共查询到20条相似文献,搜索用时 31 毫秒
1.
Singh N. Sadananda Jia Nee Foo Meng Tiak Toh Lubomira Cermakova Laia Trigueros-Motos Teddy Chan Herty Liany Jennifer A. Collins Sima Gerami Roshni R. Singaraja Michael R. Hayden Gordon A. Francis Jiri Frohlich Chiea Chuen Khor Liam R. Brunham 《Journal of lipid research》2015,56(10):1993-2001
A low level of HDL cholesterol (HDL-C) is a common clinical scenario and an important marker for increased cardiovascular risk. Many patients with very low or very high HDL-C have a rare mutation in one of several genes, but identification of the molecular abnormality in patients with extreme HDL-C is rarely performed in clinical practice. We investigated the accuracy and diagnostic yield of a targeted next-generation sequencing (NGS) assay for extreme levels of HDL-C. We developed a targeted NGS panel to capture the exons, intron/exon boundaries, and untranslated regions of 26 genes with highly penetrant effects on plasma lipid levels. We sequenced 141 patients with extreme HDL-C levels and prioritized variants in accordance with medical genetics guidelines. We identified 35 pathogenic and probably pathogenic variants in HDL genes, including 21 novel variants, and performed functional validation on a subset of these. Overall, a molecular diagnosis was established in 35.9% of patients with low HDL-C and 5.2% with high HDL-C, and all prioritized variants identified by NGS were confirmed by Sanger sequencing. Our results suggest that a molecular diagnosis can be identified in a substantial proportion of patients with low HDL-C using targeted NGS. 相似文献
2.
Julien Philippe Mehdi Derhourhi Emmanuelle Durand Emmanuel Vaillant Aurélie Dechaume Iandry Rabearivelo Véronique Dhennin Martine Vaxillaire Franck De Graeve Olivier Sand Philippe Froguel Amélie Bonnefond 《PloS one》2015,10(11)
Molecular diagnosis of monogenic diabetes and obesity is of paramount importance for both the patient and society, as it can result in personalized medicine associated with a better life and it eventually saves health care spending. Genetic clinical laboratories are currently switching from Sanger sequencing to next-generation sequencing (NGS) approaches but choosing the optimal protocols is not easy. Here, we compared the sequencing coverage of 43 genes involved in monogenic forms of diabetes and obesity, and variant detection rates, resulting from four enrichment methods based on the sonication of DNA (Agilent SureSelect, RainDance technologies), or using enzymes for DNA fragmentation (Illumina Nextera, Agilent HaloPlex). We analyzed coding exons and untranslated regions of the 43 genes involved in monogenic diabetes and obesity. We found that none of the methods achieves yet full sequencing of the gene targets. Nonetheless, the RainDance, SureSelect and HaloPlex enrichment methods led to the best sequencing coverage of the targets; while the Nextera method resulted in the poorest sequencing coverage. Although the sequencing coverage was high, we unexpectedly found that the HaloPlex method missed 20% of variants detected by the three other methods and Nextera missed 10%. The question of which NGS technique for genetic diagnosis yields the highest diagnosis rate is frequently discussed in the literature and the response is still unclear. Here, we showed that the RainDance enrichment method as well as SureSelect, which are both based on the sonication of DNA, resulted in a good sequencing quality and variant detection, while the use of enzymes to fragment DNA (HaloPlex or Nextera) might not be the best strategy to get an accurate sequencing. 相似文献
3.
Clare M. McCourt Darragh G. McArt Ken Mills Mark A. Catherwood Perry Maxwell David J. Waugh Peter Hamilton Joe M. O'Sullivan Manuel Salto-Tellez 《PloS one》2013,8(7)
Next Generation Sequencing (NGS) has the potential of becoming an important tool in clinical diagnosis and therapeutic decision-making in oncology owing to its enhanced sensitivity in DNA mutation detection, fast-turnaround of samples in comparison to current gold standard methods and the potential to sequence a large number of cancer-driving genes at the one time. We aim to test the diagnostic accuracy of current NGS technology in the analysis of mutations that represent current standard-of-care, and its reliability to generate concomitant information on other key genes in human oncogenesis. Thirteen clinical samples (8 lung adenocarcinomas, 3 colon carcinomas and 2 malignant melanomas) already genotyped for EGFR, KRAS and BRAF mutations by current standard-of-care methods (Sanger Sequencing and q-PCR), were analysed for detection of mutations in the same three genes using two NGS platforms and an additional 43 genes with one of these platforms. The results were analysed using closed platform-specific proprietary bioinformatics software as well as open third party applications. Our results indicate that the existing format of the NGS technology performed well in detecting the clinically relevant mutations stated above but may not be reliable for a broader unsupervised analysis of the wider genome in its current design. Our study represents a diagnostically lead validation of the major strengths and weaknesses of this technology before consideration for diagnostic use. 相似文献
4.
