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1.
Proteins ; 90(4): 959-972, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-34850971

RESUMEN

Protein-protein interactions (PPIs) are essential in understanding numerous aspects of protein function. Here, we significantly scaled and modified analyses of the recently developed all-vs-all sequencing (AVA-Seq) approach using a gold-standard human protein interaction set (hsPRS-v2) containing 98 proteins. Binary interaction analyses recovered 20 of 47 (43%) binary PPIs from this positive reference set (PRS), comparing favorably with other methods. However, the increase of 20× in the interaction search space for AVA-Seq analysis in this manuscript resulted in numerous changes to the method required for future use in genome-wide interaction studies. We show that standard sequencing analysis methods must be modified to consider the possible recovery of thousands of positives among millions of tested interactions in a single sequencing run. The PRS data were used to optimize data scaling, auto-activator removal, rank interaction features (such as orientation and unique fragment pairs), and statistical cutoffs. Using these modifications to the method, AVA-Seq recovered >500 known and novel PPIs, including interactions between wild-type fragments of tumor protein p53 and minichromosome maintenance complex proteins 2 and 5 (MCM2 and MCM5) that could be of interest in human disease.


Asunto(s)
Genoma , Proteínas , Humanos , Proteínas/metabolismo
2.
J Transl Med ; 20(1): 244, 2022 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-35619151

RESUMEN

BACKGROUND: Mutated and non-mutated genes interact to drive cancer growth and metastasis. While research has focused on understanding the impact of mutated genes on cancer biology, understanding non-mutated genes that are essential to tumor development could lead to new therapeutic strategies. The recent advent of high-throughput whole genome sequencing being applied to many different samples has made it possible to calculate if genes are significantly non-mutated in a specific cancer patient cohort. METHODS: We carried out random mutagenesis simulations of the human genome approximating the regions sequenced in the publicly available Cancer Growth Atlas Project for ovarian cancer (TCGA-OV). Simulated mutations were compared to the observed mutations in the TCGA-OV cohort and genes with the largest deviations from simulation were identified. Pathway analysis was performed on the non-mutated genes to better understand their biological function. We then compared gene expression, methylation and copy number distributions of non-mutated and mutated genes in cell lines and patient data from the TCGA-OV project. To directly test if non-mutated genes can affect cell proliferation, we carried out proof-of-concept RNAi silencing experiments of a panel of nine selected non-mutated genes in three ovarian cancer cell lines and one primary ovarian epithelial cell line. RESULTS: We identified a set of genes that were mutated less than expected (non-mutated genes) and mutated more than expected (mutated genes). Pathway analysis revealed that non-mutated genes interact in cancer associated pathways. We found that non-mutated genes are expressed significantly more than mutated genes while also having lower methylation and higher copy number states indicating that they could be functionally important. RNAi silencing of the panel of non-mutated genes resulted in a greater significant reduction of cell viability in the cancer cell lines than in the non-cancer cell line. Finally, as a test case, silencing ANKLE2, a significantly non-mutated gene, affected the morphology, reduced migration, and increased the chemotherapeutic response of SKOV3 cells. CONCLUSION: We show that we can identify significantly non-mutated genes in a large ovarian cancer cohort that are well-expressed in patient and cell line data and whose RNAi-induced silencing reduces viability in three ovarian cancer cell lines. Targeting non-mutated genes that are important for tumor growth and metastasis is a promising approach to expand cancer therapeutic options.


Asunto(s)
Neoplasias Ováricas , Secuencia de Bases , Carcinoma Epitelial de Ovario/genética , Femenino , Genoma , Humanos , Mutación/genética , Neoplasias Ováricas/genética
3.
Cancer Cell Int ; 22(1): 376, 2022 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-36457029

RESUMEN

BACKGROUND: Colon cancer is often driven by mutations of the adenomatous polyposis coli (APC) gene, an essential tumor suppressor gene of the Wnt ß-catenin signaling pathway. APC and its cytoplasmic interactions have been well studied. However, various groups have also observed its presence in the nucleus. Identifying novel interactions of APC in the Wnt pathway will provide an opportunity to understand APC's nuclear role better and ultimately identify potential cancer treatment targets. METHODS: We used the all-vs-all sequencing (AVA-Seq) method to interrogate the interactome of protein fragments spanning most of the 60 Wnt ß-catenin pathway proteins. Using protein fragments identified the interacting regions between the proteins with more resolution than a full-length protein approach. Pull-down assays were used to validate a subset of these interactions. RESULTS: 74 known and 703 novel Wnt ß-catenin pathway protein-protein interactions were recovered in this study. There were 8 known and 31 novel APC protein-protein interactions. Novel interactions of APC and nuclear transcription factors TCF7, JUN, FOSL1, and SOX17 were particularly interesting and confirmed in validation assays. CONCLUSION: Based on our findings of novel interactions between APC and transcription factors and previous evidence of APC localizing to the nucleus, we suggest APC may compete and repress CTNNB1. This would occur through APC binding to the transcription factors (JUN, FOSL1, TCF7) to regulate the Wnt signaling pathway including through enhanced marking of CTNNB1 for degradation in the nucleus by APC binding with SOX17. Additional novel Wnt ß-catenin pathway protein-protein interactions from this study could lead researchers to novel drug designs for cancer.

