Novel splice variants of the human kallikrein-related peptidases 11 (KLK11) and 12 (KLK12), unraveled by next-generation sequencing technology

AbstractTissue kallikrein, kallikrein-related peptidases (KLKs), and plasma kallikrein form the largest group of serine proteases in the human genome, sharing many structural and functional characteristics. In this study, we describe the molecular cloning of four novel splice variants of the humanKLK11andKLK12genes, discovered by combining 3′ rapid amplification of cDNA ends (3′ RACE), next-generation sequencing (NGS) technology, advanced bioinformatic analysis and Sanger sequencing. Expression analysis of these new transcripts in cell lines originating from 17 cancerous and two normal tissues revealed the expression pattern of each transcript. These novelKLK11andKLK12splice variants represent new potential cancer biomarkers.

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Next Generation Sequencing Identifies Five Novel Mutations in Lebanese Patients with Bardet–Biedl and Usher Syndromes

Genes ◽

10.3390/genes10121047 ◽

2019 ◽

Vol 10 (12) ◽

pp. 1047 ◽

Cited By ~ 1

Author(s):

Lama Jaffal ◽

Wissam H Joumaa ◽

Alexandre Assi ◽

Charles Helou ◽

George Cherfan ◽

...

Keyword(s):

Next Generation Sequencing ◽

Usher Syndrome ◽

Bioinformatic Analysis ◽

Next Generation ◽

Novel Mutations ◽

Pathogenic Variants ◽

Whole Exome ◽

Targeted Ngs ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Aim: To identify disease-causing mutations in four Lebanese families: three families with Bardet–Biedl and one family with Usher syndrome (BBS and USH respectively), using next generation sequencing (NGS). Methods: We applied targeted NGS in two families and whole exome sequencing (WES) in two other families. Pathogenicity of candidate mutations was evaluated according to frequency, conservation, in silico prediction tools, segregation with disease, and compatibility with inheritance pattern. The presence of pathogenic variants was confirmed via Sanger sequencing followed by segregation analysis. Results: Most likely disease-causing mutations were identified in all included patients. In BBS patients, we found (M1): c.2258A > T, p. (Glu753Val) in BBS9, (M2): c.68T > C; p. (Leu23Pro) in ARL6, (M3): c.265_266delTT; p. (Leu89Valfs*11) and (M4): c.880T > G; p. (Tyr294Asp) in BBS12. A previously known variant (M5): c.551A > G; p. (Asp184Ser) was also detected in BBS5. In the USH patient, we found (M6): c.188A > C, p. (Tyr63Ser) in CLRN1. M2, M3, M4, and M6 were novel. All of the candidate mutations were shown to be likely disease-causing through our bioinformatic analysis. They also segregated with the corresponding phenotype in available family members. Conclusion: This study expanded the mutational spectrum and showed the genetic diversity of BBS and USH. It also spotlighted the efficiency of NGS techniques in revealing mutations underlying clinically and genetically heterogeneous disorders.

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Potentially Actionable Targets: Evidence Standards for Credible Next Generation Sequencing Technology Assessment Claims

INNOVATIONS in pharmacy ◽

10.24926/iip.v8i3.534 ◽

2017 ◽

Vol 8 (3) ◽

pp. 9

Author(s):

Paul C Langley

Keyword(s):

Next Generation Sequencing ◽

Technology Assessment ◽

Critical Issue ◽

Evidence Base ◽

Cost Of Care ◽

Next Generation ◽

Next Generation Sequencing Technology ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Despite considerable resources devoted to developing databases to support competitive credible claims for next generation sequencing (NGS) claims, we have yet to meet the standards required in health technology assessment to support such claims. The purpose of this commentary is to consider options open in establishing claims for NGS recommendations. Although NGS platforms offer potential promise in improving clinical outcomes, supporting cost-effectiveness and reducing the overall cost of care in target populations, this has yet to be demonstrated on a scale that is likely to satisfy reimbursers and health care decision makers. Issues addressed include (i) the importance of credible, evaluable and replicable claims from individual NGS platforms; (ii) the difficulties in moving beyond broad-brush claims for improved survival; (iii) the standards required for an NGS evidence base; (iv) protocol designs in establishing the independent contribution of NGS actionable therapy recommendations to outcomes claims; (v) the role of NGS registries; and (vi) protocols to support ongoing credible, evaluable and replicable claims in target patient populations. The critical issue is not analytical and clinical validity but clinical utility. This has yet to be demonstrated. Type: Commentary

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LncRNA LEF1-AS1 promotes metastasis of prostatic carcinoma via the Wnt/β-catenin pathway

Cancer Cell International ◽

10.1186/s12935-020-01624-x ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Weiyuan Li ◽

Ganggang Yang ◽

Dengke Yang ◽

Dong Li ◽

Qian Sun

Keyword(s):

