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• W2912454862 abstract "Von Hippel-Lindau disease (VHL) is a monogenic disorder characterized by the development of tumors affecting the central nervous system, kidney, pancreas, or adrenal glands, and due to germline mutations in the VHL tumor suppressor gene. About 5% of patients with a typical VHL phenotype have no mutation detected by conventional techniques, so a postzygotic VHL mosaicism can be suspected. The aim of this study was therefore to implement a next-generation sequencing (NGS) strategy for VHL mosaic mutation detection, including an optimization of the original Personal Genome Machine design by enrichment with oligonucleotides corresponding to amplicons with insufficient depth of coverage. Two complementary strategies were developed for the confirmation of mosaic mutations identified by NGS, SNaPshot for variants present at an allelic ratio greater than 5%, and droplet digital PCR for allelic ratio above 1%. VHL mutant plasmids were generated to assess VHL mosaic mutation detection in different exons and to set up an internal quality control that could be included in each run or regularly to validate the assay. This strategy was applied to 47 patients with a suggestive or clinical VHL disease, and mosaic mutations were identified in 8.5% of patients. In conclusion, NGS technologies combined with SNaPshot or droplet digital PCR allow the detection and confirmation of mosaic mutations in a clinical laboratory setting. Von Hippel-Lindau disease (VHL) is a monogenic disorder characterized by the development of tumors affecting the central nervous system, kidney, pancreas, or adrenal glands, and due to germline mutations in the VHL tumor suppressor gene. About 5% of patients with a typical VHL phenotype have no mutation detected by conventional techniques, so a postzygotic VHL mosaicism can be suspected. The aim of this study was therefore to implement a next-generation sequencing (NGS) strategy for VHL mosaic mutation detection, including an optimization of the original Personal Genome Machine design by enrichment with oligonucleotides corresponding to amplicons with insufficient depth of coverage. Two complementary strategies were developed for the confirmation of mosaic mutations identified by NGS, SNaPshot for variants present at an allelic ratio greater than 5%, and droplet digital PCR for allelic ratio above 1%. VHL mutant plasmids were generated to assess VHL mosaic mutation detection in different exons and to set up an internal quality control that could be included in each run or regularly to validate the assay. This strategy was applied to 47 patients with a suggestive or clinical VHL disease, and mosaic mutations were identified in 8.5% of patients. In conclusion, NGS technologies combined with SNaPshot or droplet digital PCR allow the detection and confirmation of mosaic mutations in a clinical laboratory setting. Von Hippel Lindau (VHL) disease is an inherited neoplasia syndrome characterized by the development of several benign and malignant tumors affecting multiple organs, including cerebellar, spinal, and retinal hemangioblastoma, renal clear cell carcinoma, pheochromocytoma/paraganglioma, pancreatic cysts, and pancreatic neuroendocrine tumors. The clinical diagnosis is based on the occurrence of two characteristic VHL-related tumors, including at least one hemangioblastoma, or the presence of a single characteristic VHL-related tumor with a family history of VHL.1Gossage L. Eisen T. Maher E.R. VHL, the story of a tumour suppressor gene.Nat Rev Cancer. 2015; 1: 55-64Crossref Scopus (443) Google Scholar, 2Haddad N.M. Cavallerano J.D. Silva P.S. Von Hippel-Lindau disease: a genetic and clinical review.Semin Ophthalmol. 