4.
Clin Infect Dis ; 73(7): e1830-e1840, 2021 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-33315061

RESUMEN

BACKGROUND: Risk of reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unknown. We assessed the risk and incidence rate of documented SARS-CoV-2 reinfection in a cohort of laboratory-confirmed cases in Qatar. METHODS: All SARS-CoV-2 laboratory-confirmed cases with at least 1 polymerase chain reaction-positive swab that was ≥45 days after a first positive swab were individually investigated for evidence of reinfection. Viral genome sequencing of the paired first positive and reinfection viral specimens was conducted to confirm reinfection. RESULTS: Out of 133 266 laboratory-confirmed SARS-CoV-2 cases, 243 persons (0.18%) had at least 1 subsequent positive swab ≥45 days after the first positive swab. Of these, 54 cases (22.2%) had strong or good evidence for reinfection. Median time between the first swab and reinfection swab was 64.5 days (range, 45-129). Twenty-three of the 54 cases (42.6%) were diagnosed at a health facility, suggesting presence of symptoms, while 31 (57.4%) were identified incidentally through random testing campaigns/surveys or contact tracing. Only 1 person was hospitalized at the time of reinfection but was discharged the next day. No deaths were recorded. Viral genome sequencing confirmed 4 reinfections of 12 cases with available genetic evidence. Reinfection risk was estimated at 0.02% (95% confidence interval [CI], .01%-.02%), and reinfection incidence rate was 0.36 (95% CI, .28-.47) per 10 000 person-weeks. CONCLUSIONS: SARS-CoV-2 reinfection can occur but is a rare phenomenon suggestive of protective immunity against reinfection that lasts for at least a few months post primary infection.


Asunto(s)
COVID-19 , SARS-CoV-2 , Trazado de Contacto , Humanos , Incidencia , Reinfección
5.
PLoS Med ; 18(12): e1003879, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34914711

RESUMEN

BACKGROUND: The epidemiology of the SARS-CoV-2 B.1.1.7 (or Alpha) variant is insufficiently understood. This study's objective was to describe the introduction and expansion of this variant in Qatar and to estimate the efficacy of natural infection against reinfection with this variant. METHODS AND FINDINGS: Reinfections with the B.1.1.7 variant and variants of unknown status were investigated in a national cohort of 158,608 individuals with prior PCR-confirmed infections and a national cohort of 42,848 antibody-positive individuals. Infections with B.1.1.7 and variants of unknown status were also investigated in a national comparator cohort of 132,701 antibody-negative individuals. B.1.1.7 was first identified in Qatar on 25 December 2020. Sudden, large B.1.1.7 epidemic expansion was observed starting on 18 January 2021, triggering the onset of epidemic's second wave, 7 months after the first wave. B.1.1.7 was about 60% more infectious than the original (wild-type) circulating variants. Among persons with a prior PCR-confirmed infection, the efficacy of natural infection against reinfection was estimated to be 97.5% (95% CI: 95.7% to 98.6%) for B.1.1.7 and 92.2% (95% CI: 90.6% to 93.5%) for variants of unknown status. Among antibody-positive persons, the efficacy of natural infection against reinfection was estimated to be 97.0% (95% CI: 92.5% to 98.7%) for B.1.1.7 and 94.2% (95% CI: 91.8% to 96.0%) for variants of unknown status. A main limitation of this study is assessment of reinfections based on documented PCR-confirmed reinfections, but other reinfections could have occurred and gone undocumented. CONCLUSIONS: In this study, we observed that introduction of B.1.1.7 into a naïve population can create a major epidemic wave, but natural immunity in those previously infected was strongly associated with limited incidence of reinfection by B.1.1.7 or other variants.


