International Journal of Molecular Sciences

Research

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Open AccessArticle

Hotspot Selective Preference of the Chimeric Sequences Formed in Multiple Displacement Amplification

by Jing Tu, Na Lu, Mengqin Duan, Mengting Huang, Liang Chen, Junji Li, Jing Guo and Zuhong Lu

Int. J. Mol. Sci. 2017, 18(3), 492; https://doi.org/10.3390/ijms18030492 - 24 Feb 2017

Cited by 7 | Viewed by 4727

Abstract

Multiple displacement amplification (MDA) is considered to be a conventional approach to comprehensive amplification from low input DNA. The chimeric reads generated in MDA lead to severe disruption in some studies, including those focusing on heterogeneity, structural variation, and genetic recombination. Meanwhile, the [...] Read more.

Multiple displacement amplification (MDA) is considered to be a conventional approach to comprehensive amplification from low input DNA. The chimeric reads generated in MDA lead to severe disruption in some studies, including those focusing on heterogeneity, structural variation, and genetic recombination. Meanwhile, the generation of by-products gives a new approach to gain insights into the reaction process of φ29 polymerase. Here, we analyzed 36.7 million chimeras and screened 196 billion chimeric hotspots in the human genome, as well as evaluating the hotspot selective preference of chimeras. No significant preference was captured in the distributions of chimeras and hotspots among chromosomes. Hotspots with overlaps for 12–13 nucleotides (nt) were most likely to be selected as templates in chimera generation. Meanwhile, a regularly selective preference was noticed in overlap GC content. The preferences in overlap length and GC content was shown to be pertinent to the sequence denaturation temperature, which pointed out the optimization direction for reducing chimeras. Distance preference between two segments of chimeras was 80–280 nt. The analysis is beneficial for reducing the chimeras in MDA, and the characterization of MDA chimeras is helpful in distinguishing MDA chimeras from chimeric sequences caused by disease. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessArticle

Exome Sequencing in a Family with Luminal-Type Breast Cancer Underpinned by Variation in the Methylation Pathway

by Nicole Van der Merwe, Armand V. Peeters, Fredrieka M. Pienaar, Juanita Bezuidenhout, Susan J. Van Rensburg and Maritha J. Kotze

Int. J. Mol. Sci. 2017, 18(2), 467; https://doi.org/10.3390/ijms18020467 - 22 Feb 2017

Cited by 16 | Viewed by 7583

Abstract

Panel-based next generation sequencing (NGS) is currently preferred over whole exome sequencing (WES) for diagnosis of familial breast cancer, due to interpretation challenges caused by variants of uncertain clinical significance (VUS). There is also no consensus on the selection criteria for WES. In [...] Read more.

Panel-based next generation sequencing (NGS) is currently preferred over whole exome sequencing (WES) for diagnosis of familial breast cancer, due to interpretation challenges caused by variants of uncertain clinical significance (VUS). There is also no consensus on the selection criteria for WES. In this study, a pathology-supported genetic testing (PSGT) approach was used to select two BRCA1/2 mutation-negative breast cancer patients from the same family for WES. Homozygosity for the MTHFR 677 C>T mutation detected during this PSGT pre-screen step was considered insufficient to cause bilateral breast cancer in the index case and her daughter diagnosed with early-onset breast cancer (<30 years). Extended genetic testing using WES identified the RAD50 R385C missense mutation in both cases. This rare variant with a minor allele frequency (MAF) of <0.001 was classified as a VUS after exclusion in an affected cousin and extended genotyping in 164 unrelated breast cancer patients and 160 controls. Detection of functional polymorphisms (MAF > 5%) in the folate pathway in all three affected family members is consistent with inheritance of the luminal-type breast cancer in the family. PSGT assisted with the decision to pursue extended genetic testing and facilitated clinical interpretation of WES aimed at reduction of recurrence risk. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessArticle

Whole Exome Sequencing in Psoriasis Patients Contributes to Studies of Acitretin Treatment Difference

by Xingchen Zhou, Yijing He, Yehong Kuang, Jie Li, Jianglin Zhang, Mingliang Chen, Wangqing Chen, Juan Su, Shuang Zhao, Panpan Liu, Menglin Chen, Minxue Shen, Xiaoping Chen, Wu Zhu and Xiang Chen

Int. J. Mol. Sci. 2017, 18(2), 295; https://doi.org/10.3390/ijms18020295 - 29 Jan 2017

Cited by 10 | Viewed by 5493 | Correction

Abstract

Psoriasis vulgaris is an immune-mediated inflammatory skin disease. Although acitretin is a widely used synthetic retinoid for moderate to severe psoriasis, little is known about patients’ genetics in response to this drug. In this study, 179 patients were enrolled in either the discovery [...] Read more.

