Alternative Mutations and Isoform Dysregulation in MYD88 in Waldenstrom's Macroglobulinemia

Abstract Background Mutations in MYD88 are highly recurring in Waldenstrom's Macroglobulinemia (WM) patients and are important for establishing the diagnosis of WM. The most common mutation in MYD88 is c.978T>C resulting a proline substitution for leucine at amino acid position 265 (p.Leu265Pro). Both allele specific PCR (AS-PCR) and clinical diagnostic next generation sequencing (NGS) panels are used to detect mutated MYD88, though they differ in sensitivity and scope. In this study we screened 734 patients with WM by AS-PCR for MYD88 c.978T>C MYD88 followed by Sanger sequencing to clarify negative results for non-MYD88 p.Leu265Pro mutations and compared the findings to clinical NGS panel data from the same biopsy when available. We also investigated MYD88 isoform dysregulation and isoform specific effects of the observed mutations that may impact mutated MYD88 regulation which has not been previously studied in WM. Methods DNA from CD19-selected bone marrow mononuclear cells (BMMC) of 734 WM patients were used for the MYD88 c.978T>C AS-PCR assay previously described by us (Xu et al, Blood 2013). For patients wild-type for MYD88 c.978T>C by AS-PCR, Sanger sequencing of the open reading frame of MYD88 was performed for both DNA and RNA simultaneously isolated from CD19-selected BMMC. DNA was also used to validate the presence of c.978T>C by Sanger. Findings were compared to 222/734 (30.2%) patients who also underwent illumina miSeq based targeted next generation sequencing on a clinical diagnostic platform using unselected BMMC. NGS isoform specific expression estimates were calculated using Salmon for 77 WM patients and 34 healthy donors (Hunter et al, Blood 2016). Results 688/734 (93.7%) WM patients tested positive for the c.978T>C mutation. To confirm these results, Sanger sequencing at the DNA level covering the c.978T>C mutation was performed in 361/688 (52.5%) patients confirming the presence of the mutation in all cases. These Sanger studies revealed that one patient had two somatic mutations in addition to c.978T>C. Of the 46/734 (6.3%) that were wild-type by AS-PCR, 18 had cDNA available to screen for alternative MYD88 mutations. Of these, 13/18 (72.2%) were confirmed to be truly wild type for MYD88, and 5/18 (27.8%) harbored alternative MYD88 mutations making up 0.7% of the study population. Taken together 693/734 (94.4%) of patients were found to harbor somatic MYD88 mutations. Of the 222 patients form whom matching NGS panel data was available, the finding between the NGS and AS-PCR studies were largely concordant. The only discrepancies observed were 69 (31.1%) cases where targeted NGS gave false negative results for c.978T>C but was detected by AS-PCR. Of the four patients with alternative MYD88 mutations, one patient had a dinucleotide substitution that also resulted in p.Leu265Pro but tested as wild-type by AS-PCR, two patients each had one previously documented mutation (either pVal217Phe or p.Ser243Asn) and one patient had a mutation that was synonymous at the protein level (p.Phe277Phe). The patient with the two novel mutations in addition to c.978T>C had a mutation in the polypyrimidine track leading to the final exon and one resulting in p.Gly259Gly in the primary transcript but presents as a highly disruptive p.Val199Glu in the shorter regulatory isoforms. This is similar to c.978T>C which presents as p.L265P in the primary transcripts but acts as a stop loss in the shorter isoforms. We therefore looked for evidence of isoform level dysregulation in MYD88 using RNASeq and found highly significant and distinctive MYD88 isoform signatures for MYD88 mutant, MYD88 wild-type and healthy donor samples (Figure 1). Conclusions Using CD19-selected BMMC, MYD88 c.978T>C (p.Leu265Pro) was found in 93.7% of 734 patients, while non-c.978T>C mutations were present in <1% of WM population. Discordance with targeted NGS is common, with a false negative rate of 31%. Distinct isoform usage patterns and isoform specific mutational effects among MYD88 mutated patients was observed, highly suggestive of a functional role for MYD88 splicing in WM pathogenesis. Figure 1. Figure 1. Disclosures Castillo: Millennium: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Genentech: Consultancy; Beigene: Consultancy, Research Funding. Treon:Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; BMS: Research Funding; Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding. Hunter:Pharmacyclics: Consultancy.

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667. Next Generation Sequencing of Microbial Cell Free DNA in the Diagnosis and Treatment of Infectious Disease in Children: When Does the Result Justify the Cost?