Asunto(s)
COVID-19/epidemiología , COVID-19/virología , Reinfección/epidemiología , Reinfección/virología , SARS-CoV-2 , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Número Básico de Reproducción , Niño , Femenino , Humanos , Inmunidad Innata , Masculino , Persona de Mediana Edad , Modelos Teóricos , Reacción en Cadena de la Polimerasa , Qatar/epidemiología , Estudios Retrospectivos , Factores de Tiempo , Adulto Joven
6.
Hum Mol Genet ; 28(23): 3970-3981, 2019 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-31625567

RESUMEN

The effectiveness of next generation sequencing at solving genetic disease has motivated the rapid adoption of this technology into clinical practice around the world. In this study, we use whole exome sequencing (WES) to assess 48 patients with Mendelian disease from 30 serial families as part of the "Qatar Mendelian Disease pilot program" - a coordinated multi-center effort to build capacity and clinical expertise in genetic medicine in Qatar. By enrolling whole families (parents plus available siblings), we demonstrate significantly improved discriminatory power for candidate variant identification over trios for both de novo and recessive inheritance patterns. For the same index cases, we further demonstrate that even in the absence of families, variant prioritization is improved up to 8-fold when a modest set of population-matched controls is used vs large public databases, stressing the poor representation of Middle Eastern alleles in presently available databases. Our in-house pipeline identified candidate disease variants in 27 of 30 families (90%), 23 of which (85%) harbor novel pathogenic variants in known disease genes, pointing to significant allelic heterogeneity and founder mutations underlying Mendelian disease in the Middle East. For 6 of these families, the clinical presentation was only partially explained by the candidate gene, suggesting phenotypic expansion of known syndromes. Our pilot study demonstrates the utility of WES for Middle Eastern populations, the dramatic improvement in variant prioritization conferred by enrolling population-matched controls and/or enrolling additional unaffected siblings at the point-of-care, and 25 novel disease-causing alleles, relevant to newborn and premarital screening panels in regional populations.


Asunto(s)
Secuenciación del Exoma/métodos , Heterogeneidad Genética , Predisposición Genética a la Enfermedad/genética , Femenino , Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Linaje , Fenotipo , Proyectos Piloto , Sistemas de Atención de Punto , Qatar
7.
RNA ; 25(12): 1765-1778, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31519742

RESUMEN

Circular RNAs (circRNAs) are abundant in eukaryotic transcriptomes and have been linked to various human disorders. However, understanding genetic control of circular RNA expression is in the early stages. Here we present the first integrated analysis of circRNAs and genome sequence variation from lymphoblastoid cell lines of the 1000 Genomes Project. We identified thousands of circRNAs in the RNA-seq data and show their association with local single-nucleotide polymorphic sites, referred to as circQTLs, which influence the circRNA transcript abundance. Strikingly, we found that circQTLs exist independently of eQTLs with most circQTLs having no effect on mRNA expression. Only a fraction of the polymorphic sites are shared and linked to both circRNA and mRNA expression with mostly similar effects on circular and linear RNA. A shared intronic QTL, rs55928920, of HMSD gene drives the circular and linear expression in opposite directions, potentially modulating circRNA levels at the expense of mRNA. Finally, circQTLs and eQTLs are largely independent and exist in separate linkage disequilibrium (LD) blocks with circQTLs highly enriched for functional genomic elements and regulatory regions. This study reveals a previously uncharacterized role of DNA sequence variation in human circular RNA regulation.


Asunto(s)
Regulación de la Expresión Génica , Variación Genética , ARN Circular , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , MicroARNs/genética , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , ARN Mensajero/genética , Análisis de Secuencia de ADN , Transcriptoma
8.
J Biol Chem ; 294(30): 11549-11558, 2019 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-31182485

RESUMEN

Two-hybrid systems can be used for investigating protein-protein interactions and may provide important information about gene products with unknown function. Despite their success in mapping protein interactions, two-hybrid systems have remained mostly untouched by improvements in next-generation DNA sequencing. The two-hybrid systems rely on one-versus-all methods in which each bait is sequentially screened against an entire library. Here, we developed a screening method that joins both bait and prey as a convergent fusion into one bacterial plasmid vector that can then be amplified and paired-end sequencing by next-generation sequencing (NGS). Our method enables all-versus-all sequencing (AVA-Seq) and utilizes NGS to remove multiple bottlenecks of the two-hybrid system. AVA-Seq allows for high-resolution protein-protein interaction mapping of a small set of proteins and has the potential for lower-resolution mapping of entire proteomes. Features of the system include ORF selection to improve efficiency, high bacterial transformation efficiency, a convergent fusion vector to allow paired-end sequencing of interactors, and the use of protein fragments rather than full-length proteins to better resolve specific protein contact points. We demonstrate the system's strengths and limitations on a set of proteins known to interact in humans and provide a framework for future large-scale projects.


Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Sistemas de Lectura Abierta , Reproducibilidad de los Resultados , Técnicas del Sistema de Dos Híbridos
9.
Hum Mol Genet ; 27(6): 1106-1121, 2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29325019

RESUMEN

Epigenetic regulation of cellular function provides a mechanism for rapid organismal adaptation to changes in health, lifestyle and environment. Associations of cytosine-guanine di-nucleotide (CpG) methylation with clinical endpoints that overlap with metabolic phenotypes suggest a regulatory role for these CpG sites in the body's response to disease or environmental stress. We previously identified 20 CpG sites in an epigenome-wide association study (EWAS) with metabolomics that were also associated in recent EWASs with diabetes-, obesity-, and smoking-related endpoints. To elucidate the molecular pathways that connect these potentially regulatory CpG sites to the associated disease or lifestyle factors, we conducted a multi-omics association study including 2474 mass-spectrometry-based metabolites in plasma, urine and saliva, 225 NMR-based lipid and metabolite measures in blood, 1124 blood-circulating proteins using aptamer technology, 113 plasma protein N-glycans and 60 IgG-glyans, using 359 samples from the multi-ethnic Qatar Metabolomics Study on Diabetes (QMDiab). We report 138 multi-omics associations at these CpG sites, including diabetes biomarkers at the diabetes-associated TXNIP locus, and smoking-specific metabolites and proteins at multiple smoking-associated loci, including AHRR. Mendelian randomization suggests a causal effect of metabolite levels on methylation of obesity-associated CpG sites, i.e. of glycerophospholipid PC(O-36: 5), glycine and a very low-density lipoprotein (VLDL-A) on the methylation of the obesity-associated CpG loci DHCR24, MYO5C and CPT1A, respectively. Taken together, our study suggests that multi-omics-associated CpG methylation can provide functional read-outs for the underlying regulatory response mechanisms to disease or environmental insults.


Asunto(s)
Islas de CpG , Metilación de ADN , Trastornos del Metabolismo de la Glucosa/genética , Obesidad/genética , Fumar Tabaco/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas Portadoras/genética , Biología Computacional/métodos , Epigénesis Genética , Femenino , Estudios de Asociación Genética/métodos , Genoma Humano , Estudio de Asociación del Genoma Completo/métodos , Humanos , Lípidos/sangre , Masculino , Metaboloma , Proteínas Represoras/genética
10.
Reprod Biomed Online ; 41(4): 579-583, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32819839

RESUMEN

RESEARCH QUESTION: Long non-coding RNA (lncRNA) do not show protein translation but do have gene regulatory functions in several disease states. Studies have shown that lncRNA differ in overweight women with polycystic ovary syndrome (PCOS), increased insulin resistance and hyperandrogenaemia. The objective of this study was to determine the lncRNA in serum in age- and weight-matched non-obese women with and without PCOS. METHODS: In this prospective pilot cohort study, lncRNA were measured in serum in 13 non-obese women with PCOS and 10 control women undergoing IVF. RESULTS: There was no difference between groups in terms of age, body mass index or insulin resistance. Women with PCOS showed a higher free androgen index (FAI; P = 0.03) and anti-Müllerian hormone (AMH) concentration (P = 0.001). A total of 29 lncRNA (P ≤ 0.05) differed between PCOS groups. lncRNA AC095350.1 correlated with age (r = 0.79, P = 0.04), but no correlation was seen between the significantly different lncRNA and FAI or AMH values. Functional pathway assessment using the Ingenuity Pathway Assessment tool showed no relationships for the lncRNA. CONCLUSION: lncRNA in serum differed between non-obese women with PCOS and the control group, and the pattern of expression differed from that reported in obese women with PCOS from the same ethnic population; however, it but did not correlate with androgen or insulin resistance.


