scholarly journals Methylmalonic Acidemia with Novel MUT Gene Mutations

2017 ◽  
Vol 2017 ◽  
pp. 1-2
Author(s):  
Inusha Panigrahi ◽  
Savita Bhunwal ◽  
Harish Varma ◽  
Simranjeet Singh
Keyword(s):  
Sanger Sequencing ◽  
Gene Mutations ◽  
Methylmalonic Acidemia ◽  
Feeding Problems ◽  
Novel Variants ◽  
Mut Gene ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Poor Weight Gain

A 5-year-old boy presented with recurrent episodes of fever, feeding problems, lethargy, from the age of 11 months, and poor weight gain. He was admitted and evaluated for metabolic causes and diagnosed as having methylmalonic acidemia (MMA). He was treated with vit B12 and carnitine supplements and has been on follow-up for the last 3 years. Mutation analysis by next generation sequencing (NGS), supplemented with Sanger sequencing, revealed two novel variants in the MUT gene responsible for MMA in exon 5 and exon 3, respectively. Recently he developed dystonic movements including orofacial dyskinesia. With advent of NGS, judicious use of NGS with Sanger sequencing can help identify causative possibly pathogenic mutations.

scholarly journals Liquid Biopsy as a Diagnostic and Prognostic Tool for Women and Female Dogs with Breast Cancer

Cancers ◽  
2021 ◽  
Vol 13 (20) ◽  
pp. 5233
Author(s):  
Jucimara Colombo ◽  
Marina Gobbe Moschetta-Pinheiro ◽  
Adriana Alonso Novais ◽  
Bruna Ribeiro Stoppe ◽  
Enrico Dumbra Bonini ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Liquid Biopsy ◽  
Gene Mutations ◽  
Malignant Neoplasm ◽  
Genetic Variations ◽  
Highly Pathogenic ◽  
Genome Analysis Toolkit ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Introduction: Breast cancer (BC) is the malignant neoplasm with the highest mortality rate in women and female dogs are good models to study BC. Objective: We investigated the efficacy of liquid biopsy to detect gene mutations in the diagnosis and follow-up of women and female dogs with BC. Materials and Methods: In this study, 57 and 37 BC samples were collected from women and female dogs, respectively. After core biopsy and plasma samples were collected, the DNA and ctDNA of the tumor fragments and plasma were processed for next generation sequencing (NGS) assay. After preprocessing of the data, they were submitted to the Genome Analysis ToolKit (GATK). Results: In women, 1788 variants were identified in tumor fragments and 221 variants in plasma; 66 variants were simultaneously detected in tumors and plasma. Conversely, in female dogs, 1430 variants were found in plasma and 695 variants in tumor fragments; 59 variants were simultaneously identified in tumors and plasma. The most frequently mutated genes in the tumor fragments of women were USH2A, ATM, and IGF2R; in female dogs, they were USH2A, BRCA2, and RRM2. Plasma of women showed the most frequent genetic variations in the MAP3K1, BRCA1, and GRB7 genes, whereas plasma from female dogs had variations in the NF1, ERBB2, and KRT17 genes. Mutations in the AKT1, PIK3CA, and BRIP genes were associated with tumor recurrence, with a highly pathogenic variant in PIK3CA being particularly prominent. We also detected a gain-of-function mutation in the GRB7, MAP3K1, and MLH1 genes. Conclusion: Liquid biopsy is useful to identify specific genetic variations at the beginning of BC manifestation and may be accompanied over the entire follow-up period, thereby supporting the clinicians in refining interventions.

scholarly journals Jervell and Lange-Nielsen Syndrome due to a Novel Compound Heterozygous KCNQ1 Mutation in a Chinese Family

