sanger sequencing
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2022 ◽  
Vol 42 (2) ◽  
pp. 213-248
Young-Gon Kim ◽  
Kiwook Jung ◽  
Seunghwan Kim ◽  
Man Jin Kim ◽  
Jee-Soo Lee ◽  

2022 ◽  
Vol 23 (2) ◽  
pp. 969
Michał Wągrodzki ◽  
Andrzej Tysarowski ◽  
Katarzyna Seliga ◽  
Aneta Wojnowska ◽  
Maria Stepaniuk ◽  

To validate the reliability and implementation of an objective diagnostic method for giant cell tumour of bone (GCTB). H3-3A gene mutation testing was performed using two different methods, Sanger sequencing and immunohistochemical (IHC) assays. A total of 214 patients, including 120 with GCTB and 94 with other giant cell-rich bone lesions, participated in the study. Sanger sequencing and IHC with anti-histone H3.3 G34W and G34V antibodies were performed on formalin-fixed, paraffin-embedded tissues, which were previously decalcified in EDTA if needed. The sensitivity and specificity of the molecular method was 100% (95% CI: 96.97–100%) and 100% (95% CI: 96.15–100%), respectively. The sensitivity and specificity of IHC was 94.32% (95% CI: 87.24–98.13%) and 100% (95% CI: 93.94–100.0%), respectively. P.G35 mutations were discovered in 2/9 (22.2%) secondary malignant GCTBs and 9/13 (69.2%) GCTB after denosumab treatment. We confirmed in a large series of patients that evaluation of H3-3A mutational status using direct sequencing is a reliable tool for diagnosing GCTB, and it should be incorporated into the diagnostic algorithm. Additionally, we discovered IHC can be used as a screening tool. Proper tissue processing and decalcification are necessary. The presence of the H3-3A mutation did not exclude malignant GCTB. Denosumab did not eradicate the neoplastic cell population of GCTB.

2022 ◽  
Vol 20 (1) ◽  
Alexandra Lebedeva ◽  
Yulia Shaykhutdinova ◽  
Daria Seriak ◽  
Ekaterina Ignatova ◽  
Ekaterina Rozhavskaya ◽  

Abstract Background A fraction of patients referred for complex molecular profiling of biopsied tumors may harbor germline variants in genes associated with the development of hereditary cancer syndromes (HCS). Neither the bioinformatic analysis nor the reporting of such incidental germline findings are standardized. Methods Data from Next-Generation Sequencing (NGS) of biopsied tumor samples referred for complex molecular profiling were analyzed for germline variants in HCS-associated genes. Analysis of variant origin was performed employing bioinformatic algorithms followed by manual curation. When possible, the origin of the variant was validated by Sanger sequencing of the sample of normal tissue. The variants’ pathogenicity was assessed according to ACMG/AMP. Results Tumors were sampled from 183 patients (Males: 75 [41.0%]; Females: 108 [59.0%]; mean [SD] age, 57.7 [13.3] years) and analysed by targeted NGS. The most common tumor types were colorectal (19%), pancreatic (13%), and lung cancer (10%). A total of 56 sequence variants in genes associated with HCS were detected in 40 patients. Of them, 17 variants found in 14 patients were predicted to be of germline origin, with 6 variants interpreted as pathogenic (PV) or likely pathogenic (LPV), and 9 as variants of uncertain significance (VUS). For the 41 out of 42 (97%) missense variants in HCS-associated genes, the results of computational prediction of variant origin were concordant with that of experimental examination. We estimate that Sanger sequencing of a sample of normal tissue would be required for ~ 1–7% of the total assessed cases with PV or LPV, when necessity to follow with genetic counselling referral in ~ 2–15% of total assessed cases (PV, LPV or VUS found in HCS genes). Conclusion Incidental findings of pathogenic germline variants are common in data from cancer patients referred for complex molecular profiling. We propose an algorithm for the management of patients with newly detected variants in genes associated with HCS.

