Arif Anwar

Second generation sequencing of the mesothelioma tumor genome.

The current paradigm for elucidating the molecular etiology of cancers relies on the interrogation of small numbers of genes, which limits the scope of investigation. Emerging second-generation massively parallel DNA sequencing technologies have enabled more precise definition of the cancer genome on a global scale. We examined the genome of a human primary malignant pleural mesothelioma (MPM) tumor and matched normal tissue by using a combination of sequencing-by-synthesis and pyrosequencing methodologies to a 9.6X depth of coverage. Read density analysis uncovered significant aneuploidy and numerous rearrangements. Method-dependent informatics rules, which combined the results of different sequencing platforms, were developed to identify and validate candidate mutations of multiple types. Many more tumor-specific rearrangements than point mutations were uncovered at this depth of sequencing, resulting in novel, large-scale, inter- and intra-chromosomal deletions, inversions, and translocations. Nearly all candidate point mutations appeared to be previously unknown SNPs. Thirty tumor-specific fusions/translocations were independently validated with PCR and Sanger sequencing. Of these, 15 represented disrupted gene-encoding regions, including kinases, transcription factors, and growth factors. One large deletion in DPP10 resulted in altered transcription and expression of DPP10 transcripts in a set of 53 additional MPM tumors correlated with survival. Additionally, three point mutations were observed in the coding regions of NKX6-2, a transcription regulator, and NFRKB, a DNA-binding protein involved in modulating NFKB1. Several regions containing genes such as PCBD2 and DHFR, which are involved in growth factor signaling and nucleotide synthesis, respectively, were selectively amplified in the tumor. Second-generation sequencing uncovered all types of mutations in this MPM tumor, with DNA rearrangements representing the dominant type.

Genome-wide analysis of copy number variation identifies candidate gene loci associated with the progression of non-alcoholic fatty liver disease

Between 10 and 25% of individuals with non-alcoholic fatty liver disease (NAFLD) develop hepatic fibrosis leading to cirrhosis and hepatocellular carcinoma (HCC). To investigate the molecular basis of disease progression, we performed a genome-wide analysis of copy number variation (CNV) in a total of 49 patients with NAFLD [10 simple steatosis and 39 non-alcoholic steatohepatitis (NASH)] and 49 matched controls using high-density comparative genomic hybridization (CGH) microarrays. A total of 11 CNVs were found to be unique to individuals with simple steatosis, whilst 22 were common between simple steatosis and NASH, and 224 were unique to NASH. We postulated that these CNVs could be involved in the pathogenesis of NAFLD progression. After stringent filtering, we identified four rare and/or novel CNVs that may influence the pathogenesis of NASH. Two of these CNVs, located at 13q12.11 and 12q13.2 respectively, harbour the exportin 4 (XPO4) and phosphodiesterase 1B (PDE1B) genes which are already known to be involved in the etiology of liver cirrhosis and HCC. Cross-comparison of the genes located at these four CNV loci with genes already known to be associated with NAFLD yielded a set of genes associated with shared biological processes including cell death, the key process involved in ‘second hit’ hepatic injury. To our knowledge, this pilot study is the first to provide CNV information of potential relevance to the NAFLD spectrum. These data could prove invaluable in predicting patients at risk of developing NAFLD and more importantly, those who will subsequently progress to NASH.

Genome Wide Analysis of Chromosomal Alterations in Oral Squamous Cell Carcinomas Revealed over Expression of MGAM and ADAM9