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Yue Qiu ◽  
Sen Chen ◽  
Xia Wu ◽  
Wen-Juan Zhang ◽  
Wen Xie ◽  
...  
Keyword(s):  
Deaf Child ◽  
Gene Mutations ◽  
Deaf Children ◽  
Chinese Family ◽  
Compound Heterozygous ◽  
Qtc Interval ◽  
Cardiac Events ◽  
Life Threatening ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Jervell and Lange-Nielsen syndrome (JLNS) is a rare but severe autosomal recessive disease characterized by profound congenital deafness and a prolonged QTc interval (greater than 500 milliseconds) in the ECG waveforms. The prevalence of JLNS is about 1/1000000 to 1/200000 around the world. However, exceed 25% of JLNS patients suffered sudden cardiac death with kinds of triggers containing anesthesia. Approximately 90% of JLNS cases are caused by KCNQ1 gene mutations. Here, using next-generation sequencing (NGS), we identified a compound heterozygosity for two mutations c.1741A>T (novel) and c.477+5G>A (known) in KCNQ1 gene as the possible pathogenic cause of JLNS, which suggested a high risk of cardiac events in a deaf child. The hearing of this patient improved significantly with the help of cochlear implantation (CI). But life-threatening arrhythmias occurred with a trigger of anesthesia after the end of the CI surgery. Our findings extend the KCNQ1 gene mutation spectrum and contribute to the management of deaf children diagnosed with JLNS for otolaryngologists (especially cochlear implant teams).

scholarly journals Targeted Next Generation Sequencing Identifies Novel Genetic Mutations in Patients with Waldenstrom's Macroglobulinemia/Lymphoplasmacytic Lymphoma or IgM-Monoclonal Gammopathies of Undetermined Significance

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2928-2928 ◽  
Author(s):  
Marzia Varettoni ◽  
Silvia Zibellini ◽  
Ettore Rizzo ◽  
Luca Malcovati ◽  
Virginia Valeria Ferretti ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Somatic Mutations ◽  
Genetic Mutations ◽  
Wild Type ◽  
Targeted Next Generation Sequencing ◽  
Coefficient Of Concordance ◽  
Generation Sequencing ◽  
Myd88 L265p ◽  
Undetermined Significance