2013; 28: 377-386Crossref PubMed Scopus (37) Google Scholar VHL disease is the result of a germline mutation inactivating the VHL tumor suppressor gene (NM_000551.3, 3p25-26) and that is inherited in an autosomal dominant manner. In 20% of cases, VHL disease occurs without family history, in individuals with seemingly sporadic VHL-related tumors, and a de novo germline mutational event in the VHL gene is identified in these patients. The majority of VHL pathogenic variants are private and the spectrum is very large (about 200 distinct mutations reported to date), including missense and non-sense variants, frameshifts, splice site variants (60% to 80%), as well as small deletions or insertions and large or complete gene deletions (20% to 40%).3Decker J. Neuhaus C. Macdonald F. Brauch H. Maher E.R. Clinical utility gene card for: von Hippel-Lindau (VHL).Eur J Hum Genet. 2014; 22: 572Crossref Scopus (20) Google Scholar Conventional VHL genetic testing, based on DNA sequencing and PCR-based exonic rearrangement screening, allows the detection of a VHL pathogenic variant in approximately 95% of patients with a typical VHL disease.4Maher E.R. Neumann H.P. Richard S. Von Hippel-Lindau disease: a clinical and scientific review.Eur J Hum Genet. 2011; 19: 617-623Crossref PubMed Scopus (447) Google Scholar In the remaining 5% of patients harboring a typical VHL phenotype, a postzygotic VHL mosaicism can be suspected because of a lack of sensitivity of conventional genetic testing.5Sgambati M.T. Stolle C. Choyke P.L. Walther M.M. Zbar B. Linehan W.M. Glenn G.M. Mosaicism in von Hippel-Lindau disease: lessons from kindreds with germline mutations identified in offspring with mosaic parents.Am J Hum Genet. 2000; 66: 84-91Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar However, prior to the development of next-generation sequencing (NGS) technologies,6Mardis E.R. The impact of next-generation sequencing technology on genetics.Trends Genet. 2008; 24: 133-141Abstract Full Text Full Text PDF PubMed Scopus (1559) Google Scholar mosaic mutation screening was cumbersome to perform routinely in a clinical laboratory setting. NGS provides higher sensitivity than does conventional Sanger sequencing through a great depth of coverage that enables the detection of genetic variants in cell subpopulations.7Pagnamenta A.T. Lise S. Harrison V. Stewart H. Jayawant S. Quaghebeur G. Deng A.T. Murphy V.E. Sadighi Akha E. Rimmer A. Mathieson I. Knight S.J. Kini U. Taylor J.C. Keays D.A. Exome sequencing can detect pathogenic mosaic mutations present at low allele frequencies.J Hum Genet. 2012; 57: 70-72Crossref PubMed Scopus (53) Google Scholar, 8Qin L. Wang J. Tian X. Yu H. Truong C. Mitchell J.J. Wierenga K.J. Craigen W.J. Zhang V.W. Wong L.C. Detection and quantification of mosaic mutations in disease genes by next-generation sequencing.J Mol Diagn. 2016; 18: 446-453Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9Page K. Guttery D.S. Fernandez-Garcia D. Hills A. Hastings R.K. Luo J. Goddard K. Shahin V. Woodley-Barker L. Rosales B.M. Coombes R.C. Stebbing J. Shaw J.A. Next generation sequencing of circulating cell-free DNA for evaluating mutations and gene amplification in metastatic breast cancer.Clin Chem. 2017; 63: 532-541Crossref PubMed Scopus (58) Google Scholar The identification of a mosaic mutation present at a 1% allelic ratio with a good statistical significance by NGS requires a minimal depth of 700 reads with equilibrium between the forward and reverse strands in order to distinguish the mutation from a sequencing artefact.10Izawa K. Hijikata A. Tanaka N. Kawai T. Saito M.K. Goldbach-Mansky R. Aksentijevich I. Yasumi T. Nakahata T. Heike T. Nishikomori R. Ohara O. Detection of base substitution-type somatic mosaicism of the NLRP3 gene with >99.9% statistical confidence by massively parallel sequencing.DNA Res. 2012; 19: 142-152Crossref Scopus (47) Google Scholar A few studies have reported levels of VHL mosaic mutation around 5% and have demonstrated that this kind of event was especially detected in patients with mild phenotype.5Sgambati M.T. Stolle C. Choyke P.L. Walther M.M. Zbar B. Linehan W.M. Glenn G.M. Mosaicism in von Hippel-Lindau disease: lessons from kindreds with germline mutations identified in offspring with mosaic parents.Am J Hum Genet. 