Despite the advances in diagnosis and treatment of oral squamous cell carcinoma (OSCC), mortality and morbidity rates have not improved over the past decade. A major drawback in diagnosis and treatment of OSCC is the lack of knowledge relating to how genetic instability in oral cancer genomes affects oral carcinogenesis. Hence, the key aim of this study was to identify copy number alterations (CNAs) that may be cancer associated in OSCC using high-resolution array comparative genomic hybridization (aCGH). To our knowledge this is the first study to use ultra-high density aCGH microarrays to profile a large number of OSCC genomes (n = 46). The most frequently amplified CNAs were located on chromosome 11q11(52%), 2p22.3(52%), 1q21.3–q22(54%), 6p21.32(59%), 20p13(61%), 7q34(52% and 72%),8p11.23–p11.22(80%), 8q11.1–q24.4(54%), 9q13–q34.3(54%), 11q23.3–q25(57%); 14q21.3–q31.1(54%); 14q31.3–q32.33(57%), 20p13–p12.3(54%) and 20q11.21–q13.33(52%). The most frequently deleted chromosome region was located on 3q26.1 (54%). In order to verify the CNAs from aCGH using quantitative polymerase chain reaction (qPCR), the three top most amplified regions and their associated genes, namely ADAM5P (8p11.23–p11.22), MGAM (7q34) and SIRPB1 (20p13.1), were selected in this study. The ADAM5P locus was found to be amplified in 39 samples and deleted in one; MGAM (24 amplifications and 3 deletions); and SIRPB1 (12 amplifications, others undetermined). On the basis of putative cancer-related annotations, two genes, namely ADAM metallopeptidase domain 9 (ADAM9) and maltase-glucoamylase alpha-glucosidase (MGAM), that mapped to CNA regions were selected for further evaluation of their mRNA expression using reverse transcriptase qPCR. The over-expression of MGAM was confirmed with a 6.6 fold increase in expression at the mRNA level whereas the fold change in ADAM9 demonstrated a 1.6 fold increase. This study has identified significant regions in the OSCC genome that were amplified and resulted in consequent over-expression of the MGAM and ADAM9 genes that may be utilized as biological markers for OSCC.

Draft genome of neurotropic nematode parasite Angiostrongylus cantonensis, causative agent of human eosinophilic meningitis

Angiostrongylus cantonensis is a bursate nematode parasite that causes eosinophilic meningitis (or meningoencephalitis) in humans in many parts of the world. The genomic data from A. cantonensis will form a useful resource for comparative genomic and chemogenomic studies to aid the development of diagnostics and therapeutics. We have sequenced, assembled and annotated the genome of A. cantonensis. The genome size is estimated to be ∼260 Mb, with 17,280 genomic scaffolds, 91X coverage, 81.45% for complete and 93.95% for partial score based on CEGMA analysis of genome completeness. The number of predicted genes of ≥300 bp was 17,482. A total of 7737 predicted protein-coding genes of ≥50 amino acids were identified in the assembled genome. Among the proteins of known function, kinases are the most abundant followed by transferases. The draft genome contains 34 excretory-secretory proteins (ES), a minimum of 44 Nematode Astacin (NAS) metalloproteases, 12 Homeobox (HOX) genes, and 30 neurotransmitters. The assembled genome size (260 Mb) is larger than those of Pristionchus pacificus, Caenorhabditis elegans, Necator americanus, Caenorhabditis briggsae, Trichinella spiralis, Brugia malayi and Loa loa, but smaller than Haemonchus contortus and Ascaris suum. The repeat content (25%) is similar to H. contortus. The GC content (41.17%) is lower compared to P. pacificus (42.7%) and H. contortus (43.1%) but higher compared to C. briggsae (37.69%), A. suum (37.9%) and N. americanus (40.2%) while the scaffold N50 is 42,191. This draft genome will facilitate the understanding of many unresolved issues on the parasite and the disorder it causes.

Genome-wide analysis of oral squamous cell carcinomas revealed over expression of ISG15, Nestin and WNT11