Abstract Background. TheMYD88 (L265P) somatic mutation is present in more than 90% of patients (pts) with Waldenström's Macroglobulinemia (WM)/lymphoplasmacitic lymphoma (LPL). The second most common mutations are nonsense (NS) or frameshift (FS) mutations in the CXCR4 gene, detectable in approximately 25-30% of WM pts by Sanger sequencing. Limited data are available about other genetic mutations in WM/LPL and its precursor condition IgM-monoclonal gammopathy of undetermined significance (IgM-MGUS). Pts and methods. Using targeted next generation sequencing (NGS), we evaluated the prevalence of somatic mutations of 11 genes selected on the basis of evidences available from the literature (MYD88, CXCR4, ARID1A, KMT2D, TP53, NOTCH2, PRDM1, CD79b, TRAF3,TNFAIP3, MYDBBP1A) in 119 pts, classified as WM/LPL (n=63) or IgM-MGUS (n=56) according to International Consensus Criteria. Median age of pts (67 males, 52 females) was 65 years (range: 38-82). Samples were collected at diagnosis (n=101), after diagnosis but before any treatment (n=9) or at progression after therapy (n=9). Paired tumor and germline DNA extracted respectively from CD19-selected and CD19-depleted bone marrow (BM) mononuclear cells was available in all pts. Mean resequencing depth across gene panel was 1009x. Only mutations tagged as oncogenic or possibly oncogenic based on information derived from the literature and on in silico prediction effect were considered in the analysis. For MYD88 (L265P) and CXCR4 mutations, results obtained with NGS were compared with those obtained respectively with allele-specific PCR (AS-PCR) and Sanger sequencing. Results. Overall, we found 151 mutations in 88 pts (74%). The median number of mutations was significantly higher in WM/LPL as compared with IgM-MGUS and in pts previously treated as compared with untreated ones (median 2 versus 1, P < 0.001 for both comparisons). MYD88 mutations were found in 80/119 pts (67%), with a median allele burden of 34.2% (range: 2.5-93.3%). The prevalence of MYD88 mutations was significantly higher in WM/LPL as compared with IgM-MGUS (86% versus 46%, P <0.001). MYD88 mutations other than classical L265P (n=76) were found in 4 pts and were represented by V217F (n=2), S219C (n=1), M232T (n=1). Fifteen pts who were MYD88 (L265P) wild-type by NGS were found to be mutated by AS-PCR (K coefficient of concordance between NGS and AS-PCR: 70%, P < 0.001). CXCR4 mutations were found in 19/119 pts (16%), with a median allele burden of 34% (range: 4.2-84%). The prevalence of CXCR4 mutations was significantly higher in WM/LPL as compared with IgM-MGUS (24% versus 7%, P < 0.02). The K coefficient of concordance between NGS and Sanger was 83% (P < 0.001), with 2 pts mutated only by NGS and 2 pts mutated only by Sanger. Somatic mutations were also found in KMT2D (formerly known as MLL2) (16% of pts), TP53 (8%), NOTCH2 (7%), PRDM1 (4%), ARID1A (3%), CD79b (2%), and TRAF3 (1%). No mutations were found in MYBBP1A and TNFAI3. Overall, the prevalence of these mutations was significantly lower in pts wild-type either for MYD88 or CXCR4 as compared with those with MYD88 and/or CXCR4 mutations (15% versus 41%, P = 0.04). The prevalence of KMT2D mutations was significantly higher in WM/LPL as compared with MGUS (25% versus 5%), while for the other genes the distribution was not statistically different according to diagnosis. With a median follow-up of 20 months (range: 0-264), we did not find a statistically significant correlation between genetic mutations and pts' outcome in terms of overall survival or time to first treatment. Conclusions. In this cohort of pts with WM/LPL and IgM-MGUS studied with NGS we could demonstrate that: i) NGS identifies MYD88 mutations other than L265P in a small proportion of pts; ii) the prevalence of CXCR4 mutations by Sanger is confirmed by NGS, despite the higher sensitivity of the latter method; iii) the subgroup of pts wild type either for MYD88 or CXCR4 shows a low incidence of other genetic mutations; iv) 25% of pts with WM/LPL were found to carry KMT2D mutations, a prevalence similar to that reported in marginal zone lymphoma; v) genetic mutations are more common in WM/LPL than in IgM-MGUS in agreement with the hypothesis that multiple genetic hits are required for progression from a pre-benign condition to a neoplastic disease; vi) due to the indolent nature of these disorders, longer follow-up is probably needed to see the prognostic impact of these mutations, if any. Disclosures No relevant conflicts of interest to declare.