Next Generation Sequencing ◽

Rna Binding ◽

Next Generation ◽

Next Generation Sequencing Technology ◽

Normal Tissues ◽

Factor C ◽

Generation Sequencing

Abstract Background Long noncoding RNAs (lncRNAs) are important functional regulators of many biological processes of cancers. However, the mechanisms by which lncRNAs modulate androgen-independent prostate cancer (AIPC) development remain largely unknown. Methods Next-generation sequencing technology and RT-qPCR were used to assess LEF1-AS1 expression level in AIPC tissues and adjacent normal tissues. Functional in vitro experiments, including colony formation, EDU and transwell assays were performed to assess the role of LEF1-AS1 in AIPC. Xenograft assays were conducted to assess the effect of LEF1-AS1 on cell proliferation in vivo. Chromatin immunoprecipitation (ChIP) and RNA binding protein immunoprecipitation (RIP) assays were performed to elucidate the regulatory network of LEF1-AS1. Results The next-generation sequencing results showed that LEF1-AS1 is significantly overexpressed in AIPC. Furthermore, our RT-qPCR assay data showed that LEF1-AS1 is overexpressed in AIPC tissues. Functional experiments showed that LEF1-AS1 promotes the proliferation, migration, invasion and angiogenic ability of AIPC cells in vitro and tumour growth in vivo by recruiting the transcription factor C-myb to the promoter of FZD2, inducing its transcription. Furthermore, LEF1-AS1 was shown to function as a competing endogenous RNA (ceRNA) that sponges miR-328 to activate CD44. Conclusion In summary, the results of our present study revealed that LEF1-AS1 acts as a tumour promoter in the progression of AIPC. Furthermore, the results revealed that LEF1-AS1 functions as a ceRNA and regulates Wnt/β-catenin pathway activity via FZD2 and CD44. Our results provide new insights into the mechanism that links the function of LEF1-AS1 with AIPC and suggests that LEF1-AS1 may serve as a novel potential target for the improvement of AIPC therapy.

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LncRNA LEF1-AS1 promotes metastasis of prostatic carcinoma via the Wnt/β-catenin pathway

10.21203/rs.3.rs-40046/v4 ◽

2020 ◽

Author(s):

Weiyuan Li ◽

Ganggang Yang ◽

Dengke Yang ◽

Dong Li ◽

Qian Sun

Keyword(s):

Next Generation Sequencing ◽

Rna Binding ◽

Next Generation ◽

Next Generation Sequencing Technology ◽

Normal Tissues ◽

Factor C ◽

Generation Sequencing

Abstract Background: Long noncoding RNAs (lncRNAs) are important functional regulators of many biological processes of cancers. However, the mechanisms by which lncRNAs modulate androgen-independent prostate cancer (AIPC) development remain largely unknown.Methods: Next-generation sequencing technology and RT-qPCR were used to assess LEF1-AS1 expression level in AIPC tissues and adjacent normal tissues. Functional in vitro experiments, including colony formation, EDU and transwell assays were performed to assess the role of LEF1-AS1 in AIPC. Xenograft assays were conducted to assess the effect of LEF1-AS1 on cell proliferation in vivo. Chromatin immunoprecipitation (ChIP) and RNA binding protein immunoprecipitation (RIP) assays were performed to elucidate the regulatory network of LEF1-AS1.Results: The next-generation sequencing results showed that LEF1-AS1 is significantly overexpressed in AIPC. Furthermore, our RT-qPCR assay data showed that LEF1-AS1 is overexpressed in AIPC tissues. Functional experiments showed that LEF1-AS1 promotes the proliferation, migration, invasion and angiogenic ability of AIPC cells in vitro and tumour growth in vivo by recruiting the transcription factor C-myb to the promoter of FZD2, inducing its transcription. Furthermore, LEF1-AS1 was shown to function as a competing endogenous RNA (ceRNA) that sponges miR-328 to activate CD44.Conclusion: In summary, the results of our present study revealed that LEF1-AS1 acts as a tumour promoter in the progression of AIPC. Furthermore, the results revealed that LEF1-AS1 functions as a ceRNA and regulates Wnt/β-catenin pathway activity via FZD2 and CD44. Our results provide new insights into the mechanism that links the function of LEF1-AS1 with AIPC and suggests that LEF1-AS1 may serve as a novel potential target for the improvement of AIPC therapy.

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Genotyping of 25 Leukemia-Associated Genes in a Single Work Flow by Next-Generation Sequencing Technology with Low Amounts of Input Template DNA

Clinical Chemistry ◽

10.1373/clinchem.2013.204099 ◽

2013 ◽

Vol 59 (8) ◽

pp. 1238-1250 ◽

Cited By ~ 14

Author(s):

Jenny Rinke ◽

Vivien Schäfer ◽

Mathias Schmidt ◽

Janine Ziermann ◽

Alexander Kohlmann ◽

...

Keyword(s):

Next Generation Sequencing ◽

Target Genes ◽

Work Flow ◽

Next Generation ◽

Next Generation Sequencing Technology ◽

Wide Range ◽

Next Generation Sequencing Ngs ◽

Template Dna ◽

Ngs Data ◽

Generation Sequencing

BACKGROUND We sought to establish a convenient, sensitive next-generation sequencing (NGS) method for genotyping the 26 most commonly mutated leukemia-associated genes in a single work flow and to optimize this method for low amounts of input template DNA. METHODS We designed 184 PCR amplicons that cover all of the candidate genes. NGS was performed with genomic DNA (gDNA) from a cohort of 10 individuals with chronic myelomonocytic leukemia. The results were compared with NGS data obtained from sequencing of DNA generated by whole-genome amplification (WGA) of 20 ng template gDNA. Differences between gDNA and WGA samples in variant frequencies were determined for 2 different WGA kits. RESULTS For gDNA samples, 25 of 26 genes were successfully sequenced with a sensitivity of 5%, which was achieved by a median coverage of 492 reads (range, 308–636 reads) per amplicon. We identified 24 distinct mutations in 11 genes. With WGA samples, we reliably detected all mutations above 5% sensitivity with a median coverage of 506 reads (range, 256–653 reads) per amplicon. With all variants included in the analysis, WGA amplification by the 2 kits tested yielded differences in variant frequencies that ranged from −28.19% to +9.94% [mean (SD) difference, −0.2% (4.08%)] and from −35.03% to +18.67% [mean difference, −0.75% (5.12%)]. CONCLUSIONS Our method permits simultaneous analysis of a wide range of leukemia-associated target genes in a single sequencing run. NGS can be performed after WGA of template DNA for reliable detection of variants without introducing appreciable bias.

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