Asunto(s)
Índice de Masa Corporal , Hiperandrogenismo/metabolismo , Resistencia a la Insulina/fisiología , Síndrome del Ovario Poliquístico/metabolismo , ARN Largo no Codificante/metabolismo , Adulto , Glucemia , Estudios de Casos y Controles , Femenino , Humanos , Hiperandrogenismo/sangre , Hiperandrogenismo/genética , Insulina/sangre , Síndrome del Ovario Poliquístico/sangre , Síndrome del Ovario Poliquístico/genética , Estudios Prospectivos , ARN Largo no Codificante/genética , Testosterona/sangre
11.
BMC Genomics ; 20(1): 498, 2019 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-31208317

RESUMEN

BACKGROUND: The date palm is one of the oldest cultivated fruit trees. The tree can withstand high temperatures and low water and the fruit can be stored dry offering nutrition across the year. The first region of cultivation is believed to be near modern day Iraq, however, where and if the date palm was domesticated is still a topic of debate. Recent studies of chloroplast and genomic DNA revealed two major subpopulations of cultivars centered in both the Eastern range of date palm cultivation including Arabian Peninsula, Iraq and parts of South Asia, and the Western range, including North Africa. RESULTS: To better understand the origins of date palm cultivation we sequenced and analyzed over 200 mitochondrial and chloroplast genomes from a geographically diverse set of date palms. Here we show that, based on mitochondrial and chloroplast genome-wide genotyping data, the most common cultivated date palms contain 4 haplotypes that appear associated with geographical region of cultivar origin. CONCLUSIONS: These data suggest at least 3 and possibly 4 original maternal contributions to the current date palm population and doubles the original number. One new haplotype was found mainly in Tunisia, Algeria and Egypt and the second in Iraq, Iran and Oman. We propose that earliest date palm cultivation occurred independently in at least 3 distinct locations. This discovery will further inform understanding of the history and origins of cultivated date palm.


Asunto(s)
Orgánulos/genética , Phoeniceae/genética , Secuenciación Completa del Genoma , Secuencia de Bases , Haplotipos/genética , Phoeniceae/clasificación
12.
Genome Res ; 26(2): 151-62, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26728717

RESUMEN

An open question in the history of human migration is the identity of the earliest Eurasian populations that have left contemporary descendants. The Arabian Peninsula was the initial site of the out-of-Africa migrations that occurred between 125,000 and 60,000 yr ago, leading to the hypothesis that the first Eurasian populations were established on the Peninsula and that contemporary indigenous Arabs are direct descendants of these ancient peoples. To assess this hypothesis, we sequenced the entire genomes of 104 unrelated natives of the Arabian Peninsula at high coverage, including 56 of indigenous Arab ancestry. The indigenous Arab genomes defined a cluster distinct from other ancestral groups, and these genomes showed clear hallmarks of an ancient out-of-Africa bottleneck. Similar to other Middle Eastern populations, the indigenous Arabs had higher levels of Neanderthal admixture compared to Africans but had lower levels than Europeans and Asians. These levels of Neanderthal admixture are consistent with an early divergence of Arab ancestors after the out-of-Africa bottleneck but before the major Neanderthal admixture events in Europe and other regions of Eurasia. When compared to worldwide populations sampled in the 1000 Genomes Project, although the indigenous Arabs had a signal of admixture with Europeans, they clustered in a basal, outgroup position to all 1000 Genomes non-Africans when considering pairwise similarity across the entire genome. These results place indigenous Arabs as the most distant relatives of all other contemporary non-Africans and identify these people as direct descendants of the first Eurasian populations established by the out-of-Africa migrations.


Asunto(s)
Árabes/genética , Población Negra/genética , Migración Humana , Hombre de Neandertal/genética , Población Blanca/genética , Animales , Análisis por Conglomerados , ADN Mitocondrial/genética , Frecuencia de los Genes , Humanos , Hibridación Genética , Cadenas de Markov , Modelos Genéticos , Filogenia , Análisis de Componente Principal , Qatar , Análisis de Secuencia de ADN
13.
Clin Endocrinol (Oxf) ; 91(6): 793-797, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31482638

RESUMEN

Long noncoding RNAs (lncRNAs) are RNA transcripts over 200 nucleotides long that are not translated into protein; however, there is increasing evidence of their regulatory functions. To date, there are few studies measuring lncRNA in control women or women with polycystic ovary syndrome (PCOS). OBJECTIVE: To determine lncRNA differences between PCOS and control women. DESIGN: Cross sectional study. PATIENTS: Twenty four anovulatory women with all three diagnostic features of PCOS compared to 24 control women in the follicular phase of their menstrual cycle from a PCOS biobank. RESULTS: Women with PCOS were age and weight matched compared to the control women but were significantly insulin resistant and hyperandrogenemic (P < .01). Eight lncRNA (P < .05) were detected that differed between PCOS and control women, but only MIRLET7BHG correlated with body mass index (r = .66, P < .05). No lncRNA correlated with antimullerian hormone (AMH) levels, insulin resistance (HOMA-IR) or the free androgen index (FAI). Ingenuity pathway assessment (IPA) did not identify any functional pathways for the lncRNAs. CONCLUSION: LncRNAs differ between anovulatory PCOS and control women in the follicular phase of the menstrual cycle. It is unclear if this is due to inherent differences between PCOS and control women or due to changes in lncRNA that are menstrual cycle dependent. However, their IPA did not identify linked pathways, likely because few functions are as yet assigned to these lncRNAs.


