Identification of gene mutations associated with type 1 diabetes by next-generation sequencing in affected Palestinian families.

Introduction: Diabetes Mellitus is a group of metabolic disorders characterized by hyperglycemia secondary to insulin resistance or deficiency. It is considered a major health problem worldwide. T1DM is a result of a combination of genetics, epigenetics, and environmental factors. Several genes have been associated with T1DM, including HLA, INS, CTLA4, and PTPN22. However, none of these findings have been based on linkage analysis because it is rare to find families with several diabetic individuals. Two Palestinian families with several afflicted members with variations in the mode of inheritance were identified and selected for this study. This study aimed to identify the putative causative gene(s) responsible for T1DM development in these families to improve our understanding of the molecular genetics of the disease. Methods: One afflicted member from each family was selected for Whole-Exome Sequencing. Data were mapped to the reference of the human genome, and the resulting VCF file data were filtered. The variants with the highest phenotype correlation score were checked by Sanger sequencing for all family members. The confirmed variants were analyzed in silico by bioinformatics tools. Results: In one family, the IGF1R p.V579F variant, which follows autosomal dominant inheritance, was confirmed and segregated in the family. In another family, the NEUROD1 p.P197H variant, which follows autosomal recessive inheritance, was positively confirmed and segregated. Conclusion: IGF1R p.V579F and NEUROD1 p.P197H variants were associated with T1DM development in the two inflicted families. Further analysis and functional assays will be performed, including the generation of mutant model cell systems, to unravel their specific molecular mechanism in the disease development.

Iron metabolism in autism spectrum disorder; inference through single nucleotide polymorphisms in key iron metabolism genes.

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental problems with various genetic and environmental components. The ASD diagnosis is based on symptom expression without reliance on any biomarkers. The genetic contributions in ASD remain elusive. Various studies have linked ASD with iron. Since iron plays a crucial role in brain development, neurotransmitter synthesis, neuronal myelination and mitochondrial function, we hypothesized that iron dysregulation in the brain could play a role and contribute to the pathogenesis of ASD. In this study, we investigated single nucleotide polymorphisms in ASD in various iron metabolism genes, including the Transferrin Receptor (TFRC) gene (rs11915082), the Solute Carrier Family 11 Member 2 (SLC11A2) gene (rs1048230 and rs224589), the Solute Carrier Family 40 Member 1 (SLC40A1) gene (rs1439816), and hepcidin antimicrobial peptide (HAMP) gene (rs10421768). We recruited 48 patients with ASD and 88 matched non-ASD controls. Our results revealed a significant difference between ASD and controls in the G allele of the TFRC gene rs11915082, and in the C allele of the SLC40A1 gene rs1439816. In silico analysis demonstrated potential positive role of the indicated genetic variations in ASD development and pathogenesis. These results suggest that specific genetic variations in iron metabolism genes may represent part of early genetic markers for early diagnosis of ASD. A significant effect of SNPs, groups (ASD/control) as well as interaction between SNPs and groups was revealed. Follow-up post hoc tests showed a significant difference between the ASD and control groups in rs11915082 (TFRC gene) and rs1439816 (SLC40A1 gene). Backward conditional logistic regression using both the genotype and allele data showed similar ability in detecting ASD using allel model (Nagelkerke R2 = 0.350 p = 0.967; Variables: rs1439816, rs11915082) compared to genotype model (Nagelkerke R2 = 0.347, p = 0.430; Variables: rs1439816 G, rs1439816 C, rs10421768 A). ROC curve showed 54% sensitivity in detecting ASD compared to 47% for the genotype model. Both models differentiated controls with high accuracy; the allele model had a specificity of 91% compared to 92% for the genotype model. In conclusion, our findings suggest that specific genetic variations in iron metabolism may represent early biomarkers for a diagnosis of ASD. Further research is needed to correlate these markers with specific blood iron indicators and their contribution to brain development and behavior.

Genetic screening of familial Mediterranean fever mutations in the Palestinian population.

