Mamdooh Gari

BRAF mutations in thyroid tumors from an ethnically diverse group

BackgroundThe molecular etiology of thyroid carcinoma (TC) and other thyroid diseases which may present malignant precursor lesions is not fully explored yet. The purpose of this study was to estimate frequency, type and clinicopathological value of BRAF exon 15 mutations in different types of cancerous and non-cancerous thyroid lesions originating in an ethnically diverse population.MethodsBRAF exon 15 was sequenced in 381 cases of thyroid lesions including Hashimoto´s thyroiditis, nodular goiters, hyperplastic nodules, follicular adenomas (FA), papillary TC (PTC), follicular variant PTC (FVPTC), microcarcinomas of PTC (micro PTC; tumor size ≤ 1 cm), follicular TC (FTC), and non-well differentiated TC (non-WDTC).ResultsWe identified BRAF mutations in one of 69 FA, 72 of 115 (63%) PTC, seven of 42 (17%) FVPTC, 10 of 56 (18%) micro PTC, one of 17 (6%) FTC, and one of eight (13%) non-WDTC. Most of the cases showed the common V600E mutation. One case each of PTC, FVPTC, and FTC harbored a K601E mutation. A novel BRAF mutation was identified in a FA leading to deletion of threonine at codon 599 (p.T599del). A rare 3-base pair insertion was detected in a stage III PTC resulting in duplication of threonine at codon 599 (p.T599dup). Patients with PTC harboring no BRAF mutation (BRAFwt) were on average younger than those with a BRAF mutation (BRAFmut) in the PTC (36.6 years vs. 43.8 years). Older age (≥ 45 years) in patients with PTC was significantly associated with tumor size ≥ 4 cm (P = 0.018), vessel invasion (P = 0.004), and distant metastasis (P = 0.001). Lymph node (LN) involvement in PTC significantly correlated with tumor size (P = 0.044), and vessel invasion (P = 0.013). Of notice, taken the whole TC group, family history of thyroid disease positively correlated with capsular invasion (P = 0.025).ConclusionsOlder age is manifold associated with unfavorable tumor markers in our series. The K601E identified in a PTC, FVPTC, and FTC seems to be more distributed among different histological types of TC than previously thought. The T599del is a yet undescribed mutation and the rare T599dup has not been reported as a mutation in PTC so far.

Methylation of the Polycomb Group Target Genes Is a Possible Biomarker for Favorable Prognosis in Colorectal Cancer

Background: Colorectal cancer (CRC) is the second most common cancer in the Kingdom of Saudi Arabia with ever increasing incidence rates. DNA methylation is a common event in CRC where it is now considered an important phenomenon in CRC carcinogenesis and useful for the classification and prognosis of CRC. Methods: To gain insight into the molecular mechanisms underpinning CRC in Saudi Arabian patients, we profiled the DNA methylation frequency of key genes (MLH1, MSH2, RASSF1A, SLIT2, HIC1, MGMT, SFRP1, MYOD1, APC, CDKN2A, as well as five CIMP markers) in 120 sporadic CRC cases. CRC tumors originating from the rectum, left, and right colons are represented in this cohort of formalin-fixed paraffin-embedded tissues. Results: The most common methylation frequency was detected in the polycomb group target genes (PCGT) including SFRP1 (70%), MYOD1 (60.8%), HIC1 (61.7%), and SLIT2 (56.7%). In addition, MGMT methylation was detected at a high frequency (68.3%). RASSF1A, APC, and CDKN2A methylation frequencies were 42.5%, 25%, and 32.8%, respectively. K-means clustering analysis of the methylation events results in the clustering of the CRC samples into three groups depending on the level of methylation detected. Conclusion: Group II (PCGT methylation and CIMP-negative) methylation signature carried a favorable prognosis for male patients, whereas older patients with group I rare methylation signature have a potentially poorer clinical outcome. Impact: Methylation of the PCGT genes along with RASSF1A, APC, and MGMT can be potentially used as a new biomarker for the classification and prognosis of CRC tumors and independently of where the tumor has originated. Cancer Epidemiol Biomarkers Prev; 21(11); 2069–75. ©2012 AACR.

Next Generation Sequencing of Acute Myeloid Leukemia: Influencing Prognosis

Acute myeloid leukemia (AML) is a clonal disorder of the blood forming cells characterized by accumulation of immature blast cells in the bone marrow and peripheral blood. Being a heterogeneous disease, AML has been the subject of numerous studies that focus on unraveling the clinical, cellular and molecular variations with the aim to better understand and treat the disease. Cytogenetic-risk stratification of AML is well established and commonly used by clinicians in therapeutic management of cases with chromosomal abnormalities. Successive inclusion of novel molecular abnormalities has substantially modified the classification and understanding of AML in the past decade. With the advent of next generation sequencing (NGS) technologies the discovery of novel molecular abnormalities has accelerated. NGS has been successfully used in several studies and has provided an unprecedented overview of molecular aberrations as well as the underlying clonal evolution in AML. The extended spectrum of abnormalities discovered by NGS is currently under extensive validation for their prognostic and therapeutic values. In this review we highlight the recent advances in the understanding of AML in the NGS era.