Psoriasis vulgaris is an immune-mediated inflammatory skin disease. Although acitretin is a widely used synthetic retinoid for moderate to severe psoriasis, little is known about patients’ genetics in response to this drug. In this study, 179 patients were enrolled in either the discovery set (13 patients) or replication set (166 patients). The discovery set was sequenced by whole exome sequencing and sequential validation was conducted in the replication set by MassArray assays. Four SNPs (single nucleotide polymorphisms) (rs1105223T>C in CRB2, rs11086065A>G in ANKLE1, rs3821414T>C in ARHGEF3, rs1802073 T>G in SFRP4) were found to be significantly associated with acitretin response in either co-dominant or dominant models via multivariable logistic regression analysis, while CRB2 rs1105223CC (OR = 4.10, 95% CI = 1.46–11.5, p = 0.007) and ANKLE1 rs11086065AG/GG (OR = 2.76, 95% CI = 1.42–5.37, p = 0.003) were associated with no response to acitretin after 8-week treatment. Meanwhile, ARHGEF3 rs3821414CT/CC (OR = 0.25, 95% CI = 0.10–0.68, p = 0.006) and SFRP4 rs1802073GG/GT (OR = 2.40, 95% CI, 1.23–4.70, p = 0.011) were associated with a higher response rate. Four new genetic variations with potential influences on the response to acitretin were found in this study which may serve as genetic markers for acitretin in psoriasis patients. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessArticle

The Cause of Death of a Child in the 18th Century Solved by Bone Microbiome Typing Using Laser Microdissection and Next Generation Sequencing

by Valeria D’Argenio, Marielva Torino, Vincenza Precone, Giorgio Casaburi, Maria Valeria Esposito, Laura Iaffaldano, Umberto Malapelle, Giancarlo Troncone, Iolanda Coto, Paolina Cavalcanti, Gaetano De Rosa, Francesco Salvatore and Lucia Sacchetti

Int. J. Mol. Sci. 2017, 18(1), 109; https://doi.org/10.3390/ijms18010109 - 6 Jan 2017

Cited by 11 | Viewed by 5309

Abstract

The history of medicine abounds in cases of mysterious deaths, especially by infectious diseases, which were probably unresolved because of the lack of knowledge and of appropriate technology. The aim of this study was to exploit contemporary technologies to try to identify the [...] Read more.

The history of medicine abounds in cases of mysterious deaths, especially by infectious diseases, which were probably unresolved because of the lack of knowledge and of appropriate technology. The aim of this study was to exploit contemporary technologies to try to identify the cause of death of a young boy who died from a putative “infection” at the end of the 18th century, and for whom an extraordinarily well-preserved minute bone fragment was available. After confirming the nature of the sample, we used laser microdissection to select the most “informative” area to be examined. Tissue genotyping indicated male gender, thereby confirming the notary’s report. 16S ribosomal RNA sequencing showed that Proteobacteria and Actinobacteria were more abundant than Firmicutes and Bacteroidetes, and that Pseudomonas was the most abundant bacterial genus in the Pseudomonadaceae family. These data suggest that the patient most likely died from Pseudomonas osteomyelitis. This case is an example of how new technological approaches, like laser microdissection and next-generation sequencing, can resolve ancient cases of uncertain etiopathology. Lastly, medical samples may contain a wealth of information that may not be accessible until more sophisticated technology becomes available. Therefore, one may envisage the possibility of systematically storing medical samples for evaluation by future generations. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessArticle

A Novel Pathogenic BRCA1 Splicing Variant Produces Partial Intron Retention in the Mature Messenger RNA

by Maria Valeria Esposito, Marcella Nunziato, Flavio Starnone, Antonella Telese, Alessandra Calabrese, Giuseppe D’Aiuto, Pietro Pucci, Massimiliano D’Aiuto, Francisco Baralle, Valeria D’Argenio and Francesco Salvatore

Int. J. Mol. Sci. 2016, 17(12), 2145; https://doi.org/10.3390/ijms17122145 - 21 Dec 2016

Cited by 11 | Viewed by 5831

Abstract

About 10% of all breast cancers arise from hereditary mutations that increase the risk of breast and ovarian cancers; and about 25% of these are associated with the BRCA1 or BRCA2 genes. The identification of BRCA1/BRCA2 mutations can enable physicians to [...] Read more.