BACKGROUND Multistep pathways and mechanisms are involved in the development of oral cancer. Chromosomal alterations are one of such key mechanisms implicated oral carcinogenesis. Therefore, this study aims to determine the genomic copy number alterations (CNAs) in oral squamous cell carcinoma (OSCC) using array comparative genomic hybridization (aCGH) and in addition attempt to correlate CNAs with modified gene expression. MATERIALS AND METHODS Genome-wide screening was performed on 15 OSCCs using high-density aCGH. On the basis of pathway analysis, three genes (ISG15, Nestin and WNT11) which mapped to CNA regions were selected for further evaluation of their mRNA expression using quantitative reverse transcriptase PCR (qRT-PCR). RESULTS Copy number alterations were observed on multiple genomic regions, including amplifications on 1p, 3q, 5p, 6p, 7p, 8q, 9q, 11q, 12q, 16p, 18p and deletions on 3p, 7q, 8p, 11q, 19q and 20q. Among the three selected genes, ISG15 had the highest mRNA expression level with a 22.5-fold increase, followed by Nestin with a 4.5-fold increase and WNT11 with a 2.5-fold increase. CONCLUSIONS This study has identified several major CNAs in oral cancer genomes and indicated that this correlates with over expression of the ISG15, WNT11, and Nestin genes.

Augmentation of Autoantibodies by Helicobacter pylori in Parkinson’s Disease Patients May Be Linked to Greater Severity

Parkinsons disease (PD) is the second most common chronic and progressive neurodegenerative disorder. Its etiology remains elusive and at present only symptomatic treatments exists. Helicobacter pylori chronically colonizes the gastric mucosa of more than half of the global human population. Interestingly, H. pylori positivity has been found to be associated with greater of PD motor severity. In order to investigate the underlying cause of this association, the Sengenics Immunome protein array, which enables simultaneous screening for autoantibodies against 1636 human proteins, was used to screen the serum of 30 H. pylori-seropositive PD patients (case) and 30 age- and gender-matched H. pylori-seronegative PD patients (control) in this study. In total, 13 significant autoantibodies were identified and ranked, with 8 up-regulated and 5 down-regulated in the case group. Among autoantibodies found to be elevated in H. pylori-seropositive PD were included antibodies that recognize Nuclear factor I subtype A (NFIA), Platelet-derived growth factor B (PDGFB) and Eukaryotic translation initiation factor 4A3 (eIFA3). The presence of elevated autoantibodies against proteins essential for normal neurological functions suggest that immunomodulatory properties of H. pylori may explain the association between H. pylori positivity and greater PD motor severity.

Deciphering the Draft Genome of Toxoplasma gondii RH Strain

Toxoplasmosis is a widespread parasitic infection by Toxoplasma gondii, a parasite with at least three distinct clonal lineages. This article reports the whole genome sequencing and de novo assembly of T. gondii RH (type I representative strain), as well as genome-wide comparison across major T. gondii lineages. Genomic DNA was extracted from tachyzoites of T. gondii RH strain and its identity was verified by PCR and LAMP. Subsequently, whole genome sequencing was performed, followed by sequence filtering, genome assembly, gene annotation assignments, clustering of gene orthologs and phylogenetic tree construction. Genome comparison was done with the already archived genomes of T. gondii. From this study, the genome size of T. gondii RH strain was found to be 69.35Mb, with a mean GC content of 52%. The genome shares high similarity to the archived genomes of T. gondii GT1, ME49 and VEG strains. Nevertheless, 111 genes were found to be unique to T. gondii RH strain. Importantly, unique genes annotated to functions that are potentially critical for T. gondii virulence were found, which may explain the unique phenotypes of this particular strain. This report complements the genomic archive of T. gondii. Data obtained from this study contribute to better understanding of T. gondii and serve as a reference for future studies on this parasite.

Draft genome of Brugia pahangi: high similarity between B. pahangi and B. malayi

BackgroundEfforts to completely eradicate lymphatic filariasis from human population may be challenged by the emergence of Brugia pahangi as another zoonotic lymphatic filarial nematode. In this report, a genomic study was conducted to understand this species at molecular level.MethodsAfter blood meal on a B. pahangi-harbouring cat, the Aedes togoi mosquitoes were maintained to harvest infective third stage larvae, which were then injected into male Mongolian gerbils. Subsequently, adult B. pahangi were obtained from the infected gerbil for genomic DNA extraction. Sequencing and subsequently, construction of genomic libraries were performed. This was followed by genomic analyses and gene annotation analysis. By using archived protein sequences of B. malayi and a few other nematodes, clustering of gene orthologs and phylogenetics were conducted.ResultsA total of 9687 coding genes were predicted. The genome of B. pahangi shared high similarity to that B. malayi genome, particularly genes annotated to fundamental processes. Nevertheless, 166 genes were considered to be unique to B. pahangi, which may be responsible for the distinct properties of B. pahangi as compared to other filarial nematodes. In addition, 803 genes were deduced to be derived from Wolbachia, an endosymbiont bacterium, with 44 of these genes intercalate into the nematode genome.ConclusionsThe reporting of B. pahangi draft genome contributes to genomic archive. Albeit with high similarity to B. malayi genome, the B. pahangi-unique genes found in this study may serve as new focus to study differences in virulence, vector selection and host adaptability among different Brugia spp.