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab466.864 ◽

2021 ◽

Vol 8 (Supplement_1) ◽

pp. S436-S436

Author(s):

Rachel Downey Quick ◽

Kelli A Martinez ◽

Susan M Russo ◽

Sarah E McGwier ◽

Rachel A Quirt ◽

...

Keyword(s):

Next Generation Sequencing ◽

Antimicrobial Resistance ◽

False Negative ◽

Next Generation ◽

Cell Free Dna ◽

Negative Results ◽

Free Dna ◽

False Negative Results ◽

The Cost ◽

Generation Sequencing

Abstract Background Pathogen testing using next-generation sequencing of microbial cell-free DNA (NGS cfDNA) is a promising diagnostic tool to identify pathogens that might not be detected using conventional lab evaluation. Considering the cost of this test, it is important to determine when it is most useful to the plan of care (POC). Figure 1. Unit of admission among cases Figure 2. Patient characteristics in cases determined to be valuable and not valuable to the plan of care (POC) Methods In this retrospective study, we collected data from the medical charts of 50 consecutive NGS cfDNA tests in a free-standing children’s hospital. We evaluated patients for demographics, underlying conditions, diagnosis at time of testing, conventional laboratory testing and timing, medical treatment, and NGS cfDNA test results for clinical relevance or false negative results compared to conventional testing. The primary goal was to identify patients for whom the NGS cfDNA testing affected the POC. Charts were reviewed, and determinations regarding whether the result influenced the POC were confirmed by a provider. Results We were unable to differentiate patients with clinically valuable NGS cfDNA results (Fig 1 & 2). Among those with NGS cfDNA results valuable to the POC (n=22), both negative and positive testing guided POC (13 valuable negative vs. 9 diagnostic cases). In the total sample, 5 cases (10%) had a clinically relevant pathogen identified through conventional testing, but not through NGS cfDNA and 2 cases had antimicrobial resistance on culture, which is not detected by NGS cfDNA. Conclusion While we did not find a specific clinical profile for NGS cfDNA use, positive results were essential to the diagnosis in 18% of cases with otherwise negative laboratory evaluation for the pathogen identified in NGS cfDNA. Negative tests affected the POC in 26% of cases by avoiding unnecessary antimicrobials in high risk immunocompromised patients and patients that presented with low-risk of infection, but unclear disease process. Caution must be exercised with reliance on this test with respect to antimicrobial resistance and risk of false negative results. Disclosures All Authors: No reported disclosures

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Identification of a Disease-Causing Mutation in a Chinese Patient with Retinitis Pigmentosa by Targeted Next-Generation Sequencing

European Journal of Ophthalmology ◽

10.5301/ejo.5000971 ◽

2017 ◽

Vol 27 (6) ◽

pp. 791-796 ◽

Cited By ~ 1

Author(s):

Jianping Xiao ◽

Xueqin Guo ◽

Yong Wang ◽

Mingkun Shao ◽

Xiaoming Wei ◽

...

Keyword(s):

Next Generation Sequencing ◽

Retinitis Pigmentosa ◽

Sanger Sequencing ◽

Bioinformatics Analysis ◽

Family Members ◽

Chinese Patient ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Targeted Ngs ◽

Generation Sequencing

Purpose To identify disease-causing mutations in a Chinese patient with retinitis pigmentosa (RP). Methods A detailed clinical examination was performed on the proband. Targeted next-generation sequencing (NGS) combined with bioinformatics analysis was performed on the proband to detect candidate disease-causing mutations. Sanger sequencing was performed on all subjects to confirm the candidate mutations and assess cosegregation within the family. Results Clinical examinations of the proband showed typical characteristics of RP. Three candidate heterozygous mutations in 3 genes associated with RP were detected in the proband by targeted NGS. The 3 mutations were confirmed by Sanger sequencing and the deletion (c.357_358delAA) in PRPF31 was shown to cosegregate with RP phenotype in 7 affected family members, but not in 3 unaffected family members. Conclusions The deletion (c.357_358delAA) in PRPF31 was the disease-causing mutation for the proband and his affected family members with RP. To our knowledge, this is the second report of the deletion and the first report of the other 2 mutations in the Chinese population. Targeted NGS combined with bioinformatics analysis proved to be an effective molecular diagnostic tool for RP.

