Mohammad H. Al-Qahtani

Molecular genetics of human primary microcephaly: an overview

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder that is characterised by microcephaly present at birth and non-progressive mental retardation. Microcephaly is the outcome of a smaller but architecturally normal brain; the cerebral cortex exhibits a significant decrease in size. MCPH is a neurogenic mitotic disorder, though affected patients demonstrate normal neuronal migration, neuronal apoptosis and neural function. Twelve MCPH loci (MCPH1-MCPH12) have been mapped to date from various populations around the world and contain the following genes: Microcephalin, WDR62, CDK5RAP2, CASC5, ASPM, CENPJ, STIL, CEP135, CEP152, ZNF335, PHC1 and CDK6. It is predicted that MCPH gene mutations may lead to the disease phenotype due to a disturbed mitotic spindle orientation, premature chromosomal condensation, signalling response as a result of damaged DNA, microtubule dynamics, transcriptional control or a few other hidden centrosomal mechanisms that can regulate the number of neurons produced by neuronal precursor cells. Additional findings have further elucidated the microcephaly aetiology and pathophysiology, which has informed the clinical management of families suffering from MCPH. The provision of molecular diagnosis and genetic counselling may help to decrease the frequency of this disorder.

Oxidative stress and hepatitis C virus

The disproportionate imbalance between the systemic manifestation of reactive oxygen species and body’s ability to detoxify the reactive intermediates is referred to as oxidative stress. Several biological processes as well as infectious agents, physiological or environmental stress, and perturbed antioxidant response can promote oxidative stress. Oxidative stress usually happens when cells are exposed to more electrically charged reactive oxygen species (ROS) such as H2O2 or O2-. The cells’ ability to handle such pro-oxidant species is impeded by viral infections particularly within liver that plays an important role in metabolism and detoxification of harmful substances. During liver diseases (such as hepatocellular or cholestatic problems), the produced ROS are involved in transcriptional activation of a large number of cytokines and growth factors, and continued production of ROS and Reactive Nitrogen Species (RNS) feed into the vicious cycle. Many human viruses like HCV are evolved to manipulate this delicate pro- and antioxidant balance; thus generating the sustainable oxidative stress that not only causes hepatic damage but also stimulates the processes to reduce treatment of damage. In this review article, the oxidant and antioxidant pathways that are perturbed by HCV genes are discussed. In the first line of risk, the pathways of lipid metabolism present a clear danger in accumulation of viral induced ROS. Viral infection leads to decrease in cellular concentrations of glutathione (GSH) resulting in oxidation of important components of cells such as proteins, DNA and lipids as well as double strand breakage of DNA. These disorders have the tendency to lead the cells toward cirrhosis and hepatocellular carcinoma in adults due to constant insult. We have highlighted the importance of such pathways and revealed differences in the extent of oxidative stress caused by HCV infection.

Comparison of microarray expression profiles between follicular variant of papillary thyroid carcinomas and follicular adenomas of the thyroid

BackgroundFollicular variant of papillary thyroid carcinoma (FVPTC) and follicular adenoma (FA) are histologically closely related tumors and differential diagnosis remains challenging. RNA expression profiling is an established method to unravel molecular mechanisms underlying the histopathology of diseases.MethodsBRAF mutational status was established by direct sequencing the hotspot region of exon 15 in six FVPTCs and seven FAs. Whole-transcript arrays were employed to generate expression profiles in six FVPTCs, seven FAs and seven normal thyroid tissue samples. The threshold of significance for differential expression on the gene and exon level was a p-value with a false discovery rate (FDR) < 0.05 and a fold change cutoff > 2. Two dimensional average linkage hierarchical clustering was generated using differentially expressed genes. Network, pathway, and alternative splicing utilities were employed to interpret significance of expression data on the gene and exon level.ResultsExpression profiling in FVPTCs and FAs, all of which were negative for a BRAF mutation, revealed 55 transcripts that were significantly differentially expressed, 40 of which were upregulated and 15 downregulated in FVPTCs vs. FAs. Amongst the most significantly upregulated genes in FVPTCs were GABA B receptor, 2 (GABBR2), neuronal cell adhesion molecule (NRCAM), extracellular matrix protein 1 (ECM1), heparan sulfate 6-O-sulfotransferase 2 (HS6ST2), and retinoid X receptor, gamma (RXRG). The most significantly downregulated genes in FVPTCs included interaction protein for cytohesin exchange factors 1 (IPCEF1), G protein-coupled receptor 155 (GPR155), Purkinje cell protein 4 (PCP4), chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1), and glutamate receptor interacting protein 1 (GRIP1). Alternative splicing analysis detected 87 genes, 52 of which were also included in the list of 55 differentially expressed genes. Network analysis demonstrated multiple interactions for a number of differentially expressed molecules including vitamin D (1,25- dihydroxyvitamin D3) receptor (VDR), SMAD family member 9 (SMAD9), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), and RXRG.ConclusionsThis is one of the first studies using whole-transcript expression arrays to compare expression profiles between FVPTCs and FAs. A set of differentially expressed genes has been identified that contains valuable candidate genes to differentiate both histopathologically related tumor types on the molecular level.