Zhi-Zhou Shi Yue-Ming Zhang Li Shang Jia-Jie Hao Tong-Tong Zhang Bo-Shi Wang Jian-Wei Liang Xi Chen Ying Zhang Gui-Qi Wang Ming-Rong Wang Yu Zhang 《BMC medical genomics》2012,5(1):1-10
Background
Aortopathies are a group of disorders characterized by aneurysms, dilation, and tortuosity of the aorta. Because of the phenotypic overlap and genetic heterogeneity of diseases featuring aortopathy, molecular testing is often required for timely and correct diagnosis of affected individuals. In this setting next generation sequencing (NGS) offers several advantages over traditional molecular techniques.Methods
The purpose of our study was to compare NGS enrichment methods for a clinical assay targeting the nine genes known to be associated with aortopathy. RainDance emulsion PCR and SureSelect RNA-bait hybridization capture enrichment methods were directly compared by enriching DNA from eight samples. Enriched samples were barcoded, pooled, and sequenced on the Illumina HiSeq2000 platform. Depth of coverage, consistency of coverage across samples, and the overlap of variants identified were assessed. This data was also compared to whole-exome sequencing data from ten individuals.Results
Read depth was greater and less variable among samples that had been enriched using the RNA-bait hybridization capture enrichment method. In addition, samples enriched by hybridization capture had fewer exons with mean coverage less than 10, reducing the need for followup Sanger sequencing. Variants sets produced were 77% concordant, with both techniques yielding similar numbers of discordant variants.Conclusions
When comparing the design flexibility, performance, and cost of the targeted enrichment methods to whole-exome sequencing, the RNA-bait hybridization capture enrichment gene panel offers the better solution for interrogating the aortopathy genes in a clinical laboratory setting. 相似文献5.
Nicole Weisschuh Anja K. Mayer Tim M. Strom Susanne Kohl Nicola Gl?ckle Max Schubach Sten Andreasson Antje Bernd David G. Birch Christian P. Hamel John R. Heckenlively Samuel G. Jacobson Christina Kamme Ulrich Kellner Erdmute Kunstmann Pietro Maffei Charlotte M. Reiff Klaus Rohrschneider Thomas Rosenberg Günther Rudolph Rita Vámos Balázs Varsányi Richard G. Weleber Bernd Wissinger 《PloS one》2016,11(1)
Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes. 相似文献
6.
Guy Froyen An Broekmans Femke Hillen Karin Pat Ruth Achten Jeroen Mebis Jean-Luc Rummens Johan Willemse Brigitte Maes 《PloS one》2016,11(4)
The inevitable switch from standard molecular methods to next-generation sequencing for the molecular profiling of tumors is challenging for most diagnostic laboratories. However, fixed validation criteria for diagnostic accreditation are not in place because of the great variability in methods and aims. Here, we describe the validation of a custom panel of hotspots in 24 genes for the detection of somatic mutations in non-small cell lung carcinoma, colorectal carcinoma and malignant melanoma starting from FFPE sections, using 14, 36 and 5 cases, respectively. The targeted hotspots were selected for their present or future clinical relevance in solid tumor types. The target regions were enriched with the TruSeq approach starting from limited amounts of DNA. Cost effective sequencing of 12 pooled libraries was done using a micro flow cell on the MiSeq and subsequent data analysis with MiSeqReporter and VariantStudio. The entire workflow was diagnostically validated showing a robust performance with maximal sensitivity and specificity using as thresholds a variant allele frequency >5% and a minimal amplicon coverage of 300. We implemented this method through the analysis of 150 routine diagnostic samples and identified clinically relevant mutations in 16 genes including KRAS (32%), TP53 (32%), BRAF (12%), APC (11%), EGFR (8%) and NRAS (5%). Importantly, the highest success rate was obtained when using also the low quality DNA samples. In conclusion, we provide a workflow for the validation of targeted NGS by a custom-designed pan-solid tumor panel in a molecular diagnostic lab and demonstrate its robustness in a clinical setting. 相似文献
7.