About 10% of all breast cancers arise from hereditary mutations that increase the risk of breast and ovarian cancers; and about 25% of these are associated with the BRCA1 or BRCA2 genes. The identification of BRCA1/BRCA2 mutations can enable physicians to better tailor the clinical management of patients; and to initiate preventive measures in healthy carriers. The pathophysiological significance of newly identified variants poses challenges for genetic counseling. We characterized a new BRCA1 variant discovered in a breast cancer patient during BRCA1/2 screening by next-generation sequencing. Bioinformatic predictions; indicating that the variant is probably pathogenetic; were verified using retro-transcription of the patient’s RNA followed by PCR amplifications performed on the resulting cDNA. The variant causes the loss of a canonic donor splice site at position +2 in BRCA1 intron 21; and consequently the partial retention of 156 bp of intron 21 in the patient’s transcript; which demonstrates that this novel BRCA1 mutation plays a pathogenetic role in breast cancer. These findings enabled us to initiate appropriate counseling and to tailor the clinical management of this family. Lastly; these data reinforce the importance of studying the effects of sequence variants at the RNA level to verify their potential role in disease onset. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessArticle

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

by Giuseppe Fiume, Annarita Scialdone, Francesca Rizzo, Maria Rosaria De Filippo, Carmelo Laudanna, Francesco Albano, Gaetanina Golino, Eleonora Vecchio, Marilena Pontoriero, Selena Mimmi, Simona Ceglia, Antonio Pisano, Enrico Iaccino, Camillo Palmieri, Sergio Paduano, Giuseppe Viglietto, Alessandro Weisz, Giuseppe Scala and Ileana Quinto

Int. J. Mol. Sci. 2016, 17(11), 1848; https://doi.org/10.3390/ijms17111848 - 7 Nov 2016

Cited by 13 | Viewed by 6216

Abstract

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein [...] Read more.

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessCommunication

Pre-Analytical Considerations for Successful Next-Generation Sequencing (NGS): Challenges and Opportunities for Formalin-Fixed and Paraffin-Embedded Tumor Tissue (FFPE) Samples

by Gladys Arreaza, Ping Qiu, Ling Pang, Andrew Albright, Lewis Z. Hong, Matthew J. Marton and Diane Levitan

Int. J. Mol. Sci. 2016, 17(9), 1579; https://doi.org/10.3390/ijms17091579 - 20 Sep 2016

Cited by 64 | Viewed by 9054

Abstract

In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability [...] Read more.

In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability to measure more diverse analytes (e.g., circulating tumor cell (CTC), circulating tumor DNA (ctDNA), etc.), tumor tissue is still the most common and reliable source for biomarker investigation. Because of its worldwide use and ability to preserve samples for many decades at ambient temperature, formalin-fixed, paraffin-embedded tumor tissue (FFPE) is likely to be the preferred choice for tissue preservation in clinical practice for the foreseeable future. Multiple analyses are routinely performed on the same FFPE samples (such as Immunohistochemistry (IHC), in situ hybridization, RNAseq, DNAseq, TILseq, Methyl-Seq, etc.). Thus, specimen prioritization and optimization of the isolation of analytes is critical to ensure successful completion of each assay. FFPE is notorious for producing suboptimal DNA quality and low DNA yield. However, commercial vendors tend to request higher DNA sample mass than what is actually required for downstream assays, which restricts the breadth of biomarker work that can be performed. We evaluated multiple genomics service laboratories to assess the current state of NGS pre-analytical processing of FFPE. Significant differences in pre-analytical capabilities were observed. Key aspects are highlighted and recommendations are made to improve the current practice in translational research. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessArticle

A Next-Generation Sequencing Approach to Identify Gene Mutations in Early- and Late-Onset Hypertrophic Cardiomyopathy Patients of an Italian Cohort

by Speranza Rubattu, Cristina Bozzao, Ermelinda Pennacchini, Erika Pagannone, Beatrice Maria Musumeci, Maria Piane, Aldo Germani, Camilla Savio, Pietro Francia, Massimo Volpe, Camillo Autore and Luciana Chessa