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Proficiency Testing of Standardized Samples Shows Very High Interlaboratory Agreement for Clinical Next-Generation Sequencing–Based Oncology Assays

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2018-0336-cp ◽

2018 ◽

Vol 143 (4) ◽

pp. 463-471 ◽

Cited By ~ 9

Author(s):

Jason D. Merker ◽

Kelly Devereaux ◽

A. John Iafrate ◽

Suzanne Kamel-Reid ◽

Annette S. Kim ◽

...

Keyword(s):

Next Generation Sequencing ◽

Proficiency Testing ◽

False Negative ◽

High Specificity ◽

Variant Allele ◽

Next Generation ◽

Single Nucleotide Variants ◽

Sequencing Platform ◽

Negative Results ◽

Generation Sequencing

Context.— Next-generation sequencing–based assays are being increasingly used in the clinical setting for the detection of somatic variants in solid tumors, but limited data are available regarding the interlaboratory performance of these assays. Objective.— To examine proficiency testing data from the initial College of American Pathologists (CAP) Next-Generation Sequencing Solid Tumor survey to report on laboratory performance. Design.— CAP proficiency testing results from 111 laboratories were analyzed for accuracy and associated assay performance characteristics. Results.— The overall accuracy observed for all variants was 98.3%. Rare false-negative results could not be attributed to sequencing platform, selection method, or other assay characteristics. The median and average of the variant allele fractions reported by the laboratories were within 10% of those orthogonally determined by digital polymerase chain reaction for each variant. The median coverage reported at the variant sites ranged from 1922 to 3297. Conclusions.— Laboratories demonstrated an overall accuracy of greater than 98% with high specificity when examining 10 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 15% or greater. These initial data suggest excellent performance, but further ongoing studies are needed to evaluate the performance of lower variant allele fractions and additional variant types.

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Molecular diagnosis of inherited peripheral neuropathies by targeted next-generation sequencing: molecular spectrum delineation

BMJ Open ◽

10.1136/bmjopen-2018-021632 ◽

2018 ◽

Vol 8 (10) ◽

pp. e021632 ◽

Cited By ~ 13

Author(s):

Juliette Bacquet ◽

Tanya Stojkovic ◽

Amandine Boyer ◽

Nathalie Martini ◽

Frédérique Audic ◽

...

Keyword(s):

Next Generation Sequencing ◽

Molecular Diagnosis ◽

Sanger Sequencing ◽

Diagnostic Yield ◽

Motor Neuropathy ◽

Peripheral Neuropathies ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Targeted Ngs ◽

Generation Sequencing

PurposeInherited peripheral neuropathies (IPN) represent a large heterogenous group of hereditary diseases with more than 100 causative genes reported to date. In this context, targeted next-generation sequencing (NGS) offers the opportunity to screen all these genes with high efficiency in order to unravel the genetic basis of the disease. Here, we compare the diagnostic yield of targeted NGS with our previous gene by gene Sanger sequencing strategy. We also describe several novel likely pathogenic variants.Design and participantsWe have completed the targeted NGS of 81 IPN genes in a cohort of 123 unrelated patients affected with diverse forms of IPNs, mostly Charcot-Marie-Tooth disease (CMT): 23% CMT1, 52% CMT2, 9% distal hereditary motor neuropathy, 7% hereditary sensory and autonomic neuropathy and 6.5% intermediate CMT.ResultsWe have solved the molecular diagnosis in 49 of 123 patients (~40%). Among the identified variants, 26 variants were already reported in the literature. In our cohort, the most frequently mutated genes are respectively:MFN2,SH3TC2,GDAP1,NEFL,GAN,KIF5AandAARS. Panel-based NGS was more efficient in familial cases than in sporadic cases (diagnostic yield 49%vs19%, respectively). NGS-based search for copy number variations, allowed the identification of three duplications in three patients and raised the diagnostic yield to 41%. This yield is two times higher than the one obtained previously by gene Sanger sequencing screening. The impact of panel-based NGS screening is particularly important for demyelinating CMT (CMT1) subtypes, for which the success rate reached 87% (36% only for axonal CMT2).ConclusionNGS allowed to identify causal mutations in a shorter and cost-effective time. Actually, targeted NGS is a well-suited strategy for efficient molecular diagnosis of IPNs. However, NGS leads to the identification of numerous variants of unknown significance, which interpretation requires interdisciplinary collaborations between molecular geneticists, clinicians and (neuro)pathologists.

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FAS Mutations in Follicular Lymphoma Are Rare but Associated with Aggressive Clinical Behavior.