Isabelle Audo Kinga M Bujakowska Thierry Léveillard Saddek Mohand-Sa?d Marie-Elise Lancelot Aurore Germain Aline Antonio Christelle Michiels Jean-Paul Saraiva Mélanie Letexier José-Alain Sahel Shomi S Bhattacharya Christina Zeitz 《Orphanet journal of rare diseases》2012,7(1):1-17
Background
Inherited retinal disorders are clinically and genetically heterogeneous with more than 150 gene defects accounting for the diversity of disease phenotypes. So far, mutation detection was mainly performed by APEX technology and direct Sanger sequencing of known genes. However, these methods are time consuming, expensive and unable to provide a result if the patient carries a new gene mutation. In addition, multiplicity of phenotypes associated with the same gene defect may be overlooked.Methods
To overcome these challenges, we designed an exon sequencing array to target 254 known and candidate genes using Agilent capture. Subsequently, 20 DNA samples from 17 different families, including four patients with known mutations were sequenced using Illumina Genome Analyzer IIx next-generation-sequencing (NGS) platform. Different filtering approaches were applied to identify the genetic defect. The most likely disease causing variants were analyzed by Sanger sequencing. Co-segregation and sequencing analysis of control samples validated the pathogenicity of the observed variants.Results
The phenotype of the patients included retinitis pigmentosa, congenital stationary night blindness, Best disease, early-onset cone dystrophy and Stargardt disease. In three of four control samples with known genotypes NGS detected the expected mutations. Three known and five novel mutations were identified in NR2E3, PRPF3, EYS, PRPF8, CRB1, TRPM1 and CACNA1F. One of the control samples with a known genotype belongs to a family with two clinical phenotypes (Best and CSNB), where a novel mutation was identified for CSNB. In six families the disease associated mutations were not found, indicating that novel gene defects remain to be identified.Conclusions
In summary, this unbiased and time-efficient NGS approach allowed mutation detection in 75% of control cases and in 57% of test cases. Furthermore, it has the possibility of associating known gene defects with novel phenotypes and mode of inheritance. 相似文献8.
To date we have little knowledge of how accurate next-generation sequencing (NGS) technologies are in sequencing repetitive sequences beyond known limitations to accurately sequence homopolymers. Only a handful of previous reports have evaluated the potential of NGS for sequencing short tandem repeats (microsatellites) and no empirical study has compared and evaluated the performance of more than one NGS platform with the same dataset. Here we examined yeast microsatellite variants from both long-read (454-sequencing) and short-read (Illumina) NGS platforms and compared these to data derived through Sanger sequencing. In addition, we investigated any locus-specific biases and differences that might have resulted from variability in microsatellite repeat number, repeat motif or type of mutation. Out of 112 insertion/deletion variants identified among 45 microsatellite amplicons in our study, we found 87.5% agreement between the 454-platform and Sanger sequencing in frequency of variant detection after Benjamini-Hochberg correction for multiple tests. For a subset of 21 microsatellite amplicons derived from Illumina sequencing, the results of short-read platform were highly consistent with the other two platforms, with 100% agreement with 454-sequencing and 93.6% agreement with the Sanger method after Benjamini-Hochberg correction. We found that the microsatellite attributes copy number, repeat motif and type of mutation did not have a significant effect on differences seen between the sequencing platforms. We show that both long-read and short-read NGS platforms can be used to sequence short tandem repeats accurately, which makes it feasible to consider the use of these platforms in high-throughput genotyping. It appears the major requirement for achieving both high accuracy and rare variant detection in microsatellite genotyping is sufficient read depth coverage. This might be a challenge because each platform generates a consistent pattern of non-uniform sequence coverage, which, as our study suggests, may affect some types of tandem repeats more than others. 相似文献
9.
Germline and somatic mutation profile in Cancer patients revealed by a medium-sized pan-Cancer panel
《Genomics》2021,113(4):1930-1939
Gene mutation detection and the resulted precision-medicine therapy is transforming clinical practice. Here, we report the use of a custom-developed, medium-sized, pan-cancer probe panel for the detection of somatic and germline mutations. We used a hybridization capture-based NGS assay for targeted deep sequencing of all exons and selected introns of 181 key cancer driver genes, covering both inherited risks and somatic mutations. We performed paired-variant calling on tumor samples and their matched normal samples. We processed clinical patient samples of formalin-fixed, paraffin embedded tumors (FFPE samples) and cell-free peripheral blood (cfDNA samples). We found germline mutations of inherited cancer risk at 9%; and discovered a novel germline mutation in BRCA1. Somatic mutation rate in driver genes is at 73.1%, much higher than previously reported. On recommending precision-medicine therapeutics, we achieved 91.6% for patients with FFPE samples. 相似文献
10.