Asunto(s)
Ciclo Menstrual/fisiología , Síndrome del Ovario Poliquístico/genética , ARN Largo no Codificante/genética , Adulto , Índice de Masa Corporal , Estudios Transversales , Femenino , Humanos , Ciclo Menstrual/genética , Adulto Joven
14.
BMC Cancer ; 19(1): 565, 2019 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-31185953

RESUMEN

BACKGROUND: Circular RNAs (circRNAs) that form through non-canonical backsplicing events of pre-mRNA transcripts are evolutionarily conserved and abundantly expressed across species. However, the functional relevance of circRNAs remains a topic of debate. METHODS: We identified one of the highly expressed circRNA (circANKRD12) in cancer cell lines and characterized it validated it by Sanger sequencing, Real-Time PCR. siRNA mediated silencing of the circular junction of circANKRD12 was followed by RNA Seq analysis of circANKRD12 silenced cells and control cells to identify the differentially regulated genes. A series of cell biology and molecular biology techniques (MTS assay, Migration analysis, 3D organotypic models, Real-Time PCR, Cell cycle analysis, Western blot analysis, and Seahorse Oxygen Consumption Rate analysis) were performed to elucidate the function, and underlying mechanisms involved in circANKRD12 silenced breast and ovarian cancer cells. RESULTS: In this study, we identified and characterized a circular RNA derived from Exon 2 and Exon 8 of the ANKRD12 gene, termed here as circANKRD12. We show that this circRNA is abundantly expressed in breast and ovarian cancers. The circANKRD12 is RNase R resistant and predominantly localized in the cytoplasm in contrast to its source mRNA. We confirmed the expression of this circRNA across a variety of cancer cell lines and provided evidence for its functional relevance through downstream regulation of several tumor invasion genes. Silencing of circANKRD12 induces a strong phenotypic change by significantly regulating cell cycle, increasing invasion and migration and altering the metabolism in cancer cells. These results reveal the functional significance of circANKRD12 and provide evidence of a regulatory role for this circRNA in cancer progression. CONCLUSIONS: Our study demonstrates the functional relevance of circANKRD12 in various cancer cell types and, based on its expression pattern, has the potential to become a new clinical biomarker.


Asunto(s)
Silenciador del Gen , Invasividad Neoplásica/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Circular/genética , Biomarcadores de Tumor/genética , Mama/citología , Neoplasias de la Mama/patología , Movimiento Celular , Ciclina D1/metabolismo , Exones/genética , Puntos de Control de la Fase G1 del Ciclo Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Pulmón/citología , Neoplasias Pulmonares/patología , Células MCF-7 , Fenotipo , ARN Interferente Pequeño/genética , Transfección
15.
PLoS Genet ; 12(1): e1005755, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26735499

RESUMEN

Identifying genes where a variant allele is preferentially expressed in tumors could lead to a better understanding of cancer biology and optimization of targeted therapy. However, tumor sample heterogeneity complicates standard approaches for detecting preferential allele expression. We therefore developed a novel approach combining genome and transcriptome sequencing data from the same sample that corrects for sample heterogeneity and identifies significant preferentially expressed alleles. We applied this analysis to epithelial ovarian cancer samples consisting of matched primary ovary and peritoneum and lymph node metastasis. We find that preferentially expressed variant alleles include germline and somatic variants, are shared at a relatively high frequency between patients, and are in gene networks known to be involved in cancer processes. Analysis at a patient level identifies patient-specific preferentially expressed alleles in genes that are targets for known drugs. Analysis at a site level identifies patterns of site specific preferential allele expression with similar pathways being impacted in the primary and metastasis sites. We conclude that genes with preferentially expressed variant alleles can act as cancer drivers and that targeting those genes could lead to new therapeutic strategies.


