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Background: Moebius Syndrome (MBS) is a rare congenital neurological disorder characterized by paralysis of facial nerves, impairment of ocular abduction and other variable abnormalities. MBS has been attributed to both environmental and genetic factors as potential causes. Until now only two genes, PLXND1 and REV3L have been identified to cause MBS. Results: We present a 9-year-old male clinically diagnosed with MBS, presenting facial palsy, altered ocular mobility, microglossia, dental anomalies and congenital torticollis. Radiologically, he lacks both abducens nerves and shows altered symmetry of both facial and vestibulocochlear nerves. Whole-exome sequence identified a de novo missense variant c.643G>A; p.Gly215Arg in CHN1, encoding the α2-chimaerin protein. The p.Gly215Arg variant is located in the C1 domain of CHN1 where other pathogenic gain of function variants have been reported. Bioinformatic analysis and molecular structural modelling predict a deleterious effect of the missense variant on the protein function. Conclusion: Our findings support that pathogenic variants in the CHN1 gene may be responsible for different cranial congenital dysinnervation syndromes, including Moebius and Duane retraction syndromes. We propose to include CHN1 in the genetic diagnoses of MBS.
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BACKGROUND: Helicobacter pylori infection is the most important risk factor for gastric cancer (GC). Human gastric adenocarcinoma develops after long-term H. pylori infection via the Correa cascade. This carcinogenic pathway describes the progression from gastritis to atrophy, intestinal metaplasia (IM), dysplasia and GC. Patients with atrophy and intestinal metaplasia are considered to have precancerous lesions of GC (PLGC). H. pylori eradication and endoscopy surveillance are currently the only interventions for preventing GC. Better knowledge of the biology of human PLGC may help find stratification markers and contribute to better understanding of biological mechanisms. One way to achieve this is by using co-expression network analysis. Weighted gene co-expression network analysis (WGCNA) is often used to identify modules from co-expression networks and relate them to clinical traits. It also allows identification of driver genes that may be critical for PLGC. AIM: The purpose of this study was to identify co-expression modules and differential gene expression in dyspeptic patients at different stages of the Correa pathway. METHODS: We studied 96 gastric biopsies from 78 patients that were clinically classified as: non-active (n = 10) and chronic-active gastritis (n = 20), atrophy (n = 12), and IM (n = 36). Gene expression of coding RNAs was determined by microarrays and non-coding RNAs by RNA-seq. The WGCNA package was used for network construction, module detection, module preservation and hub and driver gene selection. RESULTS: WGCNA identified 20 modules for coding RNAs and 4 for each miRNA and small RNA class. Modules were associated with antrum and corpus gastric locations, chronic gastritis and IM. Notably, coding RNA modules correlated with the Correa cascade. One was associated with the presence of H. pylori. In three modules, the module eigengene (ME) gradually increased in the stages toward IM, while in three others the inverse relationship was found. One miRNA module was negatively correlated to IM and was used for a mRNA-miRNA integration analysis. WGCNA also uncovered driver genes. Driver genes show both high connectivity within a module and are significantly associated with clinical traits. Some of those genes have been previously involved in H. pylori carcinogenesis, but others are new. Lastly, using similar external transcriptomic data, we confirmed that the discovered mRNA modules were highly preserved. CONCLUSION: Our analysis captured co-expression modules that provide valuable information to understand the pathogenesis of the progression of PLGC.