Int. J. Mol. Sci. 2016, 17(8), 1239; https://doi.org/10.3390/ijms17081239 - 30 Jul 2016

Cited by 24 | Viewed by 6556

Abstract

Sequencing of sarcomere protein genes in patients fulfilling the clinical diagnostic criteria for hypertrophic cardiomyopathy (HCM) identifies a disease-causing mutation in 35% to 60% of cases. Age at diagnosis and family history may increase the yield of mutations screening. In order to assess [...] Read more.

Sequencing of sarcomere protein genes in patients fulfilling the clinical diagnostic criteria for hypertrophic cardiomyopathy (HCM) identifies a disease-causing mutation in 35% to 60% of cases. Age at diagnosis and family history may increase the yield of mutations screening. In order to assess whether Next-Generation Sequencing (NGS) may fulfil the molecular diagnostic needs in HCM, we included 17 HCM-related genes in a sequencing panel run on PGM IonTorrent. We selected 70 HCM patients, 35 with early (≤25 years) and 35 with late (≥65 years) diagnosis of disease onset. All samples had a 98.6% average of target regions, with coverage higher than 20× (mean coverage 620×). We identified 41 different mutations (seven of them novel) in nine genes: MYBPC3 (17/41 = 41%); MYH7 (10/41 = 24%); TNNT2, CAV3 and MYH6 (3/41 = 7.5% each); TNNI3 (2/41 = 5%); GLA, MYL2, and MYL3 (1/41=2.5% each). Mutation detection rate was 30/35 (85.7%) in early-onset and 8/35 (22.9%) in late-onset HCM patients, respectively (p < 0.0001). The overall detection rate for patients with positive family history was 84%, and 90.5% in patients with early disease onset. In our study NGS revealed higher mutations yield in patients with early onset and with a family history of HCM. Appropriate patient selection can increase the yield of genetic testing and make diagnostic testing cost-effective. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessArticle

Human Milk Cells Contain Numerous miRNAs that May Change with Milk Removal and Regulate Multiple Physiological Processes

by Mohammed Alsaweed, Ching Tat Lai, Peter E. Hartmann, Donna T. Geddes and Foteini Kakulas

Int. J. Mol. Sci. 2016, 17(6), 956; https://doi.org/10.3390/ijms17060956 - 17 Jun 2016

Cited by 63 | Viewed by 8612

Abstract

Human milk (HM) is a complex biofluid conferring nutritional, protective and developmental components for optimal infant growth. Amongst these are maternal cells, which change in response to feeding and were recently shown to be a rich source of miRNAs. We used next generation [...] Read more.

Human milk (HM) is a complex biofluid conferring nutritional, protective and developmental components for optimal infant growth. Amongst these are maternal cells, which change in response to feeding and were recently shown to be a rich source of miRNAs. We used next generation sequencing to characterize the cellular miRNA profile of HM collected before and after feeding. HM cells conserved higher miRNA content than the lipid and skim HM fractions or other body fluids, in accordance with previous studies. In total, 1467 known mature and 1996 novel miRNAs were identified, with 89 high-confidence novel miRNAs. HM cell content was higher post-feeding (p < 0.05), and was positively associated with total miRNA content (p = 0.014) and species number (p < 0.001). This coincided with upregulation of 29 known and 2 novel miRNAs, and downregulation of 4 known and 1 novel miRNAs post-feeding, but no statistically significant change in expression was found for the remaining miRNAs. These findings suggest that feeding may influence the miRNA content of HM cells. The most highly and differentially expressed miRNAs were key regulators of milk components, with potential diagnostic value in lactation performance. They are also involved in the control of body fluid balance, thirst, appetite, immune response, and development, implicating their functional significance for the infant. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessArticle

Gene Mutation Profiles in Primary Diffuse Large B Cell Lymphoma of Central Nervous System: Next Generation Sequencing Analyses

by Milena Todorovic Balint, Jelena Jelicic, Biljana Mihaljevic, Jelena Kostic, Bojana Stanic, Bela Balint, Nadja Pejanovic, Bojana Lucic, Natasa Tosic, Irena Marjanovic, Maja Stojiljkovic, Teodora Karan-Djurasevic, Ognjen Perisic, Goran Rakocevic, Milos Popovic, Sava Raicevic, Jelena Bila, Darko Antic, Bosko Andjelic and Sonja Pavlovic

Int. J. Mol. Sci. 2016, 17(5), 683; https://doi.org/10.3390/ijms17050683 - 6 May 2016

Cited by 32 | Viewed by 7764

Abstract

The existence of a potential primary central nervous system lymphoma-specific genomic signature that differs from the systemic form of diffuse large B cell lymphoma (DLBCL) has been suggested, but is still controversial. We investigated 19 patients with primary DLBCL of central nervous system [...] Read more.