Asunto(s)
Redes Reguladoras de Genes , Proteínas de Neoplasias/biosíntesis , Neoplasias Ováricas/genética , Transcriptoma , Alelos , Desequilibrio Alélico/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células Germinativas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Proteínas de Neoplasias/genética , Neoplasias Ováricas/patología
16.
Int J Mol Sci ; 20(14)2019 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-31336560

RESUMEN

Transcriptome profiling of 3D models compared to 2D models in various cancer cell lines shows differential expression of TGF-ß-mediated and cell adhesion pathways. Presence of TGF-ß in these cell lines shows an increased invasion potential which is specific to cell type. In the present study, we identified exogenous addition of TGF-ß can induce Epithelial to Mesenchymal Transition (EMT) in a few cancer cell lines. RNA sequencing and real time PCR were carried out in different ovarian cancer cell lines to identify molecular profiling and metabolic profiling. Since EMT induction by TGF-ß is cell-type specific, we decided to select two promising ovarian cancer cell lines as model systems to study EMT. TGF-ß modulation in EMT and cancer invasion were successfully depicted in both 2D and 3D models of SKOV3 and CAOV3 cell lines. Functional evaluation in 3D and 2D models demonstrates that the addition of the exogenous TGF-ß can induce EMT and invasion in cancer cells by turning them into aggressive phenotypes. TGF-ß receptor kinase I inhibitor (LY364947) can revert the TGF-ß effect in these cells. In a nutshell, TGF-ß can induce EMT and migration, increase aggressiveness, increase cell survival, alter cell characteristics, remodel the Extracellular Matrix (ECM) and increase cell metabolism favorable for tumor invasion and metastasis. We concluded that transcriptomic and phenotypic effect of TGF-ß and its inhibitor is cell-type specific and not cancer specific.


Asunto(s)
Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adenosina Trifosfato/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Matriz Extracelular , Femenino , Humanos , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas
17.
Genet Med ; 20(11): 1365-1373, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-29790874

RESUMEN

PURPOSE: Nonobstructive azoospermia (NOA) affects 1% of the male population; however, despite state-of-the-art clinical assessment, for most patients the cause is unknown. We capitalized on an analysis of multiplex families in the Middle East to identify highly penetrant genetic causes. METHODS: We used whole-exome sequencing (WES) in 8 consanguineous families and combined newly discovered genes with previously reported ones to create a NOA gene panel, which was used to identify additional variants in 75 unrelated idiopathic NOA subjects and 74 fertile controls. RESULTS: In five of eight families, we identified rare deleterious recessive variants in CCDC155, NANOS2, SPO11, TEX14, and WNK3 segregating with disease. These genes, which are novel to human NOA, have remarkable testis-specific expression, and murine functional evidence supports roles for them in spermatogenesis. Among 75 unrelated NOA subjects, we identified 4 (~5.3%) with additional recessive variants in these newly discovered genes and 6 with deleterious variants in previously reported NOA genes, yielding an overall genetic etiology for 13.3% subjects versus 0 fertile controls (p = 0.001). CONCLUSION: NOA affects millions of men, many of whom remain idiopathic despite extensive laboratory evaluation. The genetic etiology for a substantial fraction of these patients (>50% familial and >10% sporadic) may be discovered by WES at the point of care.


Asunto(s)
Azoospermia/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Infertilidad Masculina/genética , Adulto , Azoospermia/epidemiología , Azoospermia/fisiopatología , Proteínas de Ciclo Celular/genética , Consanguinidad , Endodesoxirribonucleasas/genética , Humanos , Infertilidad Masculina/epidemiología , Infertilidad Masculina/fisiopatología , Masculino , Medio Oriente , Mutación , Proteínas Nucleares/genética , Linaje , Polimorfismo de Nucleótido Simple/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Unión al ARN/genética , Espermatogénesis/genética , Factores de Transcripción/genética , Secuenciación del Exoma
18.
Ann Neurol ; 81(1): 68-78, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27863452

RESUMEN

OBJECTIVE: Exome sequences account for only 2% of the genome and may overlook mutations causing disease. To obtain a more complete view, whole genome sequencing (WGS) was analyzed in a large consanguineous family in which members displayed autosomal recessively inherited cerebellar ataxia manifesting before 2 years of age. METHODS: WGS from blood-derived genomic DNA was used for homozygosity mapping and a rare variant search. RNA from isolated blood leukocytes was used for quantitative polymerase chain reaction (PCR), RNA sequencing, and comparison of the transcriptomes of affected and unaffected family members. RESULTS: WGS revealed a point mutation in noncoding RNA RNU12 that was associated with early onset cerebellar ataxia. The U12-dependent minor spliceosome edits 879 known transcripts. Reverse transcriptase PCR demonstrated minor intron retention in all of 9 randomly selected RNAs from this group, and RNAseq showed splicing disruption specific to all U12-type introns detected in blood monocytes from affected individuals. Moreover, 144 minor intron-containing RNAs were differentially expressed, including transcripts for 3 genes previously associated with cerebellar neurodegeneration. INTERPRETATION: Interference with particular spliceosome components, including small nuclear RNAs, cause reproducible uniquely distributed phenotypic and transcript-specific effects, making this an important category of disease-associated mutation. Our approach to differential expression analysis of minor intron-containing genes is applicable to other diseases involving altered transcriptome processing. ANN NEUROL 2017;81:68-78.