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Gastritis , Infecciones por Helicobacter , Helicobacter pylori , MicroARNs , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/epidemiología , Mucosa Gástrica/patología , Gastritis/complicaciones , Gastritis/epidemiología , Gastritis/patología , Atrofia/complicaciones , Atrofia/patología , MicroARNs/genética , Metaplasia/genética , Metaplasia/complicaciones , Metaplasia/patología , ARN MensajeroRESUMEN
Long non-coding RNAs (lncRNAs) play important roles in cancer and are potential new biomarkers or targets for therapy. However, given the low and tissue-specific expression of lncRNAs, linking these molecules to particular cancer types and processes through transcriptional profiling is challenging. Formalin-fixed, paraffin-embedded (FFPE) tissues are abundant resources for research but are prone to nucleic acid degradation, thereby complicating the study of lncRNAs. Here, we designed and validated a probe-based enrichment strategy to efficiently profile lncRNA expression in FFPE samples, and we applied it for the detection of lncRNAs associated with colorectal cancer (CRC). Our approach efficiently enriched targeted lncRNAs from FFPE samples, while preserving their relative abundance, and enabled the detection of tumor-specific mutations. We identified 379 lncRNAs differentially expressed between CRC tumors and matched healthy tissues and found tumor-specific lncRNA variants. Our results show that numerous lncRNAs are differentially expressed and/or accumulate variants in CRC tumors, thereby suggesting a role in CRC progression. More generally, our approach unlocks the study of lncRNAs in FFPE samples, thus enabling the retrospective use of abundant, well documented material available in hospital biobanks.
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Helicobacter pylori infects 4.4 billion individuals worldwide and is considered the most important etiologic agent for peptic ulcers and gastric cancer. Individual response to H. pylori infection is complex and depends on complex interactions between host and environmental factors. The pathway towards gastric cancer is a sequence of events known as Correa's model of gastric carcinogenesis, a stepwise inflammatory process from normal mucosa to chronic-active gastritis, atrophy, metaplasia and gastric adenocarcinoma. This study examines gastric clinical specimens representing different steps of the Correa pathway with the aim of identifying the expression profiles of coding- and non-coding RNAs that may have a role in Correa's model of gastric carcinogenesis. We screened for differentially expressed genes in gastric biopsies by employing RNAseq, microarrays and qRT-PCR. Here we provide a detailed description of the experiments, methods and results generated. The datasets may help other scientists and clinicians to find new clues to the pathogenesis of H. pylori and the mechanisms of progression of the infection to more severe gastric diseases. Data is available via ArrayExpress.
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Infecciones por Helicobacter/genética , ARN no Traducido/análisis , ARN/análisis , Estudios Transversales , Humanos , Análisis por Micromatrices , RNA-Seq , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The gastrointestinal tract harbors most of the microbiota associated with humans. In recent years, there has been a surge of interest in assessing the relationships between the gut microbiota and several gut alterations, including colorectal cancer. Changes in the gut microbiota in patients suffering colorectal cancer suggest a possible role of host-microbe interactions in the origin and development of this malignancy and, at the same time, open the door for novel ways of preventing, diagnosing, or treating this disease. In this review we survey current knowledge on the healthy microbiome of the gut and how it is altered in colorectal cancer and other related disease conditions. In describing past studies we will critically assess technical limitations of different approaches and point to existing challenges in microbiome research. We will have a special focus on host-microbiome interaction mechanisms that may be important to explain how dysbiosis can lead to chronic inflammation and drive processes that influence carcinogenesis and tumor progression in colon cancer. Finally, we will discuss the potential of recent developments of novel microbiota-based therapeutics and diagnostic tools for colorectal cancer.