Blood ◽

10.1182/blood.v114.22.3967.3967 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3967-3967

Author(s):

Nathalie A. Johnson ◽

Ryan Morin ◽

Tesa Severson ◽

Andrew J. Mungall ◽

Yongjun Zhao ◽

...

Keyword(s):

Next Generation Sequencing ◽

Follicular Lymphoma ◽

Research Funding ◽

Sanger Sequencing ◽

Clinical Course ◽

Death Domain ◽

Next Generation ◽

Genomic Alterations ◽

Aggressive Clinical Course ◽

Generation Sequencing

Abstract Abstract 3967 Poster Board III-903 Background Follicular lymphoma (FL) is considered an indolent but incurable lymphoma. Treatment with cyclophosphamide, vincristine, prednisone and rituximab (CVP-R) provides complete or partial responses in most patients. The BCL2 translocation t(14;18) is present in 85% of the cases, but additional genomic alterations must occur to induce overt FL. We have sequenced the genome and transcriptome of a cytogenetically normal FL sample taken from a patient (pt 1) that had an unusually aggressive clinical course with the aim of identifying genomic alterations that could contribute to FL pathogenesis. We identified a mutation in FAS/CD95, a key component of the extrinsic apoptotic pathway. We hypothesized that FAS mutations may contribute to treatment resistance in FL by inhibiting apoptosis. This study investigates the prevalence and clinical outcome of patients with FL harbouring mutations in the exons coding for the functional “death domain” of the FAS gene. Methods The initial FL sample was subjected to cytogenetic analysis and whole genome tiling array comparative genomic hybridization followed by next-generation sequencing of the tumour genome and tumor transcriptome following the manufacturer's protocol (Illumina). FAS emerged as a potential candidate that could explain the unusually aggressive FL. We performed PCR amplification of exons 7,8,9 and the 3'UTR of the FAS gene with universal M13F(-21) and M13 primer extensions on pre-treatment FL samples derived from 214 patients including 33 diffuse large B cell lymphoma samples that had evolved from a prior FL (i.e. paired FL and DLBCL). PCR products were purified using AMPpure magnetic beads and bi-directionally sequenced using BigDye® Terminator v3.1 and an ABI 3730 XL sequencer. Analysis was performed using Mutation Surveyor. Mutations were considered present if they were observed in both forward and reverse reads. Results Patient 1 had a t(14;18) negative, grade 1 FL, that progressed rapidly despite CVP-R and second line chemotherapy but is now in complete remission following an allogeneic stem cell transplant. The FL immunophenotype was CD19+, CD10+, BCL2+, BCL6+ and lambda clonal. Minimal genomic gains and losses were observed by aCGH. After filtering known single nucleotide polymorphisms (SNPs), a total of 320 candidate novel protein-altering changes were identified (affecting 298 genes) in the tumor genome and transcriptome sequence data. Validation of the mutated genes by Sanger sequencing revealed a novel, somatic and coding mutation in FAS at genomic position chr10:90764005, changing C to T, resulting in a premature truncation of the protein. We then sequenced exons 7, 8, 9 and the 3'UTR of the FAS gene from 214 FL patients. Ten novel FAS mutations were detected, of these six were coding, three of which produced a truncated protein. Of the six coding mutations, two were observed in the transformed DLBCL sample. Coding mutations in FAS appeared to be associated with an aggressive clinical course (median time to progression=12 months for coding mutations (n=6) vs 34 months for non-coding or wild type (n=208), P=0.06). 2 of the 6 patients developed early transformation to DLBCL, 2 had treatment resistant FL that required allogeneic bone marrow transplant and 2 have already died due to progressive treatment refractory FL. Conclusion Next generation sequencing technology revealed novel somatic mutations in a cytogenetically normal FL sample, one of which was a mutation in the FAS gene. Sanger sequencing of a large cohort of FL samples revealed that 5% of cases harboured novel mutations in the death domain of FAS (2% coding, 3% non-coding). Coding mutations were rare but when present were associated with atypically aggressive disease. Disclosures: Connors: Roche Canada: Research Funding. Gascoyne:Roche Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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SP174, NRAS Q61R Mutant-Specific Antibody, Cross-Reacts With KRAS Q61R Mutant Protein in Colorectal Carcinoma

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2016-0147-oa ◽

2017 ◽

Vol 141 (4) ◽

pp. 564-568 ◽

Cited By ~ 7

Author(s):

Jerzy Lasota ◽

Artur Kowalik ◽

Anna Felisiak-Golabek ◽

Shingo Inaguma ◽

Zeng-Feng Wang ◽

...