Lee-Jun C. Wong 《Mitochondrion》2013,13(4):379-387
Mitochondrial disorders are by far the most genetically heterogeneous group of diseases, involving two genomes, the 16.6 kb mitochondrial genome and ~ 1500 genes encoded in the nuclear genome. For maternally inherited mitochondrial DNA disorders, a complete molecular diagnosis requires several different methods for the detection and quantification of mtDNA point mutations and large deletions. For mitochondrial disorders caused by autosomal recessive, dominant, and X-linked nuclear genes, the diagnosis has relied on clinical, biochemical, and molecular studies to point to a group of candidate genes followed by stepwise Sanger sequencing of the candidate genes one-by-one. The development of Next Generation Sequencing (NGS) has revolutionized the diagnostic approach. Using massively parallel sequencing (MPS) analysis of the entire mitochondrial genome, mtDNA point mutations and deletions can be detected and quantified in one single step. The NGS approach also allows simultaneous analyses of a group of genes or the whole exome, thus, the mutations in causative gene(s) can be identified in one-step. New approaches make genetic analyses much faster and more efficient. Huge amounts of sequencing data produced by the new technologies brought new challenges to bioinformatics, analytical pipelines, and interpretation of numerous novel variants. This article reviews the clinical utility of next generation sequencing for the molecular diagnoses of complex dual genome mitochondrial disorders. 相似文献
11.
12.
The congenital muscular dystrophies (CMDs) are a clinically and genetically heterogeneous group of muscle disorders. Clinically hypotonia is present from birth, with progressive muscle weakness and wasting through development. For the most part, CMDs can mechanistically be attributed to failure of basement membrane protein laminin-α2 sufficiently binding with correctly glycosylated α-dystroglycan. The majority of CMDs therefore arise as the result of either a deficiency of laminin-α2 (MDC1A) or hypoglycosylation of α-dystroglycan (dystroglycanopathy). Here we consider whether by filling a regenerative medicine niche, the zebrafish model can address the present challenge of delivering novel therapeutic solutions for CMD. In the first instance the readiness and appropriateness of the zebrafish as a model organism for pioneering regenerative medicine therapies in CMD is analysed, in particular for MDC1A and the dystroglycanopathies. Despite the recent rapid progress made in gene editing technology, these approaches have yet to yield any novel zebrafish models of CMD. Currently the most genetically relevant zebrafish models to the field of CMD, have all been created by N-ethyl-N-nitrosourea (ENU) mutagenesis. Once genetically relevant models have been established the zebrafish has several important facets for investigating the mechanistic cause of CMD, including rapid ex vivo development, optical transparency up to the larval stages of development and relative ease in creating transgenic reporter lines. Together, these tools are well suited for use in live-imaging studies such as in vivo modelling of muscle fibre detachment. Secondly, the zebrafish's contribution to progress in effective treatment of CMD was analysed. Two approaches were identified in which zebrafish could potentially contribute to effective therapies. The first hinges on the augmentation of functional redundancy within the system, such as upregulating alternative laminin chains in the candyfloss fish, a model of MDC1A. Secondly high-throughput small molecule screens not only provide effective therapies, but also an alternative strategy for investigating CMD in zebrafish. In this instance insight into disease mechanism is derived in reverse. Zebrafish models are therefore clearly of critical importance in the advancement of regenerative medicine strategies in CMD.This article is part of a Directed Issue entitled: Regenerative Medicine: The challenge of translation. 相似文献
13.