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Neoplasias Colorrectales/etiología , Susceptibilidad a Enfermedades , Microbiota , Carcinogénesis , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/metabolismo , Detección Precoz del Cáncer , Ambiente , Microbioma Gastrointestinal , Interacciones Huésped-Patógeno , Humanos , Investigación Biomédica TraslacionalRESUMEN
BACKGROUND: Helicobacter pylori infection causes long-term chronic active gastritis, a risk factor for the intestinal and diffuse forms of gastric cancer. Most gastric cancers develop in a stepwise progression from chronic active gastritis to precursor lesions of gastric cancer. The early detection of gastric cancer improves survival. Studies with recent evidence have proposed circulating-microRNAs as biomarkers of cancer. OBJECTIVE: The purpose of this study was to explore the circulating-microRNA profile from H. pylori infection to gastric adenocarcinoma. METHODS: One hundred and twenty-three patients were enrolled and assigned to the discovery or the validation sets. In the discovery phase, circulating-microRNAs were measured by dye-based quantitative polymerase chain reaction and a selection of circulating-microRNAs was validated by probe-based quantitative polymerase chain reaction. A quality control protocol was used. RESULTS: One hundred and sixty-seven circulating-microRNAs were detected. Precursor lesions of gastric cancer and gastric cancer patients showed the downregulation of eight and five circulating-microRNAs, respectively. We further validated the deregulation of miR-196a-5p in precursor lesions of gastric cancer and the deregulation of miR-134-5p, miR-144-3p and miR-451a in gastric cancer. However, circulating-microRNAs exhibited moderate diagnostic performance due to the overlap of circulating-microRNA expression between non-cancer and cancer patients. miR-144-3p/miR-451a expression levels were correlated. Interestingly, these microRNAs are in 17q11.2, a site of rearrangements associated with gastric cancer. CONCLUSION: Circulating-microRNAs are deregulated in precancerous and gastric cancer patients but efforts are needed to improve their diagnostic accuracy.
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Although circulating miRNAs are promising candidates for biomarkers, several challenges must be overcome before miRNAs can be used for diagnosis and monitoring. One is quality control for the RNA extraction and quantification process. RNA quality control techniques are unsuitable as circulating miRNAs are in the fM range. Additionally, biofluids may contain inhibitors of the reverse transcriptase and polymerase enzymes, which may survive RNA purification. Herein, we describe the protocol we have used to check the robustness of miRNA purification and measurement by the addition of spike-ins and by evaluating the quality of the qPCR data, respectively.
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MicroARN Circulante/sangre , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Biomarcadores/sangre , MicroARN Circulante/genética , Humanos , Control de Calidad , Programas Informáticos , Estadística como AsuntoRESUMEN
Gastric carcinogenesis is a multifactorial process described as a stepwise progression from non-active gastritis (NAG), chronic active gastritis (CAG), precursor lesions of gastric cancer (PLGC) and gastric adenocarcinoma. Gastric cancer (GC) 5-year survival rate is highly dependent upon stage of disease at diagnosis, which is based on endoscopy, biopsy and pathological examinations. Non-invasive GC biomarkers would facilitate its diagnosis at early stages leading to improved GC prognosis. We analyzed plasma samples collected from 80 patients diagnosed with NAG without H. pylori infection (NAG-), CAG with H. pylori infection (CAG+), PLGC and GC. A panel of 208 metabolites including acylcarnitines, amino acids and biogenic amines, sphingolipids, glycerophospholipids, hexoses, and tryptophan and phenylalanine metabolites were quantified using two complementary quantitative approaches: Biocrates AbsoluteIDQ®p180 kit and a LC-MS method designed for the analysis of 29 tryptophan pathway and phenylalanine metabolites. Significantly altered metabolic profiles were found in GC patients that allowing discrimination from NAG-, CAG+ and PLGC patients. Pathway analysis showed significantly altered tryptophan and nitrogen metabolic pathways (FDR P < 0.01). Three metabolites (histidine, tryprophan and phenylacetylglutamine) discriminated between non-GC and GC groups. These metabolic signatures open new possibilities to improve surveillance of PLGC patients using a minimally invasive blood analysis.