The existence of a potential primary central nervous system lymphoma-specific genomic signature that differs from the systemic form of diffuse large B cell lymphoma (DLBCL) has been suggested, but is still controversial. We investigated 19 patients with primary DLBCL of central nervous system (DLBCL CNS) using the TruSeq Amplicon Cancer Panel (TSACP) for 48 cancer-related genes. Next generation sequencing (NGS) analyses have revealed that over 80% of potentially protein-changing mutations were located in eight genes (CTNNB1, PIK3CA, PTEN, ATM, KRAS, PTPN11, TP53 and JAK3), pointing to the potential role of these genes in lymphomagenesis. TP53 was the only gene harboring mutations in all 19 patients. In addition, the presence of mutated TP53 and ATM genes correlated with a higher total number of mutations in other analyzed genes. Furthermore, the presence of mutated ATM correlated with poorer event-free survival (EFS) (p = 0.036). The presence of the mutated SMO gene correlated with earlier disease relapse (p = 0.023), inferior event-free survival (p = 0.011) and overall survival (OS) (p = 0.017), while mutations in the PTEN gene were associated with inferior OS (p = 0.048). Our findings suggest that the TP53 and ATM genes could be involved in the molecular pathophysiology of primary DLBCL CNS, whereas mutations in the PTEN and SMO genes could affect survival regardless of the initial treatment approach. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessCommunication

Data Interoperability of Whole Exome Sequencing (WES) Based Mutational Burden Estimates from Different Laboratories

by Ping Qiu, Ling Pang, Gladys Arreaza, Maureen Maguire, Ken C. N. Chang, Matthew J. Marton and Diane Levitan

Int. J. Mol. Sci. 2016, 17(5), 651; https://doi.org/10.3390/ijms17050651 - 29 Apr 2016

Cited by 19 | Viewed by 6203

Abstract

Immune checkpoint inhibitors, which unleash a patient’s own T cells to kill tumors, are revolutionizing cancer treatment. Several independent studies suggest that higher non-synonymous mutational burden assessed by whole exome sequencing (WES) in tumors is associated with improved objective response, durable clinical benefit, [...] Read more.

Immune checkpoint inhibitors, which unleash a patient’s own T cells to kill tumors, are revolutionizing cancer treatment. Several independent studies suggest that higher non-synonymous mutational burden assessed by whole exome sequencing (WES) in tumors is associated with improved objective response, durable clinical benefit, and progression-free survival in immune checkpoint inhibitors treatment. Next-generation sequencing (NGS) is a promising technology being used in the clinic to direct patient treatment. Cancer genome WES poses a unique challenge due to tumor heterogeneity and sequencing artifacts introduced by formalin-fixed, paraffin-embedded (FFPE) tissue. In order to evaluate the data interoperability of WES data from different sources to survey tumor mutational landscape, we compared WES data of several tumor/normal matched samples from five commercial vendors. A large data discrepancy was observed from vendors’ self-reported data. Independent data analysis from vendors’ raw NGS data shows that whole exome sequencing data from qualified vendors can be combined and analyzed uniformly to derive comparable quantitative estimates of tumor mutational burden. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessArticle

Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches

by Sandra Preuner, Agnes Barna, Florian Frommlet, Stefan Czurda, Byrgazov Konstantin, Mary Alikian, Katerina Machova Polakova, Tomasz Sacha, Johan Richter, Thomas Lion and Christian Gabriel

Int. J. Mol. Sci. 2016, 17(5), 642; https://doi.org/10.3390/ijms17050642 - 29 Apr 2016

Cited by 5 | Viewed by 5142

Abstract

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring [...] Read more.