Molecular Basis of β-Thalassemia in the Western Province of Saudi Arabia: Identification of Rare β-Thalassemia Mutations

This study aimed at the identification of the spectrum of mutations in patients with β-thalassemia (β-thal) in the western province of Saudi Arabia. Screening for the mutations was done using the polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) technique to test for 12 mutations, and direct automated DNA sequencing for the unknown samples. The study included 172 patients; of these 15 patients had sickle cell anemia and one Hb S [β6(A3)Glu→Val, GAG>GTG]/β-thal. A total of 23 mutations were identified to cause the disease in the western area. Seven common mutations were responsible for the β-thal alleles in 78% of patients and could be detected by the ARMS technique: IVS-II-1 (G>A), IVS-I-110 (G>A), IVS-I-5 (G>C), codon 39 (C>T), codon 26 (G>A) [Hb E or β26(B8)Glu→Lys, GAG>AAG], frameshift codons (FSC) 8/9 (+G), and IVS-I-1 (G>A). DNA sequencing of uncharacterized alleles detected eight less common mutations: FSC 41/42 (–TCTT), IVS-I 25 bp deletion, codon 37 (G>A), FSC 44 (–C), Cap site +1 (A>C), IVS-I-6 (T>C), FSC 5 (–CT) and IVS-I-1 (G>T), and eight rare mutations: −87 (C>G), initiation codon −1 (T>G), codon 15 (G>A), FSC 16 (–C), FSC 20/21 (+G), codon 27 (G>A), IVS-I-130 (G>C) and IVS-II-837 (A>C). Four alleles were normal by DNA sequencing. Genetic heterogeneity was observed in this study, 10 mutations were of Asian or Asian/Indian origin, two were Kurdish, one Chinese, one Turkish, one Saudi, and the remainder were of Mediterranean origin. The presence of a large population of immigrants in the western province is responsible for the great heterogeneity at the molecular level, and for the difference observed in the frequencies of mutations from those reported in the eastern province of Saudi Arabia. Screening for β-thal mutations using PCR-ARMS for the seven most frequent mutations in the Saudi population followed by DNA sequencing of the unknown alleles could be useful for the implementation of a strategy for carrier detection and preimplantation genetic diagnosis in high risk families.

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.

A novel mutation in PGAP2 gene causes developmental delay, intellectual disability, epilepsy and microcephaly in consanguineous Saudi family

PGAP2 (Post-GPI Attachment to Proteins 2) gene is involved in lipid remodeling steps of Glycosylphosphatidylinositol (GPI)-anchor maturation. At the surface of the cell this gene is required for proper expression of GPI-anchored proteins. Hyperphosphatasia with mental retardation syndrome-3 is an autosomal recessive disorder usually characterized by severe mental retardation. Mutations in the PGAP2 gene cause hyperphosphatasia mental retardation syndrome-3. We have identified a large consanguineous family from Saudi origin segregating developmental delay, intellectual disability, epilepsy and microcephaly. Whole exome sequencing with 100× coverage was performed on two affected siblings of the family. Data analysis in the patient revealed a novel missense mutation c.191C>T in PGAP2 gene resulting in Alanine to Valine substitution (Ala64Val). The mutation was reconfirmed and validated by subsequent Sanger sequencing method. The mutation was ruled out in 100 unrelated healthy controls. We suggest that this pathogenic mutation disrupts the proper function of the gene proteins resulting in the disease state.

Microcephaly‐capillary malformation syndrome: Brothers with a homozygous STAMBP mutation, uncovered by exome sequencing

We describe two brothers from a consanguineous family of Egyptian ancestry, presenting with microcephaly, apparent global developmental delay, seizures, spasticity, congenital blindness, and multiple cutaneous capillary malformations. Through exome sequencing, we uncovered a homozygous missense variant in STAMBP (p.K303R) in the two siblings, inherited from heterozygous carrier parents. Mutations in STAMBP are known to cause microcephaly‐capillary malformation syndrome (MIC‐CAP) and the phenotype in this family is consistent with this diagnosis. We compared the findings in the present brothers with those of earlier reported patients.