OBJECTIVE: To investigate the spectrum of mutations and genotypes in the pyrin gene in familial Mediterranean fever (FMF) patients. METHODS: Blood samples of 511 suspected FMF patients, received from the Molecular Genetics Laboratory, Makassed Islamic Charitable Hospital, Mount Olives, Jerusalem during the period from June 1999 to August 2004, were investigated by genotyping 24 different MEFV mutations. RESULTS: Our work revealed the presence of 14 different mutations from the identified 24 mutations in the gene which are assembled in 6 homozygous, 9 heterozygous and 16 compound heterozygous genotypes. The homozygous genotypes represent the predominant format among our patients representing approximately 38% of the revealed genotypes. Interestingly, in 94 (31.4%) of the tested subjects, only one mutation in the pyrin gene could be identified while the other mutant allele remains unidentified. Moreover, the genotype of 3 (1%) patients revealed the presence of triplet mutations in the pyrin gene. CONCLUSION: The results of our study clearly suggest that the origin of FMF among the Palestinian population is mostly homozygous. The identification of a significant number of patients with one known mutation indicates potentially the presence of new mutations in the gene which will be investigated in the future.

Diabetes mellitus: The epidemic of the century.

The epidemic nature of diabetes mellitus in different regions is reviewed. The Middle East and North Africa region has the highest prevalence of diabetes in adults (10.9%) whereas, the Western Pacific region has the highest number of adults diagnosed with diabetes and has countries with the highest prevalence of diabetes (37.5%). Different classes of diabetes mellitus, type 1, type 2, gestational diabetes and other types of diabetes mellitus are compared in terms of diagnostic criteria, etiology and genetics. The molecular genetics of diabetes received extensive attention in recent years by many prominent investigators and research groups in the biomedical field. A large array of mutations and single nucleotide polymorphisms in genes that play a role in the various steps and pathways involved in glucose metabolism and the development, control and function of pancreatic cells at various levels are reviewed. The major advances in the molecular understanding of diabetes in relation to the different types of diabetes in comparison to the previous understanding in this field are briefly reviewed here. Despite the accumulation of extensive data at the molecular and cellular levels, the mechanism of diabetes development and complications are still not fully understood. Definitely, more extensive research is needed in this field that will eventually reflect on the ultimate objective to improve diagnoses, therapy and minimize the chance of chronic complications development.

Total Antioxidant Status in Type 2 Diabetic Patients in Palestine.

The objective of this study was to compare the level of total antioxidant status (TAS) in type 2 diabetic and normal Palestinian subjects as well as the major factors influencing TAS levels. A sample of convenience composed of 212 type 2 diabetic and 208 normal subjects above the age of 40 were recruited. Only 9.8% of the subjects had normal body mass index (BMI) levels (<25), 29% were overweight (≥25 to <30), and 61.2% were obese (≥30). The mean levels of TAS were significantly higher in diabetic compared to control subjects (2.18 versus 1.84 mM Trolox, P = 0.001) and in hypertensive subjects compared to subjects with normal blood pressure (BP). Mean TAS levels were higher in obese compared to nonobese subjects (2.12 versus 1.85 mM Trolox, P = 0.001). Mean TAS levels were similarly higher in subjects with high fasting plasma glucose (FPG) compared to normal FPG (2.19 versus 1.90 mM Trolox) and high HbA1c (≥6.5%) compared to HbA1c < 6.5% (2.14 versus 1.91 mM Trolox). Multivariate analysis revealed that only diabetic status (P = 0.032) and the level of education (P = 0.036) were significantly associated with TAS. In conclusion diabetic patients had 18.5% increase in TAS levels compared to control subjects.

Evaluation of glycated hemoglobin (HbA1c) for diagnosing type 2 diabetes and prediabetes among Palestinian Arab population.

