mechanics analysis of B30.2/SPRY-p20 complex harboring Lys671Met, Ser703Ile, and Ala744Ser variants showed dynamic disequilibrium of B30.2/SPRY-casp1/p20complex in context of the studied variants that could be a new computational model for FMF pathogenicity. This study also highlighted the specific biochemical markers that could be useful to adjust the colchicine dose in FMF patients.