Mi-Hyun Park Hwanseok Rhee Jung Hoon Park Hae-Mi Woo Byung-Ok Choi Bo-Young Kim Ki Wha Chung Yoo-Bok Cho Hyung Jin Kim Ji-Won Jung Soo Kyung Koo 《PloS one》2014,9(1)
Next-generation sequencing (NGS) has enabled the high-throughput discovery of germline and somatic mutations. However, NGS-based variant detection is still prone to errors, resulting in inaccurate variant calls. Here, we categorized the variants detected by NGS according to total read depth (TD) and SNP quality (SNPQ), and performed Sanger sequencing with 348 selected non-synonymous single nucleotide variants (SNVs) for validation. Using the SAMtools and GATK algorithms, the validation rate was positively correlated with SNPQ but showed no correlation with TD. In addition, common variants called by both programs had a higher validation rate than caller-specific variants. We further examined several parameters to improve the validation rate, and found that strand bias (SB) was a key parameter. SB in NGS data showed a strong difference between the variants passing validation and those that failed validation, showing a validation rate of more than 92% (filtering cutoff value: alternate allele forward [AF]≥20 and AF<80 in SAMtools, SB<–10 in GATK). Moreover, the validation rate increased significantly (up to 97–99%) when the variant was filtered together with the suggested values of mapping quality (MQ), SNPQ and SB. This detailed and systematic study provides comprehensive recommendations for improving validation rates, saving time and lowering cost in NGS analyses. 相似文献
14.
Next‐generation sequencing (NGS)‐based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population
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Prateek Gupta Sameera Devulapalli Bradley John Till Yellamaraju Sreelakshmi Rameshwar Sharma 《The Plant journal : for cell and molecular biology》2017,92(3):495-508
The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next‐generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty‐five genes belonging to carotenoids and folate metabolism were PCR‐amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600‐bp amplicons were directly sequenced in a non‐overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128‐fold pooling. An evaluation of six different software programs (camba , crisp , gatk unified genotyper , lofreq , snver and vipr ) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate. 相似文献
15.
Christopher T. Johansen Joseph B. Dubé Melissa N. Loyzer Austin MacDonald David E. Carter Adam D. McIntyre Henian Cao Jian Wang John F. Robinson Robert A. Hegele 《Journal of lipid research》2014,55(4):765-772
We report the design of a targeted resequencing panel for monogenic dyslipidemias, LipidSeq, for the purpose of replacing Sanger sequencing in the clinical detection of dyslipidemia-causing variants. We also evaluate the performance of the LipidSeq approach versus Sanger sequencing in 84 patients with a range of phenotypes including extreme blood lipid concentrations as well as additional dyslipidemias and related metabolic disorders. The panel performs well, with high concordance (95.2%) in samples with known mutations based on Sanger sequencing and a high detection rate (57.9%) of mutations likely to be causative for disease in samples not previously sequenced. Clinical implementation of LipidSeq has the potential to aid in the molecular diagnosis of patients with monogenic dyslipidemias with a high degree of speed and accuracy and at lower cost than either Sanger sequencing or whole exome sequencing. Furthermore, LipidSeq will help to provide a more focused picture of monogenic and polygenic contributors that underlie dyslipidemia while excluding the discovery of incidental pathogenic clinically actionable variants in nonmetabolism-related genes, such as oncogenes, that would otherwise be identified by a whole exome approach, thus minimizing potential ethical issues. 相似文献
16.
Hansook Kim Chong Tao Wang Hsiao-Mei Lu Sara Seidler Hong Lu Steven Keiles Elizabeth C. Chao A. J. Stuenkel Xiang Li Aaron M. Elliott 《PloS one》2014,9(5)
Breast cancer is the most commonly diagnosed cancer in women, with 10% of disease attributed to hereditary factors. Although BRCA1 and BRCA2 account for a high percentage of hereditary cases, there are more than 25 susceptibility genes that differentially impact the risk for breast cancer. Traditionally, germline testing for breast cancer was performed by Sanger dideoxy terminator sequencing in a reflexive manner, beginning with BRCA1 and BRCA2. The introduction of next-generation sequencing (NGS) has enabled the simultaneous testing of all genes implicated in breast cancer resulting in diagnostic labs offering large, comprehensive gene panels. However, some physicians prefer to only test for those genes in which established surveillance and treatment protocol exists. The NGS based BRCAplus test utilizes a custom tiled PCR based target enrichment design and bioinformatics pipeline coupled with array comparative genomic hybridization (aCGH) to identify mutations in the six high-risk genes: BRCA1, BRCA2, PTEN, TP53, CDH1, and STK11. Validation of the assay with 250 previously characterized samples resulted in 100% detection of 3,025 known variants and analytical specificity of 99.99%. Analysis of the clinical performance of the first 3,000 BRCAplus samples referred for testing revealed an average coverage greater than 9,000X per target base pair resulting in excellent specificity and the sensitivity to detect low level mosaicism and allele-drop out. The unique design of the assay enabled the detection of pathogenic mutations missed by previous testing. With the abundance of NGS diagnostic tests being released, it is essential that clinicians understand the advantages and limitations of different test designs. 相似文献
17.