Backtracking Subclonal Mutations Of TP53 In Myelodysplasia (MDS) With Del(5q) With Next-Generation Sequencing (NGS)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 520-520
Author(s):  
Laurence Lodé ◽  
Audrey Ménard ◽  
Marion Loirat ◽  
Maxime Halliez ◽  
Steven Richebourg ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Lower Limit ◽  
Clonal Evolution ◽  
Applied Science ◽  
Tp53 Mutations ◽  
Variant Detection ◽  
Serial Samples ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Landscape analyses of mutational patterns have shown that virtually all myelodysplastic syndromes (MDS) harbor somatic mutations in >80% of cases. These molecular alterations provide useful clonality markers with a potential for early diagnosis of MDS when only cytopenia without marked dysplasia is observed. These markers have been proposed as future prognostic tools to guide therapeutic strategies (Bejar et al., 2011; Itzykson et al, 2013; Mufti et al 2013). Mutational analysis is finally a good way to track disease complexity by deciphering oligoclonality in MDS and better understand clonal evolution. Alterations in the TP53 gene are the most common cause of tumor escape from apoptosis. The aim of this study was to identify TP53 mutations in consecutive samples of lower-risk MDS (IPSS ≤1) with del(5q)obtained at follow-up or progression after sequential classical treatments. Next-generation sequencing (NGS) was used to backtrack the mutant clone(s) identified in late samples. The study was performed both by conventional Sanger sequencing and NGS on a GS Junior Instrument (Roche Applied Science, Mannheim, Germany). For each sample, eight exons (4-11) were amplified from 320 ng of DNA with preconfigured primer plates provided within the IRON II study network. PCR reactions were performed using the FastStart High Fidelity PCR System kit (Roche Applied Science). After double purification with Agencourt AMPure XP beads (Beckman Coulter, Miami, FL), exon-specific amplicon pools were generated and quantified using the Quant-iT™ Broad-Range PicoGreen DNA Assay Kit (Invitrogen, Carlsbad, CA). Emulsion PCR was performed with GS Junior emPCR Reagents (Lib-A) (Roche Applied Science) using 5 x 106 beads at a copy per bead ratio of 0.6. Finally, a fraction of 5-7% enriched beads was loaded on GS Junior Titanium sequencing PicoTiterPlate kit (Roche Applied Science). Data were analyzed for sequence alignment and variant detection using the GS Junior Sequencer and GS Amplicon Variant Analyzer softwares, versions 2.7 and 2.9 (Roche Applied Science). The results were further processed using the Sequence Pilot software version 4.0.1 (JSI Medical Systems, Kippenheim, Germany). The sensitivity of variant detection was set to a lower limit of >1% for bidirectional reads. This threshold was chosen according to a recent study investigating the assay's lower limit of detection (Grossmann et al., 2013), thus underlining the strength of NGS to identify subclones at a low frequency, not detectable by conventional Sanger analysis. A total of 89 DNA samples were extracted from the cytogenetics pellets of a cohort of 40 MDS with del(5q). TP53 mutation analysis was performed on 40 initial and 49 follow-up or progression samples including serial samples for 23 subjects. The depth of coverage was at least 500X and up to 8,444X per amplicon. Of those samples obtained and analysed at time of last follow-up or progression, 14 (61%) had TP53 mutations, mostly in the DNA-binding domain. Performing backtracking on previously collected serial samples, TP53 mutations were retrieved by NGS in 43% of initial samples (n=6), which is different from what was previously described by Jädersten et al (2011). A complete scenario of clonal evolution was retrieved in 11 cases, evidenced by TP53 mutations and/or cytogenetics. These were always consecutive to treatment with lenalidomide, yet 6 of the 12 cases without clonal evolution were also consecutive lenalidomide. Figure 1 provides the example of a complete follow-up including nine time points. More correlation with treatment will be provided. Although lenalidomide remains the treatment of choice for MDS with del(5q), resistant subclones may survive and culminate even following therapy initiation. This theory was recently suggested by Landau et al. in CLL (2013) and our test results support this. Early detection of emerging subclones could lead to initiation of alternative treatment, and we thus propose that a monitoring of TP53 alleles is performed annually after the onset of therapy for MDS using NGS. Figure 1. Figure 1. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Moreau:CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau.

scholarly journals Genetic predisposition to neural crest-derived tumors: revisiting the role of KIF1B

2020 ◽  
Vol 9 (10) ◽  
pp. 1042-1050
Author(s):  
Catherine Cardot Bauters ◽  
Emmanuelle Leteurtre ◽  
Bruno Carnaille ◽  
Christine Do Cao ◽  
Stéphanie Espiard ◽  
...  
Keyword(s):  
Germline Variant ◽  
Whole Exome ◽  
Familial Cancers ◽  
Heterozygous Variant ◽  
Unknown Significance ◽  
Next Generation Sequencing Ngs ◽  
Design And Methods ◽  
Generation Sequencing