Keyword(s):

Genetic Testing ◽

Next Generation Sequencing ◽

Sanger Sequencing ◽

Molecular Genetic ◽

Mutant Protein ◽

Colorectal Carcinomas ◽

Molecular Genetic Testing ◽

Next Generation ◽

Wild Type ◽

Generation Sequencing

Context.— NRAS is a member of the RAS family oncoproteins implicated in cancer. Gain-of-function NRAS mutations were reported in a subset of colorectal cancers. These mutations occur at codons 12, 13, and 61 and are detected by molecular genetic testing. Recently, an antibody (clone SP174) became available to immunohistochemically pinpoint NRAS Q61R mutant protein. In malignant melanoma, NRAS Q61R mutant–specific immunohistochemistry was shown to be a valuable supplement to traditional genetic testing. Objective.— To evaluate the significance of NRAS Q61R mutant–specific immunohistochemistry in a cohort of colorectal carcinomas. Design.— A total of 1185 colorectal carcinomas were immunohistochemically evaluated with SP174 antibody. NRAS Q61R mutant–specific immunohistochemistry was validated by molecular genetic testing including Sanger sequencing, quantitative polymerase chain reaction (qPCR), and next-generation sequencing. Results.— Twelve tumors showed strong SP174 immunoreactivity. Sanger sequencing detected an identical c.182A>G substitution, causing NRAS Q61R mutation at the protein level, only in 8 SP174-positive cases. These results were confirmed by qPCR study. Subsequently, NRAS wild-type tumors with strong SP174 staining were evaluated by next-generation sequencing and revealed KRAS c.182A>G substitutions predicted to cause KRAS Q61R mutation. Review of colorectal carcinomas with known KRAS and NRAS genotype revealed that none of 62 wild-type tumors or 47 mutants other than Q61R were SP174 positive. Conclusion.— SP174 immunohistochemistry allows sensitive detection of NRAS and KRAS Q61R mutants. However, molecular genetic testing is necessary to determine specifically which RAS gene is mutated.

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Towards an External Quality Assessment for Next Generation Sequencing in the Diagnosis of Rare Inherited Anaemias

Blood ◽

10.1182/blood-2018-99-111609 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4936-4936

Author(s):

Noemi Roy ◽

Melanie Proven ◽

Irene Roberts ◽

Hannah Tamary ◽

Dorine W. Swinkels ◽

...

Keyword(s):