Reference genes for expression studies in hypoxia and hyperglycemia models in human umbilical vein endothelial cells.

Human umbilical vein endothelial cell (HUVEC)-based gene expression studies performed under hypoxia and/or hyperglycemia show huge potential for modeling endothelial cell response in cardiovascular disease and diabetes. However, such studies require reference genes that are stable across the whole range of experimental conditions. These reference genes have not been comprehensively defined to date. We applied human genome-wide microarrays and quantitative real-time PCR (qRT-PCR) on RNA obtained from primary HUVEC cultures that were incubated for 24 hr either in euglycemic or in hyperglycemic conditions and then subjected to short-term CoCl2-induced hypoxia for 1, 3, or 12 hr. Using whole-transcript arrays, we selected 10 commonly used reference genes with no significant expression variation across eight different conditions. These genes were ranked using NormFinder software according to their stability values. Consequently, five genes were selected for validation by qRT-PCR. These were ribosomal protein large P0 (RPLP0), transferrin receptor (TFRC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), and β-actin (ACTB). All five genes displayed stable expression under hyperglycemia. However, only RPLP0 and TFRC genes were stable under hypoxia up to 12 hr. Under hyperglycemia combined with hypoxia up to 12 hr, the expression of RPLP0, TFRC, GUSB, and ACTB genes remained unchanged. Our findings strongly confirm that RPLP0 and TFRC are the most suitable reference genes for HUVEC gene expression experiments subjected to hypoxia and/or hyperglycemia for the given experimental conditions. We provide further evidence that even commonly known references genes require experimental validation for all conditions involved.

Detection of FLT3 Oncogene Mutations in Acute Myeloid Leukemia Using Conformation Sensitive Gel Electrophoresis

FLT3 (fms-related tyrosine kinase 3) is a receptor tyrosine kinase class III that is expressed on by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. FLT3 is also expressed on leukemia blasts in most cases of acute myeloid leukemia (AML). In order to determine the frequency of FLT3 oncogene mutations, we analyzed genomic DNA of adult de novo acute myeloid leukemia (AML). Polymerase chain reaction (PCR) and conformation-sensitive gel electrophoresis (CSGE) were used for FLT3 exons 11, 14, and 15, followed by direct DNA sequencing. Two different types of functionally important FLT 3 mutations have been identified. Those mutations were unique to patients with inv(16), t(15:17) or t(8;21) and comprised fifteen cases with internal tandem duplication (ITD) mutation in the juxtamembrane domain and eleven cases with point mutation (exon 20, Asp835Tyr). The high frequency of the flt3 proto-oncogene mutations in acute myeloid leukemia AML suggests a key role for the receptor function. The association of FLT3 mutations with chromosomal abnormalities invites speculation as to the link between these two changes in the pathogenesis of acute myeloid leukemiaAML. Furthermore, CSGE method has shown to be a rapid and sensitive screening method for detection of nucleotide alteration in FLT3 gene. Finally, this study reports, for the first time in Saudi Arabia, mutations in the human FLT3 gene in acute myeloid leukemia AML patients.

Effect of BRAF mutational status on expression profiles in conventional papillary thyroid carcinomas

BackgroundWhereas 40 % to 70 % of papillary thyroid carcinomas (PTCs) are characterized by a BRAF mutation (BRAFmut), unified biomarkers for the genetically heterogeneous group of BRAF wild type (BRAFwt) PTCs are not established yet. Using state-of-the-art technology we compared RNA expression profiles between conventional BRAFwt and BRAFmut PTCs.MethodsMicroarrays covering 36,079 reference sequences were used to generate whole transcript expression profiles in 11 BRAFwt PTCs including five micro PTCs, 14 BRAFmut PTCs, and 7 normal thyroid specimens. A p-value with a false discovery rate (FDR) < 0.05 and a fold change > 2 were used as a threshold of significance for differential expression. Network and pathway utilities were employed to interpret significance of expression data. BRAF mutational status was established by direct sequencing the hotspot region of exon 15.ResultsWe identified 237 annotated genes that were significantly differentially expressed between BRAFwt and BRAFmut PTCs. Of these, 110 genes were down- and 127 were upregulated in BRAFwt compared to BRAFmut PTCs. A number of molecules involved in thyroid hormone metabolism including thyroid peroxidase (TPO) were differentially expressed between both groups. Among cancer-associated molecules were ERBB3 that was downregulated and ERBB4 that was upregulated in BRAFwt PTCs. Two microRNAs were significantly differentially expressed of which miR492 bears predicted functions relevant to thyroid-specific molecules. The protein kinase A (PKA) and the G protein-coupled receptor pathways were identified as significantly related signaling cascades to the gene set of 237 genes. Furthermore, a network of interacting molecules was predicted on basis of the differentially expressed gene set.ConclusionsThe expression study focusing on affected genes that are differentially expressed between BRAFwt and BRAFmut conventional PTCs identified a number of molecules which are connected in a network and affect important canonical pathways. The identified gene set adds to our understanding of the tumor biology of BRAFwt and BRAFmut PTCs and contains genes/biomarkers of interest.