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessArticle

Potentially Treatable Disorder Diagnosed Post Mortem by Exome Analysis in a Boy with Respiratory Distress

by Valentina Imperatore, Maria Antonietta Mencarelli, Chiara Fallerini, Laura Bianciardi, Francesca Ariani, Simone Furini, Alessandra Renieri, Francesca Mari and Elisa Frullanti

Int. J. Mol. Sci. 2016, 17(3), 306; https://doi.org/10.3390/ijms17030306 - 27 Feb 2016

Cited by 7 | Viewed by 5324

Abstract

We highlight the importance of exome sequencing in solving a clinical case of a child who died at 14 months after a series of respiratory crises. He was the half-brother of a girl diagnosed at 7 years with the early-onset seizure variant of [...] Read more.

We highlight the importance of exome sequencing in solving a clinical case of a child who died at 14 months after a series of respiratory crises. He was the half-brother of a girl diagnosed at 7 years with the early-onset seizure variant of Rett syndrome due to CDKL5 mutation. We performed a test for CDKL5 in the boy, which came back negative. Driven by the mother’s compelling need for a diagnosis, we moved forward performing whole exome sequencing analysis. Surprisingly, two missense mutations in compound heterozygosity were identified in the RAPSN gene encoding a receptor-associated protein with a key role in clustering and anchoring nicotinic acetylcholine receptors at synaptic sites. This gene is responsible for a congenital form of myasthenic syndrome, a disease potentially treatable with cholinesterase inhibitors. Therefore, an earlier diagnosis in this boy would have led to a better clinical management and prognosis. Our study supports the key role of exome sequencing in achieving a definite diagnosis in severe perinatal diseases, an essential step especially when a specific therapy is available. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Review

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Open AccessReview

Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification

by Rick Kamps, Rita D. Brandão, Bianca J. van den Bosch, Aimee D. C. Paulussen, Sofia Xanthoulea, Marinus J. Blok and Andrea Romano

Int. J. Mol. Sci. 2017, 18(2), 308; https://doi.org/10.3390/ijms18020308 - 31 Jan 2017

Cited by 354 | Viewed by 33903

Abstract

Next-generation sequencing (NGS) technology has expanded in the last decades with significant improvements in the reliability, sequencing chemistry, pipeline analyses, data interpretation and costs. Such advances make the use of NGS feasible in clinical practice today. This review describes the recent technological developments [...] Read more.

Next-generation sequencing (NGS) technology has expanded in the last decades with significant improvements in the reliability, sequencing chemistry, pipeline analyses, data interpretation and costs. Such advances make the use of NGS feasible in clinical practice today. This review describes the recent technological developments in NGS applied to the field of oncology. A number of clinical applications are reviewed, i.e., mutation detection in inherited cancer syndromes based on DNA-sequencing, detection of spliceogenic variants based on RNA-sequencing, DNA-sequencing to identify risk modifiers and application for pre-implantation genetic diagnosis, cancer somatic mutation analysis, pharmacogenetics and liquid biopsy. Conclusive remarks, clinical limitations, implications and ethical considerations that relate to the different applications are provided. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessReview

From Clinical Standards to Translating Next-Generation Sequencing Research into Patient Care Improvement for Hepatobiliary and Pancreatic Cancers

by Ioannis D. Kyrochristos, Georgios K. Glantzounis, Demosthenes E. Ziogas, Ioannis Gizas, Dimitrios Schizas, Efstathios G. Lykoudis, Evangelos Felekouras, Anastasios Machairas, Christos Katsios, Theodoros Liakakos, William C. Cho and Dimitrios H. Roukos

Int. J. Mol. Sci. 2017, 18(1), 180; https://doi.org/10.3390/ijms18010180 - 18 Jan 2017

Cited by 27 | Viewed by 6422

Abstract

Hepatobiliary and pancreatic (HBP) cancers are associated with high cancer-related death rates. Surgery aiming for complete tumor resection (R0) remains the cornerstone of the treatment for HBP cancers. The current progress in the adjuvant treatment is quite slow, with gemcitabine chemotherapy available only [...] Read more.