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Plasma/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Progresión de la Enfermedad , Femenino , Gastritis/metabolismo , Gastritis/patología , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/patología , Humanos , Masculino , Redes y Vías Metabólicas/fisiología , Metaboloma/fisiología , Metabolómica/métodos , Persona de Mediana Edad , Fenilalanina/metabolismo , Estómago/patología , Triptófano/metabolismoRESUMEN
Gastric cancer (GC) is among the most common cancers worldwide. Gastric carcinogenesis is a multistep and multifactorial process beginning with chronic gastritis induced by Helicobacter pylori (H. pylori) infection. This process is often described via a sequence of events known as Correas's cascade, a stepwise progression from nonactive gastritis, chronic active gastritis, precursor lesions of gastric cancer (atrophy, intestinal metaplasia, and dysplasia), and finally adenocarcinoma. Our aim was to identify a plasma metabolic pattern characteristic of GC through disease progression within the Correa's cascade. This study involved the analysis of plasma samples collected from 143 patients classified in four groups: patients with nonactive gastritis and no H. pylori infection, H. pylori infected patients with chronic active gastritis, infected or noninfected patients with precursor lesions of gastric cancer, and GC. Independent partial least-squares-discriminant binary models of UPLC-ESI(+)-TOFMS metabolic profiles, implemented in a decision-directed acyclic graph, allowed the identification of tryptophan and kynurenine as discriminant metabolites that could be attributed to indoleamine-2,3-dioxygenase upregulation in cancer patients leading to tryptophan depletion and kynurenine metabolites generation. Furthermore, phenylacetylglutamine was also classified as a discriminant metabolite. Our data suggest the use of tryptophan, kynurenine, and phenylacetylglutamine as potential GC biomarkers.
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Metabolómica/métodos , Neoplasias Gástricas/diagnóstico , Adenocarcinoma/metabolismo , Biomarcadores de Tumor/sangre , Cromatografía Líquida de Alta Presión , Progresión de la Enfermedad , Femenino , Gastritis/metabolismo , Glutamina/análogos & derivados , Glutamina/análisis , Glutamina/metabolismo , Infecciones por Helicobacter , Helicobacter pylori , Humanos , Quinurenina/análisis , Quinurenina/metabolismo , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Plasma/metabolismo , Lesiones Precancerosas/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Triptófano/análisis , Triptófano/metabolismoRESUMEN
Colorectal cancer (CRC) is the fourth most common cause of death worldwide. Surgery is usually the first line of treatment for patients with CRC but many tumors with similar histopathological features show significantly different clinical outcomes. The discovery of robust prognostic biomarkers in patients with CRC is imperative to achieve more effective treatment strategies and improve patient's care. Recent progress in next generation sequencing methods and transcriptome analysis has revealed that a much larger part of the genome is transcribed into RNA than previously assumed. Collectively referred to as non-coding RNAs (ncRNAs), some of these RNA molecules such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have been shown to be altered and to play critical roles in tumor biology. This discovery leads to exciting possibilities for personalized cancer diagnosis, and therapy. Many lncRNAs are tissue and cancer-type specific and have already revealed to be useful as prognostic markers. In this review, we focus on recent findings concerning aberrant expression of lncRNAs in CRC tumors and emphasize their prognostic potential in CRC. Further studies focused on the mechanisms of action of lncRNAs will contribute to the development of novel biomarkers for diagnosis and disease progression.
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This data article contains data related to the research article entitled "Variability in microRNA recovery from plasma: Comparison of five commercial kits, doi:10.1016/j.ab.2015.07.018" Brunet-Vega (2015) [1]. PCR efficiency, along with RNA and cDNA quality, are the most important factors affecting the quality of qPCR results. Constant amplification efficiency in all compared samples is indispensable when relative quantification is used to measure changes in gene expression. An easy way to measure PCR efficiency, without the need of a standard curve, is LinRegPCR software. Individual PCR efficiency can be determined as a part of qPCR quality control. This is especially important when the initial RNA quantity is so low that cannot be accurately quantified, such as in circulating RNA extractions. This data article reports the Cqs and PCR efficiencies of 5 miRNAs quantified in RNA isolated from 4 patients with colorectal cancer (CRC) and 4 healthy donors using five commercially available kits.