Next generation sequencing reveals the antibiotic resistant variants in the genome of Pseudomonas aeruginosa

Rapid progress in next generation sequencing and allied computational tools have aided in identification of single nucleotide variants in genomes of several organisms. In the present study, we have investigated single nucleotide polymorphism (SNP) in ten multi-antibiotic resistant Pseudomonas aeruginosa clinical isolates. All the draft genomes were submitted to Rapid Annotations using Subsystems Technology (RAST) web server and the predicted protein sequences were used for comparison. Non-synonymous single nucleotide polymorphism (nsSNP) found in the clinical isolates compared to the reference genome (PAO1), and the comparison of nsSNPs between antibiotic resistant and susceptible clinical isolates revealed insights into the genome variation. These nsSNPs identified in the multi-drug resistant clinical isolates were found to be altering a single amino acid in several antibiotic resistant genes. We found mutations in genes encoding efflux pump systems, cell wall, DNA replication and genes involved in repair mechanism. In addition, nucleotide deletions in the genome and mutations leading to generation of stop codons were also observed in the antibiotic resistant clinical isolates. Next generation sequencing is a powerful tool to compare the whole genomes and analyse the single base pair variations found within the antibiotic resistant genes. We identified specific mutations within antibiotic resistant genes compared to the susceptible strain of the same bacterial species and these findings may provide insights to understand the role of single nucleotide variants in antibiotic resistance.

Genome wide profiling in oral squamous cell carcinoma identifies a four genetic marker signature of prognostic significance

Background Cancers of the oral cavity are primarily oral squamous cell carcinomas (OSCCs). Many of the OSCCs present at late stages with an exceptionally poor prognosis. A probable limitation in management of patients with OSCC lies in the insufficient knowledge pertaining to the linkage between copy number alterations in OSCC and oral tumourigenesis thereby resulting in an inability to deliver targeted therapy. Objectives The current study aimed to identify copy number alterations (CNAs) in OSCC using array comparative genomic hybridization (array CGH) and to correlate the CNAs with clinico-pathologic parameters and clinical outcomes. Materials and methods Using array CGH, genome-wide profiling was performed on 75 OSCCs. Selected genes that were harboured in the frequently amplified and deleted regions were validated using quantitative polymerase chain reaction (qPCR). Thereafter, pathway and network functional analysis were carried out using Ingenuity Pathway Analysis (IPA) software. Results Multiple chromosomal regions including 3q, 5p, 7p, 8q, 9p, 10p, 11q were frequently amplified, while 3p and 8p chromosomal regions were frequently deleted. These findings were in confirmation with our previous study using ultra-dense array CGH. In addition, amplification of 8q, 11q, 7p and 9p and deletion of 8p chromosomal regions showed a significant correlation with clinico-pathologic parameters such as the size of the tumour, metastatic lymph nodes and pathological staging. Co-amplification of 7p, 8q, 9p and 11q regions that harbored amplified genes namely CCND1, EGFR, TPM2 and LRP12 respectively, when combined, continues to be an independent prognostic factor in OSCC. Conclusion Amplification of 3q, 5p, 7p, 8q, 9p, 10p, 11q and deletion of 3p and 8p chromosomal regions were recurrent among OSCC patients. Co-alteration of 7p, 8q, 9p and 11q was found to be associated with clinico-pathologic parameters and poor survival. These regions contain genes that play critical roles in tumourigenesis pathways.