Hepatobiliary and pancreatic (HBP) cancers are associated with high cancer-related death rates. Surgery aiming for complete tumor resection (R0) remains the cornerstone of the treatment for HBP cancers. The current progress in the adjuvant treatment is quite slow, with gemcitabine chemotherapy available only for pancreatic ductal adenocarcinoma (PDA). In the advanced and metastatic setting, only two targeted drugs have been approved by the Food & Drug Administration (FDA), which are sorafenib for hepatocellular carcinoma and erlotinib for PDA. It is a pity that multiple Phase III randomized control trials testing the efficacy of targeted agents have negative results. Failure in the development of effective drugs probably reflects the poor understanding of genome-wide alterations and molecular mechanisms orchestrating therapeutic resistance and recurrence. In the post-ENCODE (Encyclopedia of DNA Elements) era, cancer is referred to as a highly heterogeneous and systemic disease of the genome. The unprecedented potential of next-generation sequencing (NGS) technologies to accurately identify genetic and genomic variations has attracted major research and clinical interest. The applications of NGS include targeted NGS with potential clinical implications, while whole-exome and whole-genome sequencing focus on the discovery of both novel cancer driver genes and therapeutic targets. These advances dictate new designs for clinical trials to validate biomarkers and drugs. This review discusses the findings of available NGS studies on HBP cancers and the limitations of genome sequencing analysis to translate genome-based biomarkers and drugs into patient care in the clinic. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Open AccessReview

Clinical Application of Targeted Next Generation Sequencing for Colorectal Cancers

by Quitterie Fontanges, Ricardo De Mendonca, Isabelle Salmon, Marie Le Mercier and Nicky D’Haene

Int. J. Mol. Sci. 2016, 17(12), 2117; https://doi.org/10.3390/ijms17122117 - 16 Dec 2016

Cited by 19 | Viewed by 5587

Abstract

Promising targeted therapy and personalized medicine are making molecular profiling of tumours a priority. For colorectal cancer (CRC) patients, international guidelines made RAS (KRAS and NRAS) status a prerequisite for the use of anti-epidermal growth factor receptor agents (anti-EGFR). Daily, new [...] Read more.

Promising targeted therapy and personalized medicine are making molecular profiling of tumours a priority. For colorectal cancer (CRC) patients, international guidelines made RAS (KRAS and NRAS) status a prerequisite for the use of anti-epidermal growth factor receptor agents (anti-EGFR). Daily, new data emerge on the theranostic and prognostic role of molecular biomarkers, which is a strong incentive for a validated, sensitive and broadly available molecular screening test in order to implement and improve multi-modal therapy strategy and clinical trials. Next generation sequencing (NGS) has begun to supplant other technologies for genomic profiling. Targeted NGS is a method that allows parallel sequencing of thousands of short DNA sequences in a single test offering a cost-effective approach for detecting multiple genetic alterations with a minimum amount of DNA. In the present review, we collected data concerning the clinical application of NGS technology in the setting of colorectal cancer. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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Next Generation Sequencing Approach in a Prenatal Case of Cardio-Facio-Cutaneus Syndrome

by Mafalda Mucciolo, Claudio Dello Russo, Laura D’Emidio, Alvaro Mesoraca and Claudio Giorlandino

Int. J. Mol. Sci. 2016, 17(6), 952; https://doi.org/10.3390/ijms17060952 - 16 Jun 2016

Cited by 5 | Viewed by 5698

Abstract

Cardiofaciocutaneous syndrome (CFCS) belongs to a group of developmental disorders due to defects in the Ras/Mitogen-Activated Protein Kinase (RAS/MAPK) signaling pathway named RASophaties. While postnatal presentation of these disorders is well known, the prenatal and neonatal characteristics are less recognized. Noonan syndrome, Costello [...] Read more.

Cardiofaciocutaneous syndrome (CFCS) belongs to a group of developmental disorders due to defects in the Ras/Mitogen-Activated Protein Kinase (RAS/MAPK) signaling pathway named RASophaties. While postnatal presentation of these disorders is well known, the prenatal and neonatal characteristics are less recognized. Noonan syndrome, Costello syndrome, and CFCS diagnosis should be considered in pregnancies with a normal karyotype and in the case of ultrasound findings such as increased nuchal translucency, polyhydramnios, macrosomia and cardiac defect. Because all the RASopathies share similar clinical features, their molecular characterization is complex, time consuming and expensive. Here we report a case of CFCS prenatally diagnosed through Next Generation Prenatal Diagnosis (NGPD), a new targeted approach that allows us to concurrently investigate all the genes involved in the RASophaties. Full article

(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)

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