Taniguchi M Kurahashi H Noguchi S Sese J Okinaga T Tsukahara T Guicheney P Ozono K Nishino I Morishita S Toda T 《Biochemical and biophysical research communications》2006,342(2):489-502
Fukuyama-type congenital muscular dystrophy (FCMD) and laminin-alpha2 deficient congenital muscular dystrophy (MDC1A) are congenital muscular dystrophies (CMDs) and they both are categorized into the same clinical entity of muscular dystrophy as Duchenne muscular dystrophy (DMD). All three disorders share a common etiologic defect in the dystrophin-glycoprotein complex, which connects muscle structural proteins with the extracellular basement membrane. To investigate the pathophysiology of these CMDs, we generated microarray gene expression profiles of skeletal muscle from patients in various clinical stages. Despite diverse pathological changes, the correlation coefficient of overall gene expression among these samples was considerably high. We performed a multi-dimensional statistical analysis, the Distillation, to extract determinant genes that distinguish CMD muscle from normal controls. Up-regulated genes were primarily extracellular matrix (ECM) components, whereas down-regulated genes included structural components of mature muscle. These observations reflect active interstitial fibrosis with less active regeneration of muscle cell components in the CMDs, characteristics that are clearly distinct from those of DMD. Although the severity of fibrosis varied among the specimens tested, ECM gene expression was consistently high without substantial changes through the clinical course. Further, in situ hybridization showed more prominent ECM gene expression on muscle cells than on interstitial tissue cells, suggesting that ECM components are induced by regeneration process rather than by 'dystrophy.' These data imply that the etiology of FCMD and MDC1A differs from that of the chronic phase of classical muscular dystrophy, and the major pathophysiologic change in CMDs might instead result from primary active fibrosis. 相似文献
18.
Tobias Eisenberger Christian Decker Milan Hiersche Ruben C. Hamann Eva Decker Steffen Neuber Valeska Frank Hanno J. Bolz Henry Fehrenbach Lars Pape Burkhard Toenshoff Christoph Mache Kay Latta Carsten Bergmann 《PloS one》2015,10(2)
Renal cysts are clinically and genetically heterogeneous conditions. Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent life-threatening genetic disease and mainly caused by mutations in PKD1. The presence of six PKD1 pseudogenes and tremendous allelic heterogeneity make molecular genetic testing challenging requiring laborious locus-specific amplification. Increasing evidence suggests a major role for PKD1 in early and severe cases of ADPKD and some patients with a recessive form. Furthermore it is becoming obvious that clinical manifestations can be mimicked by mutations in a number of other genes with the necessity for broader genetic testing. We established and validated a sequence capture based NGS testing approach for all genes known for cystic and polycystic kidney disease including PKD1. Thereby, we demonstrate that the applied standard mapping algorithm specifically aligns reads to the PKD1 locus and overcomes the complication of unspecific capture of pseudogenes. Employing careful and experienced assessment of NGS data, the method is shown to be very specific and equally sensitive as established methods. An additional advantage over conventional Sanger sequencing is the detection of copy number variations (CNVs). Sophisticated bioinformatic read simulation increased the high analytical depth of the validation study and further demonstrated the strength of the approach. We further raise some awareness of limitations and pitfalls of common NGS workflows when applied in complex regions like PKD1 demonstrating that quality of NGS needs more than high coverage of the target region. By this, we propose a time- and cost-efficient diagnostic strategy for comprehensive molecular genetic testing of polycystic kidney disease which is highly automatable and will be of particular value when therapeutic options for PKD emerge and genetic testing is needed for larger numbers of patients. 相似文献
19.
Wendy W. J. de Leng Christa G. Gadellaa-van Hooijdonk Fran?oise A. S. Barendregt-Smouter Marco J. Koudijs Ies Nijman John W. J. Hinrichs Edwin Cuppen Stef van Lieshout Robert D. Loberg Maja de Jonge Emile E. Voest Roel A. de Weger Neeltje Steeghs Marlies H. G. Langenberg Stefan Sleijfer Stefan M. Willems Martijn P. Lolkema 《PloS one》2016,11(2)
Background
Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.Method
We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.Results
Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.Conclusion
Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA. 相似文献20.
Xinzhong Li Andrew J. Buckton Samuel L. Wilkinson Shibu John Roddy Walsh Tomas Novotny Iveta Valaskova Manu Gupta Laurence Game Paul J R. Barton Stuart A. Cook James S. Ware 《PloS one》2013,8(7)