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Numerous studies have indicated that microRNAs (miRNAs) are present and stable in multiple biological fluids, suggesting a great potential as biomarkers for molecular diagnostics and prognostics. Variations in the amount of starting material and isolation method to obtain miRNA may introduce bias and contribute to quantification errors. Given these concerns, we compared five commercially available kits for serum/plasma miRNA isolation to determine whether the plasma miRNA profile varies with the isolation method. We isolated miRNAs in blood plasma from colorectal cancer patients and healthy donors with five commercially available kits: Exiqon, Norgen, Macherey-Nagel, Qiagen, and Zymo Research. First, we assessed the robustness of the RNA isolation process and the quality of isolated miRNAs with the miRCURY microRNA QC PCR Panel (Exiqon), which contains six RNA spike-ins for quality control of RNA isolation (UniSp2, -4, and -5), complementary DNA (cDNA) synthesis (UniSp6 and cel-miR-39-3p), and polymerase chain reaction (PCR) amplification (UniSp3). This panel also includes circulating human miR-103, miR-191, miR-23a, and miR-451. Second, to evaluate the variability in miRNA profiling in relation to the extraction method, we analyzed plasma levels of candidate miRNA biomarkers for colorectal cancer (miR-18a, miR-21, and miR-29a). To determine PCR efficiencies per amplicon and per sample, we used LinRegPCR software. We found that all isolation methods were suitable for extracting miRNA from plasma samples and that all had similar Cq values in the three steps analyzed: RNA isolation, cDNA synthesis, and quantitative reverse transcription (qRT)-PCR. However, although the PCR replicates were excellent, the intersample variability of the spike-ins was unsatisfactorily high and all kits yielded suboptimal PCR efficiencies for some amplicons. Overall, our results underline the great difficulties involved in measuring miRNAs in plasma. The use of spike-ins is critical to control technical factors that affect final miRNA levels. We recommend that researchers investigating circulating miRNAs verify the PCR efficiency for each amplicon because quantification may be influenced by sample and PCR components.
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Neoplasias Colorrectales/sangre , MicroARNs/aislamiento & purificación , Biomarcadores/sangre , Neoplasias Colorrectales/metabolismo , ADN Complementario/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , MicroARNs/sangre , MicroARNs/metabolismo , Control de Calidad , Juego de Reactivos para Diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , EspañaRESUMEN
Biomarkers that can facilitate disease detection, staging and prediction of outcome are highly desirable to improve survival and to help determine optimized treatment for colorectal cancer patients. microRNAs (miRNAs) are small non-coding RNAs that play a crucial role in gene regulatory networks. The deregulation of miRNA expression has been found in several types of cancer and may represent a novel class of cancer biomarkers. Our aim was to determine the miRNA signature of stage III colorectal cancer (CRC) tumors and to identify potential circulating miRNAs that may represent non-invasive biomarkers in CRC patients. Genome-wide microarray analysis of miRNA expression was performed on 12 paired tumor and non-tumor formalin-fixed paraffin-embedded tissues from stage III CRC patients. A selection of differentially overexpressed miRNAs was validated by quantitative real-time polymerase chain reaction (qRT-PCR) and determined in the serum of a set of 56 individuals (30 stage III CRC patients and 26 healthy individuals). Using 1.5-fold expression difference as a cut-off level, 43 miRNAs were identified as differentially expressed in tumor versus normal tissue. Using reverse transcription and qRT-PCR, 11 miRNAs (miR-135b, miR-141, miR-18a, miR-20a, miR-21, miR-224, miR-29a, miR-31, miR-34a, miR-92a and miR-96) were confirmed as significantly overexpressed in tumor samples when compared with normal samples. We were able to detect 9 of these 11 miRNAs in serum samples from CRC patients and healthy individuals. Serum levels of miR-18a and miR-29a were significantly higher in CRC patients when compared to levels in the controls (p<0.05). In conclusion, this study identified a substantial number of miRNAs which were differentially expressed in stage III colorectal tumors. Moreover, the findings provide relevant information concerning overexpressed tumoral miRNAs as potential circulating biomarkers and highlight serum miR-18a and miR-29a as promising biomarkers for the screening and monitoring of CRC patients.