2022 ◽  
pp. 088532822110658
Keying Xue ◽  
Bingqing Luo ◽  
Xiaoqing Li ◽  
Weixian Deng ◽  
Chunyan Zeng ◽  

This study was designed to investigate the feasibility of genetic testing using circulating tumor cells (CTCs) instead of tumor tissues in lung adenocarcinoma to break through its limitation. Separation system for epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), and Vimentin expressing CTCs was constructed and used to capture CTCs in the blood samples of 57 patients with lung adenocarcinoma. Genetic mutations of genes involved in targeted therapies were detected by next-generation sequencing (NGS) in tissues from these patients. Blood CTC samples with the gene mutations identified by tissue-NGS were selected and corresponding gene mutations were detected by Sanger sequencing. The specificity of the CTC separation system was 95.48% and the sensitivity was 90.85%. The average number of CTCs in the blood of patients with lung adenocarcinoma was 12.47/7.5 mL. Comparison of the tissue-NGS with the CTC-Sanger sequencing showed that the consistencies of genetic mutations of EGFR ( n = 24), KRAS ( n = 9), TP53 ( n = 19), BRAF ( n = 1), ERBB2 ( n = 2), and PIK3CA ( n = 3) were 92.31%, 75.00%, 86.36%, 50.00%, 66.67%, and 75.00%, with an overall consistency of 84.06%. The CTC separation system established in this study shows high specificity and sensitivity. CTCs can be used as a suitable alternative to tumor tissues that are difficult to obtain in clinical practice and overcome the difficulties in tumor tissue collection, which is of significance in guiding clinical medication and individualized treatment with significant clinical application value in terms of genetic testing for targeted therapies in tumor treatment.

2022 ◽  
Vol 8 ◽  
Weiqian Yan ◽  
Zhiping Hu ◽  
Yingchi Zhang ◽  
Xiaomei Wu ◽  
Hainan Zhang

PurposeThe objective of our study was to report a case of encephalitis and endophthalmitis caused by pseudorabies virus (PRV), identified using metagenomic next-generation sequencing (mNGS).Case PresentationA 54-year-old worker, from a swine slaughterhouse, developed signs of severe encephalitis, including fever, disturbance of consciousness, hypopnea, and status epilepticus, after finger injury at work. The PRV sequences were successfully identified from the blood, cerebrospinal fluid (CSF), and aqueous humor of the patient through mNGS, which was further verified using a Sanger sequencing.ConclusionOur case emphasizes the importance of mNGS in early diagnoses of infectious diseases, and gives a clue that PRV can spread across species and infect human. It is necessary to carry out a skin protection and education about disease prevention for people who have close contact with swine.

Lin Wang ◽  
Bin He ◽  
Qiujie Jin ◽  
Ruimiao Bai ◽  
Wenwen Yu ◽  

Abstract Objectives Phenylketonuria (PKU) is an inherited autosomal recessive disorder of phenylalanine metabolism. It is mainly caused by a deficiency in phenylalanine hydroxylase (PAH) and frequently diagnosed with Sanger sequencing. To some extent, allelic dropout can explain the inconsistency in genotype and phenotype. Methods Three families were evaluated through DNA sequence analysis, multiplex ligation-dependent probe amplification (MLPA) and prenatal diagnosis technologies. The possibility of inconsistency in phenotype and genotype with c.331C>T variant was analysed. Results Through pedigree analysis, three mothers carried a homozygous c.331C>T variant, which was a false-positive result. New primers were used, and this error was caused by allelic dropout. In this case, c.158G>A was likely a benign variant. Conclusions Sequence variants in primer-binding regions could cause allelic dropout, creating unpredictable errors in genotyping. Our results emphasised the need for careful measures to treat genotype–phenotype inconsistencies.

2022 ◽  
pp. 1-8
Liliana Fernández Hernández ◽  
Miguel A. Alcántara Ortigoza ◽  
Sandra E. Ramos Angeles ◽  
Ariadna González-del Angel

5q14.3 deletion syndrome (MIM#613443) is an uncommon but well-known syndrome characterized by intellectual disability, epilepsy, hypotonia, brain malformations, and facial dysmorphism. Most patients with this syndrome have lost one copy of the <i>MEF2C</i> gene (MIM*600662), whose haploinsufficiency is considered to be responsible for the distinctive phenotype. To date, nearly 40 cases have been reported; the deletion size and clinical spectrum are variable, and at least 6 cases without <i>MEF2C</i> involvement have been documented. We herein report the clinical and cytogenomic findings of an 11-year-old girl who has a 5q14.3q21.1 de novo deletion that does not involve <i>MEF2C</i> but shares the clinical features described in other reported patients. Moreover, she additionally presents with bilateral cleft-lip palate (CLP), which has not been previously reported as a feature of the syndrome. The most frequent syndromic forms of CLP were ruled out in our patient mainly by clinical examination, and Sanger sequencing was performed to discard the presence of a <i>TBX22</i> gene (MIM*300307) defect. Our report suggests CLP as a possible unreported feature and redefines the critical phenotypic regions of 5q14.3 deletion syndrome.