Next Generation Sequencing ◽

Quality Assessment ◽

Research Funding ◽

Sanger Sequencing ◽

External Quality Assessment ◽

Next Generation ◽

External Quality ◽

Clinical Vignettes ◽

Generation Sequencing ◽

Disease Specific

Abstract The diagnosis of patients with inherited anemias is increasingly made in conjunction with high-throughput 'next-generation' sequencing (NGS) analysis, largely using targeted resequencing panels validated for clinical use in diagnostic labs. While there is a joint UK NEQAS and European Molecular Quality Network pilot scheme for Molecular Genetics to assess NGS quality control, this is not disease-specific. Patients with inherited anaemias can have multiple mutations with complex genotype phenotype interactions therefore a scheme assessing interpretation of these results could be of value. Likewise, guidelines for variant reporting (eg ACMG- American College of Medical Genetics and Genomics) provide excellent advice on how to interpret the likely pathogenicity of genetic variants, but no disease-specific guidance exists to assist in the clinical interpretation of NGS findings for individuals with rare inherited anemias. The European Hematology Association Scientific Working Group on Red Cells and Iron carried out a survey among its members to investigate variability in current practice and determine the need for, and feasibility of, a formal external quality assessment (EQA) scheme. Surveys and two clinical vignettes with sample variant call files (VCFs) were distributed among 14 participating labs from 9 countries; 13/14 labs used a targeted panel and 1/14 lab (8%) used a 366-gene virtual panel derived from whole exome sequencing data. Accreditation: 12/14 labs had ISO (International Organisation for Standardisation) accreditation for their NGS; 2/12,used local accreditation schemes. The number of genes per targeted panel ranged from 18 to 215 genes (median 64), covering: congenital dyserythropoietic anemias, Diamond-Blackfan anemia, sideroblastic anemia, red cell membrane and enzyme disorders. Some panels included other related bone marrow failure or iron metabolism disorders. 7/13 labs with targeted panels sequenced only exons, with variable padding into introns, while 6/13 routinely sequenced 3' and 5' untranslated regions. Capture methods were variable between labs and 11/14 labs used Illumina platforms for sequencing and 3/14 Ion Torrent. Sanger sequencing was used for confirmation of NGS variants in 12/14 labs, but used for gap-filling of uncovered regions in 10/14 labs. Reporting of variants followed ACMG guidelines in 10/14 labs, ACGS (Association for Clinical Genomics Science) guidelines in 2/14 and no published guidelines in 2/14. Reporting of Tier 3 (variants of uncertain significance): 8/14 labs reported strict adherence to ACMG guidelines, including only Class 4 and Class 5 variants in clinical reports, while 6/14 labs admitted looser adherence and reporting of Class 3 variants depending on circumstances. The number of samples analysed per year was highly variable between labs (10-600, median 60). Two mini-EQA VCFs were sent with clinical vignettes, for which 100% of labs correctly identified a case of autosomal recessive sideroblastic anaemia due to compound heterozygous mutations in SLC25A38, and 100% correctly identified a case negative by NGS. Class 3 variants were not reported at all in 50% of clinical reports, reported in the main body of the report in 20% and in a separate table in 30% of labs. In conclusion, we have identified common approaches to NGS sequencing in 14 diagnostic laboratories but highlighted variability in accreditation, use of Sanger sequencing and adherence to ACMG guidelines. The feasibility of carrying out an EQA scheme has been established and work will continue with UK NEQAS to formally create such a scheme, with the aim of ensuring improved patient care through the use of objective quality assessments. Disclosures Colombatti: ADDMEDICA: Consultancy; BlueBirdBio: Consultancy; Global Blood Therapeutics: Consultancy; NOVARTIS: Consultancy. Viprakasit:Protagonist Therapeutics: Consultancy, Research Funding; Agios: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

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The Interlaboratory RObustness of Next-Generation Sequencing (IRON) Study: Deep-Sequencing Investigating TET2, CBL, and KRAS Mutations In 4464 Amplicons by An International Group Involving 8 Laboratories.

Blood ◽

10.1182/blood.v116.21.1665.1665 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1665-1665

Author(s):

Alexander Kohlmann ◽

Hans-Ulrich Klein ◽

Silvia Bresolin ◽

Tracy Chaplin ◽

Harry Cuppens ◽

...

Keyword(s):