Objective We previously described a family in which predisposition to pheochromocytoma (PCC) segregates with a germline heterozygous KIF1B nucleotide variant (c.4442G>A, p.Ser1481Asn) in three generations. During the clinical follow-up, one proband’s brother, negative for the KIF1B nucleotide variant, developed a bilateral PCC at 31 years. This prompted us to reconsider the genetic analysis. Design and methods Germline DNA was analyzed by next-generation sequencing (NGS) using a multi-gene panel plus MLPA or by whole exome sequencing (WES). Tumor-derived DNA was analyzed by SnapShot, Sanger sequencing or NGS to identify loss-of-heterozygosity (LOH) or additional somatic mutations. Results A germline heterozygous variant of unknown significance in MAX (c.145T>C, p.Ser49Pro) was identified in the proband’s brother. Loss of the wild-type MAX allele occurred in his PCCs thus demonstrating that this variant was responsible for the bilateral PCC in this patient. The proband and her affected grandfather also carried the MAX variant but no second hit could be found at the somatic level. No other pathogenic mutations were detected in 36 genes predisposing to familial PCC/PGL or familial cancers by WES of the proband germline. Germline variants detected in other genes, TFAP2E and TMEM214, may contribute to the multiple tumors of the proband. Conclusion In this family, the heritability of PCC is linked to the MAX germline variant and not to the KIF1B germline variant which, however, may have contributed to the occurrence of neuroblastoma (NB) in the proband.

scholarly journals Limited marginal utility of deep sequencing for HIV drug resistance testing in the age of integrase inhibitors

2018 ◽  
Author(s):  
Ronit Dalmat ◽  
Negar Makhsous ◽  
Gregory Pepper ◽  
Amalia Magaret ◽  
Keith R. Jerome ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Virological Failure ◽  
Sanger Sequencing ◽  
Low Frequency ◽  
Resistance Testing ◽  
Antiviral Resistance ◽  
Resistance Mutations ◽  
Hiv Drug Resistance ◽  
Next Generation Sequencing Ngs

AbstractHIV drug resistance genotyping is a critical tool in the clinical management of HIV infections. Although resistance genotyping has traditionally been conducted using Sanger sequencing, next-generation sequencing (NGS) is emerging as a powerful tool due to its ability to detect lower frequency alleles. However, the value added from NGS approaches to antiviral resistance testing remains to be demonstrated. We compared the variant detection capacity of NGS versus Sanger sequencing methods for resistance genotyping of 144 drug resistance tests (105 protease-reverse transcriptase tests and 39 integrase tests) submitted to our clinical virology laboratory over a four-month period in 2016 for Sanger-based HIV drug resistance testing. NGS detected all true high frequency drug resistance mutations (>20% frequency) found by Sanger sequencing, with greater accuracy in one instance of a Sanger-detected false positive. Freely available online NGS variant callers Hydra and PASeq were superior to Sanger methods for interpretations of allele linkage and automated variant calling. NGS additionally detected low frequency mutations (1-20% frequency) associated with higher levels of drug resistance in 30/105 (29%) of protease-reverse transcriptase tests and 4/39 (10%) of integrase tests. Clinical follow-up of 69 individuals for a median of 674 days found no difference in rates of virological failure between individuals with and without low frequency mutations, although rates of virological failure were higher for individuals with drug-relevant low frequency mutations. However, all 27 individuals who experienced virological failure reported poor adherence to their drug regimen during preceding follow-up time, and all 19 who subsequently improved their adherence achieved viral suppression at later time points consistent with a lack of clinical resistance. In conclusion, in a population with low antiviral resistance emergence, NGS methods detected numerous instances of minor alleles that did not result in subsequent bona fide virological failure due to antiviral resistance.ImportanceGenotypic antiviral resistance testing for HIV is an essential component of the clinical microbiology and virology laboratory. Next-generation sequencing (NGS) has emerged as a powerful tool for the detection of low frequency sequence variants (allele frequencies <20%). Whether detecting these low frequency mutations in HIV contributes to improved patient health, however, remains unclear. We compared NGS to conventional Sanger sequencing for detecting resistance mutations for 144 HIV drug resistance tests submitted to our clinical virology laboratory and detected low frequency mutations in 24% of tests. Over approximately two years of follow-up for 69 patients for which we had access to electronic health records, no patients had virological failure due to antiviral resistance. Instead, virological failure was entirely explained by medication non-adherence. While larger studies are required, we suggest that detection of low frequency variants by NGS presents limited marginal clinical utility when compared to standard of care.

scholarly journals 40 minutes RT-qPCR Assay for Screening Spike N501Y and HV69-70del Mutations