Genome wide analysis of novel copy number variations duplications/deletions of different epileptic patients in Saudi Arabia

BackgroundEpilepsy is genetically complex neurological disorder affecting millions of people of different age groups varying in its type and severity. Copy number variants (CNVs) are key players in the genetic etiology of numerous neurodevelopmental disorders and prior findings also revealed that chromosomal aberrations are more susceptible against the pathogenesis of epilepsy. Novel technologies, such as array comparative genomic hybridization (array-CGH), may help to uncover the pathogenic CNVs in patients with epilepsy.ResultsThis study was carried out by high density whole genome array-CGH analysis with blood DNA samples from a cohort of 22 epilepsy patients to search for CNVs associated with epilepsy. Pathogenic rearrangements which include 6p12.1 microduplications in 5 patients covering a total region of 99.9kb and 7q32.3 microdeletions in 3 patients covering a total region of 63.9kb were detected. Two genes BMP5 and PODXL were located in the predicted duplicated and deleted regions respectively. Furthermore, these CNV findings were confirmed by qPCR.ConclusionWe have described, for the first time, several novel CNVs/genes implicated in epilepsy in the Saudi population. These findings enable us to better describe the genetic variations in epilepsy, and could provide a foundation for understanding the critical regions of the genome which might be involved in the development of epilepsy.

Synthesis and Characterization of Cefotaxime Conjugated Gold Nanoparticles and Their Use to Target Drug-Resistant CTX-M-Producing Bacterial Pathogens

Multidrug‐resistance due to “β lactamases having the expanded spectrum” (ESBLs) in members of Enterobacteriaceae is a matter of continued clinical concern. CTX‐M is among the most common ESBLs in Enterobacteriaceae family. In the present study, a nanoformulation of cefotaxime was prepared using gold nanoparticles to combat drug‐resistance in ESBL producing strains. Here, two CTX‐M‐15 positive cefotaxime resistant bacterial strains (i.e., one Escherichia coli and one Klebsiella pneumoniae strain) were used for testing the efficacy of “cefotaxime loaded gold‐nanoparticles.” Bromelain was used for both reduction and capping in the process of synthesis of gold‐nanoparticles. Thereafter, cefotaxime was conjugated onto it with the help of activator 1‐Ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide. For characterization of both unconjugated and cefotaxime conjugated gold nanoparticles; UV‐Visible spectroscopy, Scanning, and Transmission type Electron Microscopy methods accompanied with Dynamic Light Scattering were used. We used agar diffusion method plus microbroth‐dilution method for the estimation of the antibacterial‐activity and determination of minimum inhibitory concentration or MIC values, respectively. MIC values of cefotaxime loaded gold nanoparticles against E. coli and K. pneumoniae were obtained as 1.009 and 2.018 mg/L, respectively. These bacterial strains were completely resistant to cefotaxime alone. These results reinforce the utility of conjugating an old unresponsive antibiotic with gold nanoparticles to restore its efficacy against otherwise resistant bacterial pathogens. J. Cell. Biochem. 118: 2802–2808, 2017.

Prognostic significance of VEGFR1/Flt-1 immunoexpression in colorectal carcinoma.

Colorectal carcinoma (CRC) is a major cause of morbidity and mortality. Vascular endothelial growth factor 1/Fms-like tyrosine kinase 1 (VEGFR1/Flt-1) regulates monocyte migration, recruits endothelial cell progenitors, increases the adhesive properties of natural killer cells and induces of growth factors. Flt-1 is expressed on tumour cells and has been implicated in tumour growth and progression. The objective of this study is to address the relation of Flt-1 expression to tumour prognostication. Paraffin blocks from 143 primary CRC and 48 regional nodal metastases were retrieved from the archives of the Department of Pathology at King Abdulaziz University. Tissue microarrays were designed and constructed. Immunohistochemistry for Flt-1 was performed. Staining intensity and extent of staining were assessed and combined. Results were dichotomised as low expression and high expression. Flt-1 was overexpressed in primary tumours and nodal metastasis (p < 0.001 and 0.001) with no difference between primary and nodal metastasis (p = 0.690). Flt-1 immunoexpression was not associated with the clinicopathological parameters. Flt-1 overexpression was an independent predictor of positive margin status, positive lymphovascular invasion and local disease recurrence (p < 0.001, p < 0.001 and p = 0.003, respectively). Flt-1 was not associated with survival (log-rank = 0.003, p = 0.959). Flt-1 was overexpressed in primary CRC and their nodal metastases. Flt-1 expression was an independent predictor of margin status, lymphovascular invasion and local disease recurrence. Therefore, expression profiling of Flt-1 seems to have a prognostic potential in CRC. However, to elucidate the association of overexpression of Flt-1 with tumour characteristics and prognostication, more in vivo and in vitro molecular investigations are recommended.