Characterization of familial breast cancer in Saudi Arabia

BackgroundThe contribution of genetic factors to the development of breast cancer in the admixed and consanguineous population of the western region of Saudi Arabia is thought to be significant as the disease is early onset. The current protocols of continuous clinical follow-up of relatives of such patients are costly and cause a burden on the usually over-stretched medical resources. Discovering the significant contribution of BRCA1/2 mutations to breast cancer susceptibility allowed for the design of genetic tests that allows the medical practitioner to focus the care for those who need it most. However, BRCA1/2 mutations do not account for all breast cancer susceptibility genes and there are other genetic factors, known and unknown that may play a role in the development of such disease. The advent of whole-exome sequencing is offering a unique opportunity to identify the breast cancer susceptibility genes in each family of sufferers. The polymorphisms/mutations identified will then allow for personalizing the genetic screening tests accordingly. To this end, we have performed whole-exome sequencing of seven breast cancer patients with positive family history of the disease using the Agilent SureSelect™ Whole-Exome Enrichment kit and sequencing on the SOLiD™ platform.ResultsWe have identified several coding single nucleotide variations that were either novel or rare affecting genes controlling DNA repair in the BRCA1/2 pathway.ConclusionThe disruption of DNA repair pathways is very likely to contribute to breast cancer susceptibility in the Saudi population.

Consanguinity in Saudi Arabia: a unique opportunity for pediatric kidney research.

Identification of disease-related genes is a critical step in understanding the molecular basis of disease and developing targeted therapies. The genetic study of diseases occurring in the offspring of consanguineous unions is a powerful way to discover new disease genes. Pediatric nephrology provides an excellent example because ∼70% of cases of kidney disease in childhood are congenital with a likely genetic basis. This percentage is likely to be even higher in countries with a high consanguinity rate, such as the Kingdom of Saudi Arabia. However, there are a number of challenges, such as cultural, legal, and religious restrictions, that should be appreciated before carrying out genetic research in a tradition-bound country. In this article, we discuss the background, opportunities, and challenges involved with this unique opportunity to conduct studies of such genetic disorders. Keys to success include collaboration and an understanding of local traditions and laws.

Five novel glucose-6-phosphate dehydrogenase deficiency haplotypes correlating with disease severity

BackgroundGlucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) deficiency is caused by one or more mutations in the G6PD gene on chromosome X. An association between enzyme levels and gene haplotypes remains to be established.MethodsIn this study, we determined G6PD enzyme levels and sequenced the coding region, including the intron-exon boundaries, in a group of individuals (163 males and 86 females) who were referred to the clinic with suspected G6PD deficiency. The sequence data were analysed by physical linkage analysis and PHASE haplotype reconstruction.ResultsAll previously reported G6PD missense changes, including the AURES, MEDITERRANEAN, A-, SIBARI, VIANGCHAN and ANANT, were identified in our cohort. The AURES mutation (p.Ile48Thr) was the most common variant in the cohort (30% in males patients) followed by the Mediterranean variant (p.Ser188Phe) detectable in 17.79% in male patients. Variant forms of the A- mutation (p.Val68Met, p.Asn126Asp or a combination of both) were detectable in 15.33% of the male patients. However, unique to this study, several of such mutations co-existed in the same patient as shown by physical linkage in males or PHASE haplotype reconstruction in females. Based on 6 non-synonymous variants of G6PD, 13 different haplotypes (13 in males, 8 in females) were identified. Five of these were previously unreported (Jeddah A, B, C, D and E) and were defined by previously unreported combinations of extant mutations where patients harbouring these haplotypes exhibited severe G6PD deficiency.ConclusionsOur findings will help design a focused population screening approach and provide better management for G6PD deficiency patients.

The Case | Renal tubular acidosis and eye findings

The patient presented at 4.5 years of age with marked developmental delay and stunted growth. She could only walk with support and also had delayed speech development. She was born at full term to consanguineous parents with no prenatal complications. She has four healthy brothers.