Next Generation Sequencing ◽

Deep Sequencing ◽

Research Funding ◽

Sanger Sequencing ◽

Myeloproliferative Neoplasms ◽

Life Sciences ◽

Silent Mutation ◽

Next Generation ◽

Kras Mutations ◽

Generation Sequencing

Abstract Abstract 1665 Massively parallel pyrosequencing in picoliter-sized wells is an innovative technique and allows highly-sensitive deep-sequencing to detect molecular aberrations. Thus far, limited data is available on the technical performance in a clinical diagnostic setting. Here, we investigated - as an international consortium - the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 8 laboratories from 6 countries. As a first candidate gene we selected TET2, a frequently mutated gene in myeloproliferative neoplasms. In total, 31 primer pairs including a 10-base molecular barcode sequence were designed and evaluated: All coding exons of TET2 were represented by 27 amplicons. In addition, 2 primer pairs were amplifying hotspot regions to characterize the RING finger domain and linker sequence for CBL and 2 amplicons covered KRAS exons 2 and 3. To execute our study, we used the small volume Titanium emulsion PCR setup (454 Life Sciences, Branford, CT). A cohort of 18 chronic myelomonocytic leukemia (CMML) patient samples were centrally collected by the Munich Leukemia Laboratory and characterized by conventional sequencing for mutations in TET2, CBL, and KRAS. In this selected cohort 33 distinct mutations in TET2, 7 mutations in CBL, and 3 mutations in KRAS, respectively, were detected by Sanger sequencing (plus 10 SNPs and one silent mutation). Each of the participating laboratories received anonymized aliquots of 1.6 μg of genomic DNA to be processed for the generation of PCR amplicons suitable for 454 deep-sequencing. In detail, a total of 31 × 18 (n=558) PCRs were locally performed at each laboratory, i.e. a total of 4464 PCR reactions across 8 centers. Subsequently, at each site each PCR product was individually purified and quantified and corresponding pools were generated by combining 31 amplicons in an equimolar ratio for each patient sample. After processing the samples using the 454 workflow, 3 patients each were loaded per lane on an 8-lane PicoTiterPlate on the GS FLX sequencer instrument. Overall, each of the 8 participating laboratories generated in median 432,606 reads across the 31 PCR amplicons (“Passed Filter Wells”). The median coverage per amplicon was 713-fold, ranging from 553-fold to 878-fold. Dropouts of single amplicons with no coverage obtained were observed in 4/8 laboratories in 61 of 4464 PCR products (1.4%). After alignment of the obtained sequences against the reference genome a total of 92 variants (44 distinct mutations and 10 SNPs) were observed across 22 amplicons. For this analysis, a given variant was scored if, in median, both forward and reverse reads were harboring the variant in at least 20% of reads, i.e. in line with the Sanger sequencing detection limit (GS FLX Amplicon Variant Analysis software v.2.3). In comparison to data available from Sanger sequencing, 454 amplicon deep-sequencing detected all mutations and SNPs that were previously known (few comparisons not possible due to single amplicon dropouts). In 90/92 variant comparisons all eight laboratories consistently detected the variant (two KRAS mutations being detected with a range from 18.0% - 22.6% of reads carrying the mutation). We did not observe a considerable bias in the measurements of the 92 variants between any two centers. Based on paired t-tests for equivalence, with equivalence limits for the standardized expected differences between two centers of -+ε (ε=0.5), the null hypothesis of dissimilar measurements was rejected for all pairs of centers (alpha=0.05). The estimated standard deviation of the measurements across centers was 3.1% (95% CI: [2.9%, 3.2%]), demonstrating the high precision of 454 sequencing to detect mutations. Additionally, we took advantage of the high sensitivity of deep-sequencing. As such, we observed 7 distinct novel mutations (n=2 TET2, n=3 CBL, n=2 KRAS) with frequencies below the Sanger sequencing cut-off value of 20% (median values ranging from 2.8% - 12.6%). These low-level mutations were consistently detected in all laboratories (one CBL mutation with <3% frequency detected in only 5/8 centers). In conclusion, we here demonstrate in a multicenter analysis that amplicon-based deep-sequencing is technically feasible, achieves a high concordance across multiple laboratories, and therefore allows a broad and in-depth molecular characterization of hematological malignancies with high diagnostic sensitivity. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Garicochea:Roche Diagnostics: Research Funding. Grossmann:MLL Munich Leukemia Laboratory: Employment. Hanczaruk:454 Life Sciences: Employment. Jansen:Roche Diagnostics: Research Funding. te Kronnie:Roche Diagnostics: Research Funding. Martinelli:Roche Diagnostics: Research Funding. McGowan:454 Life Sciences: Employment. Stabentheiner:Roche Diagnostics: Research Funding. Timmermann:Roche Diagnostics: Research Funding. Vandenberghe:Roche Diagnostics: Research Funding. Young:Roche Diagnostics: Research Funding. Dugas:Roche Diagnostics: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Roche Diagnostics: Research Funding.

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Identification of Genetic Hereditary Predisposition to Hematologic Malignancies By Clinical Next-Generation Sequencing

Blood ◽

10.1182/blood.v126.23.3854.3854 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3854-3854 ◽

Cited By ~ 2

Author(s):

Amy E Knight Johnson ◽

Lucia Guidugli ◽

Kelly Arndt ◽

Gorka Alkorta-Aranburu ◽

Viswateja Nelakuditi ◽

...

Keyword(s):