Asunto(s)
Predisposición Genética a la Enfermedad/genética , ARN Nuclear Pequeño/genética , ARN no Traducido/genética , Degeneraciones Espinocerebelosas/genética , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Mutación Puntual , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ARN , Adulto Joven
19.
BMC Genomics ; 16: 834, 2015 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-26490036

RESUMEN

BACKGROUND: The populations of the Arabian Peninsula remain the least represented in public genetic databases, both in terms of single nucleotide variants and of larger genomic mutations. We present the first high-resolution copy number variation (CNV) map for a Gulf Arab population, using a hybrid approach that integrates array genotyping intensity data and next-generation sequencing reads to call CNVs in the Qatari population. METHODS: CNVs were detected in 97 unrelated Qatari individuals by running two calling algorithms on each of two primary datasets: high-resolution genotyping (Illumina Omni 2.5M) and high depth whole-genome sequencing (Illumina PE 100bp). The four call-sets were integrated to identify high confidence CNV regions, which were subsequently annotated for putative functional effect and compared to public databases of CNVs in other populations. The availability of genome sequence was leveraged to identify tagging SNPs in high LD with common deletions in this population, enabling their imputation from genotyping experiments in the future. RESULTS: Genotyping intensities and genome sequencing data from 97 Qataris were analyzed with four different algorithms and integrated to discover 16,660 high confidence CNV regions (CNVRs) in the total population, affecting ~28 Mb in the median Qatari genome. Up to 40% of all CNVs affected genes, including novel CNVs affecting Mendelian disease genes, segregating at different frequencies in the 3 major Qatari subpopulations, including those with Bedouin, Persian/South Asian, and African ancestry. Consistent with high consanguinity levels in the Bedouin subpopulation, we found an increased burden for homozygous deletions in this group. In comparison to known CNVs in the comprehensive Database of Genomic Variants, we found that 5% of all CNVRs in Qataris were completely novel, with an enrichment of CNVs affecting several known chromosomal disorder loci and genes known to regulate sugar metabolism and type 2 diabetes in the Qatari cohort. Finally, we leveraged the availability of genome sequence to find suitable tagging SNPs for common deletions in this population. CONCLUSION: We combine four independently generated datasets from 97 individuals to study CNVs for the first time at high-resolution in a Gulf Arab population.


Asunto(s)
Variaciones en el Número de Copia de ADN , Dosificación de Gen , Genética de Población , Genoma Humano , Genómica , Biología Computacional/métodos , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Anotación de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Qatar
20.
Hum Mutat ; 35(1): 105-16, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24123366

RESUMEN

Exome sequencing of families of related individuals has been highly successful in identifying genetic polymorphisms responsible for Mendelian disorders. Here, we demonstrate the value of the reverse approach, where we use exome sequencing of a sample of unrelated individuals to analyze allele frequencies of known causal mutations for Mendelian diseases. We sequenced the exomes of 100 individuals representing the three major genetic subgroups of the Qatari population (Q1 Bedouin, Q2 Persian-South Asian, Q3 African) and identified 37 variants in 33 genes with effects on 36 clinically significant Mendelian diseases. These include variants not present in 1000 Genomes and variants at high frequency when compared with 1000 Genomes populations. Several of these Mendelian variants were only segregating in one Qatari subpopulation, where the observed subpopulation specificity trends were confirmed in an independent population of 386 Qataris. Premarital genetic screening in Qatar tests for only four out of the 37, such that this study provides a set of Mendelian disease variants with potential impact on the epidemiological profile of the population that could be incorporated into the testing program if further experimental and clinical characterization confirms high penetrance.


Asunto(s)
Cromosomas Humanos/genética , Enfermedades Genéticas Congénitas/genética , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Variación Genética , Análisis de Secuencia de ADN , Bases de Datos Genéticas , Exoma , Femenino , Frecuencia de los Genes , Enfermedades Genéticas Congénitas/epidemiología , Humanos , Masculino , Prevalencia , Qatar/epidemiología
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