2021 ◽  
Author(s):  
Gulay Korukluoglu ◽  
Mustafa Kolukirik ◽  
Fatma Bayrakdar ◽  
Gozde Girgin Ozgumus ◽  
Ayse Basak Altas ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Internal Control ◽  
Sanger Sequencing ◽  
Limit Of Detection ◽  
Qpcr Assay ◽  
Rapid Screening ◽  
Clinical Samples ◽  
One Step ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ABSTRACTA one-step reverse transcription and real-time PCR (RT-qPCR) test was developed for rapid screening (40 minutes) of the Spike N501Y and HV69-70del mutations in SARS-CoV-2 positive samples. The test also targets a conserved region of SARS-CoV-2 Orf1ab as an internal control. The samples containing both the N501Y and HV69-70del mutations are concluded as VOC-202012/01 positive. Samples suspected to be positive for B.1.351 or P.1 are the N501Y positive and HV69-70del negative cases. Limit of detection (LOD) of the kit for Orf1ab target is 500 copies/mL, while that of the N501, Y501 and HV69-70del targets are 5000 copies/mL. The developed assay was applied to 165 clinical samples containing SARS-CoV-2 from 32 different lineages. The SARS-CoV-2 lineages were determined via the next-generation sequencing (NGS). The RT-qPCR results were in 100% agreement with the NGS results that 19 samples were N501Y and HV69-70del positive, 10 samples were N501Y positive and HV69-70del negative, 1 sample was N501Y negative and HV69-70del positive, and 135 samples were N501Y and HV69-70del negative. All the VOC-202012/01 positive samples were detected in people who have traveled from England to Turkey. The RT-qPCR test and the Sanger sequencing was further applied to 1000 SARS-CoV-2 positive clinical samples collected in Jan2021 from the 81 different provinces of Turkey. The RT-qPCR results were in 100% agreement with the Sanger sequencing results that 32 samples were N501Y positive and HV69-70del negative, 4 samples were N501Y negative and HV69-70del positive, 964 samples were N501Y and HV69-70del negative. The specificity of the 40 minutes RT-qPCR assay relative to the sequencing-based technologies is 100%. The developed assay is an advantageous tool for timely and representative estimation of the N501Y positive variants’ prevalence because it allows testing a much higher portion of the SARS-CoV-2 positives in much lower time compared to the sequencing-based technologies.

scholarly journals Next-Generation Sequencing Combined With Conventional Sanger Sequencing Reveals High Molecular Diversity in Actinidia Virus 1 Populations From Kiwifruit Grown in China

2020 ◽  
Vol 11 ◽  
Author(s):  
Shaohua Wen ◽  
Guoping Wang ◽  
Zuokun Yang ◽  
Yanxiang Wang ◽  
Min Rao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Molecular Diversity ◽  
Genome Structure ◽  
Viral Disease ◽  
Open Reading Frames ◽  
Next Generation ◽  
Next Generation Sequencing Ngs ◽  
First Time ◽  
Generation Sequencing

Kiwifruit (Actinidia spp.) is native to China. Viral disease–like symptoms are common on kiwifruit plants. In this study, six libraries prepared from total RNA of leaf samples from 69 kiwifruit plants were subjected to next-generation sequencing (NGS). Actinidia virus 1 (AcV-1), a tentative species in the family Closteroviridae, was discovered in the six libraries. Two full-length and two near-full genome sequences of AcV-1 variants were determined by Sanger sequencing. The genome structure of these Chinese AcV-1 variants was identical to that of isolate K75 and consisted of 12 open reading frames (ORFs). Analyses of these sequences together with the NGS-derived contig sequences revealed high molecular diversity in AcV-1 populations, with the highest sequence variation occurring at ORF1a, ORF2, and ORF3, and the available variants clustered into three phylogenetic clades. For the first time, our study revealed different domain compositions in the viral ORF1a and molecular recombination events among AcV-1 variants. Specific reverse transcriptase–polymerase chain reaction assays disclosed the presence of AcV-1 in plants of four kiwifruit species and unknown Actinidia spp. in seven provinces and one city.

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