The purpose of the study is to compare the potential of HbA1c to diagnose diabetes among Palestinian Arabs compared to fasting plasma glucose (FPG). A cross-sectional sample of 1370 Palestinian men (468) and women (902) without known diabetes and above the age of 30 years were recruited. Whole blood was used to estimate HbA(1c) and plasma for FPG and total lipid profile. Fasting plasma glucose was used as a reference to diagnose diabetes (≥ 126 mg/dL) and prediabetes (100-125 mg/dL). The area under the receiver operating characteristic curve (AUC) for HbA(1c) was 81.9% to diagnose diabetes and 63.9% for prediabetes. The agreement between HbA(1c) and diabetes as diagnosed by FPG was moderate (ĸ  =  0.498) and low with prediabetes (ĸ = 0.142). The optimal cut-off value for HbA1c to diagnose diabetes was ≥ 6.3% (45 mmol/mol). The sensitivity, specificity and the discriminant ability were 65.6% (53.1-76.3%), 94.5% (93.1-95.6%), 80.0% (72.8-87.3%), respectively. However, using cut-off value of ≥ 6.5% (48 mmol/mol) improved specificity. At this cut-off value, the sensitivity, specificity and the discriminant ability were 57.4% (44.9-69.0%), 97.1% (96.0-97.9%) and 77.3% (71.0-83.5%). For diagnosing prediabetes with HbA1c between 5.7-6.4% (39-46 mmol/mol), the sensitivity, specificity and the discriminant ability were 62.7% (57.1-67.9%), 56.3% (53.1-59.4%) and 59.5% (56.3-62.5%), respectively. HbA(1c) at cut-off value of ≥ 6.5% (48 mmol/mol) by itself diagnosed 5.3% and 48.3% as having diabetes and prediabetes compared to 4.5% and 24.2% using FPG, respectively. Mean HbA(1c) and FPG increase significantly with increasing body mass index. In conclusion, the ROC curves showed HbA1c could be used for diagnosing diabetes when compared to FPG but not for prediabetes in Palestinians Arabs even though only about 50% of the diabetic subjects were identified by the both HbA1c and FPG.

Spectrum of beta-globin gene mutations among thalassemia patients in the West Bank region of Palestine.

beta-Thalassemia (thal) is an autosomal recessive disorder that results in hypochromic hemolytic anemia in affected patients. In the West Bank area of Palestine, the prevalence of beta-thal trait is approximately 3.5% among the population, with an estimated 120,000 carriers. Seventeen beta-globin gene mutations could be identified in 148 patients using polymerase chain reaction (PCR), amplification refractory mutation system (ARMS)-PCR and direct sequencing. The predominant mutations included: IVS-I-6 (T --> C) (28.7%), IVS-I-110 (G --> A) (17.6%), codon 37 (G --> A) (10.4%), IVS-I-1 (G --> A) (9%), codons 106/107 (+ G) (6.8%) and codon 39 (C --> T) (4.6%). Other less frequent and rare mutations included: IVS-II-1 (G --> A), codon 5 (-CT), IVS-II-848 (C --> A), -30 (T --> A), codons 8/9 (+ G), IVS-I-5 (G --> C), -28 (A --> C), IVS-II-745 (C --> G), codon 6(-A), codon 27 (G --> T) and codon 30 (AGG --> ACG). Most patients (62.2%) were homozygous for one type of mutation, while the rest (27.3%) were compound heterozygotes. Some patients were heterozygous for beta-thal and sickle cell anemia traits. No mutations could be detected in both alleles of eight patients, while in seven patients only one mutant allele could be detected. Further investigations are needed to resolve the corresponding genotypes of these patients. This study represents a comprehensive investigation of the type, frequency, and distribution of thalassemia mutations among the Palestinian population in the West Bank region of Palestine. A degree of similarity and significant variations was evident in the type and frequency of mutations when the present mutations profile was compared with similar ones among various Arab and non Arab populations. The association between the identified mutations and the corresponding genotypes of our patients with specific polymorphism frameworks in the beta-globin gene was performed and the results revealed linkage disequilibrium.