Next Generation Sequencing ◽

Sanger Sequencing ◽

Family Members ◽

Hematologic Malignancies ◽

Dyskeratosis Congenita ◽

Molecular Diagnostic ◽

Next Generation ◽

Hereditary Predisposition ◽

Ngs Data ◽

Generation Sequencing

Abstract Introduction: Myelodysplastic syndrome (MDS) and acute leukemia (AL) are a clinically diverse and genetically heterogeneous group of hematologic malignancies. Familial forms of MDS/AL have been increasingly recognized in recent years, and can occur as a primary event or secondary to genetic syndromes, such as inherited bone marrow failure syndromes (IBMFS). It is critical to confirm a genetic diagnosis in patients with hereditary predisposition to hematologic malignancies in order to provide prognostic information and cancer risk assessment, and to aid in identification of at-risk or affected family members. In addition, a molecular diagnosis can help tailor medical management including informing the selection of family members for allogeneic stem cell transplantation donors. Until recently, clinical testing options for this diverse group of hematologic malignancy predisposition genes were limited to the evaluation of single genes by Sanger sequencing, which is a time consuming and expensive process. To improve the diagnosis of hereditary predisposition to hematologic malignancies, our CLIA-licensed laboratory has recently developed Next-Generation Sequencing (NGS) panel-based testing for these genes. Methods: Thirty six patients with personal and/or family history of aplastic anemia, MDS or AL were referred for clinical diagnostic testing. DNA from the referred patients was obtained from cultured skin fibroblasts or peripheral blood and was utilized for preparing libraries with the SureSelectXT Enrichment System. Libraries were sequenced on an Illumina MiSeq instrument and the NGS data was analyzed with a custom bioinformatic pipeline, targeting a panel of 76 genes associated with IBMFS and/or familial MDS/AL. Results: Pathogenic and highly likely pathogenic variants were identified in 7 out of 36 patients analyzed, providing a positive molecular diagnostic rate of 20%. Overall, 6 out of the 7 pathogenic changes identified were novel. In 2 unrelated patients with MDS, heterozygous pathogenic sequence changes were identified in the GATA2 gene. Heterozygous pathogenic changes in the following autosomal dominant genes were each identified in a single patient: RPS26 (Diamond-Blackfan anemia 10), RUNX1 (familial platelet disorder with propensity to myeloid malignancy), TERT (dyskeratosis congenita 4) and TINF2 (dyskeratosis congenita 3). In addition, one novel heterozygous sequence change (c.826+5_826+9del, p.?) in the Fanconi anemia associated gene FANCA was identified. . The RNA analysis demonstrated this variant causes skipping of exon 9 and results in a premature stop codon in exon 10. Further review of the NGS data provided evidence of an additional large heterozygous multi-exon deletion in FANCA in the same patient. This large deletion was confirmed using array-CGH (comparative genomic hybridization). Conclusions: This study demonstrates the effectiveness of using NGS technology to identify patients with a hereditary predisposition to hematologic malignancies. As many of the genes associated with hereditary predisposition to hematologic malignancies have similar or overlapping clinical presentations, analysis of a diverse panel of genes is an efficient and cost-effective approach to molecular diagnostics for these disorders. Unlike Sanger sequencing, NGS technology also has the potential to identify large exonic deletions and duplications. In addition, RNA splicing assay has proven to be helpful in clarifying the pathogenicity of variants suspected to affect splicing. This approach will also allow for identification of a molecular defect in patients who may have atypical presentation of disease. Disclosures No relevant conflicts of interest to declare.

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Assay of frequency and spectrum of genetic variants in TTN in healthy russian population

Translational Medicine ◽

10.18705/2311-4495-2021-8-5-29-37 ◽

2021 ◽

Vol 8 (5) ◽

pp. 29-37

Author(s):

Yu. A. Vakhrushev ◽

A. A. Kozyreva ◽

S. V. Zhuk ◽

O. P. Rotar ◽

A. A. Kostareva

Keyword(s):

Next Generation Sequencing ◽

Genetic Variants ◽

Sanger Sequencing ◽

Heart Diseases ◽

Genetic Research ◽

Russian Population ◽

Next Generation ◽

Data Bases ◽

International Data ◽

Generation Sequencing

Background. Gene TTN associated with all types of cardiomyopathy, however its large size (294 b.p.) warrants a lot of individual unique genetic variants or variants with low frequency, that aggravates their interpretation. Besides that nowadays there is no data about spectrum of variants in this gene in healthy Russian population. Recognition frequency and spectrum of variants in gene TTN in healthy Russian population will allow us to use it for interpretation results of molecular genetic research for patients with different heart pathology, and define prognosis for different heart diseases.Objective. Recognize frequency and spectrum of single nucleotide and truncating variants in gene TTN in healthy Russian population and compare it with international data bases, and evaluate level of pathogenicity these variants and their distributing across titin structure.Design and methods. 192 men in age 55,8±6,6 years were tested with next-generation sequencing. Identified genetic variants were confirmed by Sanger sequencing. Results. Allele missense variant frequency (with frequency less than 0.1%) in TTN in healthy Russian population amount to 15.1 %, and truncating variants — 0.52 %. 37,9 % of them were variants of unknown significance, 62 % — likely-benign and 0.1 % — benign. There was no pathological and likely-pathological variants. Identified genetic variants distributed throughout the titin structure.Conclusion. Received result is congruent с international data bases and researches. Expended laboratory method (Next generation sequencing and confirmation with Sanger sequencing) can be used both in clinical practice, and in creating data bases of genetic variants in healthy Russian population.

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