Viruses ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 123
Nicolò Musso ◽  
Paolo Giuseppe Bonacci ◽  
Dafne Bongiorno ◽  
Stefano Stracquadanio ◽  
Dalida Angela Bivona ◽  

Background: The SARS-CoV-2 virus has assumed considerable importance during the COVID-19 pandemic. Its mutation rate is high, involving the spike (S) gene and thus there has been a rapid spread of new variants. Herein, we describe a rapid, easy, adaptable, and affordable workflow to uniquely identify all currently known variants through as few analyses. Our method only requires two conventional PCRs of the S gene and two Sanger sequencing reactions, and possibly another PCR/sequencing assay on a N gene portion to identify the B.1.160 lineage. Methods: We selected an S gene 1312 bp portion containing a set of SNPs useful for discriminating all variants. Mathematical, statistical, and bioinformatic analyses demonstrated that our choice allowed us to identify all variants even without looking for all related mutations, as some of them are shared by different variants (e.g., N501Y is found in the Alpha, Beta, and Gamma variants) whereas others, that are more informative, are unique (e.g., A57 distinctive to the Alpha variant). Results: A “weight” could be assigned to each mutation that may be present in the selected portion of the S gene. The method’s robustness was confirmed by analyzing 80 SARS-CoV-2-positive samples. Conclusions: Our workflow identified the variants without the need for whole-genome sequencing and with greater reliability than with commercial kits.

2022 ◽  
Vol 23 (2) ◽  
pp. 667
Ahmed Bouras ◽  
Cyril Lafaye ◽  
Melanie Leone ◽  
Zine-Eddine Kherraf ◽  
Tanguy Martin-Denavit ◽  

PALB2 (partner and localizer of BRCA2), as indicated by its name, is a BRCA2-interacting protein that plays an important role in homologous recombination (HR) and DNA double-strand break (DSB) repair. While pathogenic variants of PALB2 have been well proven to confer an increased risk of breast cancer, data on its involvement in prostate cancer (PrC) have not been clearly demonstrated. We investigated, using targeted next generation sequencing (NGS), a 59-year-old Caucasian man who developed synchronous breast and prostate cancers. This genetic investigation allowed to identify an intragenic germline heterozygous duplication in PALB2, implicating intronic repetitive sequences spanning exon 11. This variant was confirmed by multiplex ligation probe amplification (MLPA), and genomic breakpoints have been identified and characterized at the nucleotide level (c.3114-811_3202-1756dup) using an approach based on walking PCR, long range PCR, and Sanger sequencing. RT-PCR using mRNA extracted from lymphocytes and followed by Sanger sequencing revealed a tandem duplication r.3114_3201dup; p.(Gly1068Glufs * 14). This duplication results in the synthesis of a truncated, and most-likely, non-functional protein. These findings expand the phenotypic spectrum of PALB2 variants and may improve the yield of genetic diagnoses in this field.

2022 ◽  
José Díaz-Chávez ◽  
Olga Gutiérrez-Hernández ◽  
Lucia Taja-Chayeb ◽  
Sindy Gutiérrez-Chavarría ◽  
Alejandro Aviles-Salas ◽  

Abstract Background: The epigenetic regulator EZH2 is a subunit of the polycomb repressive complex 2 (PRC2), methylates H3K27, resulting in transcriptional silencing. The mutation at Y646 amino acid in the EZH2 gene is mutated in up to 40 % of B-cell lymphomas. Methods: We compared the presence of exon 16 EZH2 mutations in tumor samples and ctDNA in a prospective trial. The mutations were determined by sanger sequencing, and by ddPCR. We also evaluated the impact of these mutations on response, relapse, and survival. Results: One hundred and thirty-eight cases were included. Ninety-eight were germinal center, and twenty had EZH2 mutations. Mean follow-up (IQR 25-75) was 23 (7- 42) months. The tumor samples were considered the standard of reference. Considering the results of the mutation in ctDNA by Sanger sequencing, the sensibility (Se) and specificity (Sp) were 52 % and 99 %, respectively. After adding the ddPCR analysis, the Se and Sp increased to 95 and 100 %, respectively. After bivariate analysis, only the presence of double-hit lymphoma (p=0.04), or EZH2 mutations were associated with relapse. The median PFS (95 % Interval confidence) was 27.7 (95 % IC: 14-40) vs 44.1 (95 % IC: 40-47.6) months for the mutated vs wt patients. Conclusions: The ctDNA is usefull to analyse EZH2 mutations, which have an impact in PFS.

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