2000; 66: 84-91Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 11Murgia A. Martella M. Vinanzi C. Polli R. Perilongo G. Opocher G. Somatic mosaicism in von Hippel-Lindau disease.Hum Mutat. 2000; 15: 114Crossref PubMed Scopus (58) Google Scholar, 12Santarpia L. Sarlis N.J. Santarpia M. Sherman S.I. Trimarchi F. Benvenga S. Mosaicism in von Hippel-Lindau disease: an event important to recognize.J Cell Mol Med. 2007; 11: 1408-1415Crossref PubMed Scopus (20) Google Scholar, 13Wu P. Zhang N. Wang X. Li T. Ning X. Bu D. Gong K. Mosaicism in von Hippel-Lindau disease with severe renal manifestations.Clin Genet. 2013; 84: 581-584Crossref PubMed Scopus (14) Google Scholar We previously described VHL mosaic mutation detection by 454 GS Junior System sequencing technology (Roche Diagnostics, Meylan, France), and demonstrated that mosaicism in VHL disease is not restricted to patients with a mild phenotype.14Coppin L. Grutzmacher C. Crépin M. Destailleur E. Giraud S. Cardot-Bauters C. Porchet N. Pigny P. VHL mosaicism can be detected by clinical next-generation sequencing and is not restricted to patients with a mild phenotype.Eur J Hum Genet. 2014; 22: 1149-1152Crossref PubMed Scopus (21) Google Scholar The aims of this study were: i) to evaluate the efficiency of the Ion Torrent Personal Genome Machine (PGM) NGS-platform (based on semiconductor sequencing) for VHL mosaic mutation detection, ii) to develop confirmation methods for low allelic ratio mutations [droplet digital (dd)-PCR and SNaPshot], iii) to set up an internal quality control (iQc) adapted to mosaic mutation screening, and iv) to screen for VHL mosaic mutation in the largest cohort (n = 47) of VHL patients ever reported. So, we propose an overall technical protocol for VHL mosaic detection that can be implemented in a clinical laboratory setting. Forty-seven patients with a phenotype evocative of VHL disease and who previously tested negative for heterozygous VHL mutation were screened for VHL mosaic mutation. Forty-six cases were sporadic (no familial history) and one case was familial (the patient had an affected daughter). Among these 47 patients, two DNA samples previously identified with VHL mosaic mutations were included as positive controls [P1: c.481C>T, p.(Arg161*); P2: c.500G>A, p.(Arg167Gln)].14Coppin L. Grutzmacher C. Crépin M. Destailleur E. Giraud S. Cardot-Bauters C. Porchet N. Pigny P. VHL mosaicism can be detected by clinical next-generation sequencing and is not restricted to patients with a mild phenotype.Eur J Hum Genet. 2014; 22: 1149-1152Crossref PubMed Scopus (21) Google Scholar DNA was extracted from peripheral blood mononuclear cells with the EZ1 DNA Blood Kit (Qiagen, Courtaboeuf, France). Tumor DNA was isolated from formalin-fixed, paraffin-embedded tumor tissues using QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer's instructions. All patients provided informed consent allowing germline DNA analysis. High-throughput sequencing was performed using PGM platform (Ion Torrent; Thermo Fisher Scientific, Courtaboeuf, France), and primers for amplification were designed using Ion AmpliSeq Designer software version 2.2.1 (coding regions plus 25 bp corresponding to intronic flanking regions; Thermo Fisher Scientific). This design was part of a multigene panel previously designed to routinely perform the genetic testing of familial endocrine neoplasia syndromes. Primer pairs were split by the manufacturer into two mixes according to the compatibility between the different primers, so that primers for overlapping amplicons were in different tubes. Sequences of the primer pairs were separated into two pools: exon1: amplicon 1710843567 (213 pb), 5′-GCCTCCGGCCGGCTATT-3′ (forward) and 5′-CCCCGCCGTCTTCTTCA-3′ (reverse); amplicon 1809669404 (198 pb), 5′-AGGCAGGCGTCGAAGAGTA-3′ (forward) and 5′-GCTCGCCGTCGAAGTTG-3′ (reverse); and amplicon 1512083324 (114 pb), 5′-GTGCTGCCCGTATGGCT-3′ (forward) and 5′-GGGCTTCAGACCGTGCTATC-3′ (reverse); exon 2: amplicon 5141954337 (250 pb), 5′-CGGTGTGGCTCTTTAACAACC-3′ (forward) and 5′-GGGCTTAATTTTTCAAGTGGTCTATCCT-3′ (reverse); and exon 3: amplicon 5364451121 (218 pb), 5′-CCCTAGTCTGCCACTGAGGAT-3′ (forward) and 5′-ACCATCAAAAGCTGAGATGAAACAGT-3′ (reverse). The in silico coverage score predicted for this design was 100%. Libraries were constructed using the Ion AmpliSeq Panels, Ion AmpliSeq Library Kit, Ion Xpress Barcodes (for multiplexing), and 10 ng of patient DNA per pool. The amounts of DNA for VHL pCR2.1 constructions (wild type and mutated) were adapted as described in Library Construction for Plasmids. The libraries were transferred to the Ion OneTouch 2 System for emulsion PCR (Thermo Fisher Scientific). Libraries were sequenced on the Ion PGM Sequencer (314 Chip for eight samples or 316 Chip for 16 samples), and automated data analysis was performed with SeqNext Module version 4.1.2 of the Sequencing Pilot software (JSI Medical Systems GmbH, Ettenheim, Germany) with parameters allowing the detection of variants with a minimum of 1% of reads on both directions. An in-house script was used to specifically analyze mutation loci. In this script, reference or alternate sequence plus anchor bases on both the 5′ and 3′ ends of each mutation were used as motifs. Those motifs were searched and counted in raw read sequences from .fastq files of all samples. It was assumed that no more than one patient sample per run could be positive for each variant. For each tested variant, the candidate sample was compared to other samples—considered as controls—using the pairwise Fisher exact test on 2 × 2 contingency tables. This allowed systematic comparison in the worst scenario for background noise (cross-sample contamination, sequencing errors, etc). A mutation was considered as detected when the Fisher test–derived P value was less than 0.001 in all comparisons for a given candidate. Several plasmids harboring different VHL mutations were generated. The first step consisted of the creation of VHL plasmids for each VHL exon. Amplicons corresponding to exons 1, 2, and 3 of VHL were generated from genomic DNA amplified with the following primers: exon 1, 5′-TGGTCTGGATCGCGGAGG-3′ (forward) and 5′-CCCGTCTGCAAAATGGACCC-3′ (reverse) (450 pb); exon 2, 5′-ACCGGTGTGGCTCTTTAACA-3′ (forward) and 5′-GCCCAAAGTGCTTTTGAGAC-3′ (reverse) (376 pb); and exon 3, 5′-TGGCAAAGCCTCTTGTTCG-3′ (forward) and 5′-GCCCCTAAACATCACAATGCC-3′ (reverse) (433 pb). Proportions of the insert cloned into pCR2.1 were respectively 10.28%, 8.73%, and 9.92% for exons 1, 2, and 3. Primers were designed so that AmpliSeq primers for NGS could hybridize onto these amplicons to enable their use in subsequent library preparation. Then, each amplicon was cloned into a pCR2.1 vector (3.929 kb) with the original TA Cloning Kit (Thermo Fisher Scientific), and plasmids were prepared using the NucleoBond Xtra Maxi Plus kit (Macherey Nagel, Hoerdt, France). Then, starting from these wild-type plasmids, VHL mutant plasmids were generated. They corresponded to eight VHL pathogenic variants distributed across the whole VHL gene: three variants in exon 1 [c.194C>G, p.(Ser65Trp); c.226_228del, p.(Phe76del); and c.232A>C, p.(Asn78His)]; one in intron 1 exon 2 (c.341-1G>C, p.?); and four in exon 3 [c.467A>G, p.(Tyr156Cys); c.481C>T, p.(Arg161*); c.482G>A, p.(Arg161Gln); and c.500G>A, p.(Arg167Gln)]. Site-directed mutagenesis was performed with the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). Primers were as follows: VHL1-p.(Ser6Trp), 5′-CCGTGCTGCGCTGGGTGAACTCGCG-3′ (forward) and reverse 5′-CGCGAGTTCACCCAGCGCAGCACGG-3′; VHL1-p.(Phe76del), 5′-CCCAGGTCATCTGCAATCGCAGTCC-3′ (forward) and 5′-GGACTGCGATTGCAGATGACCTGGG-3′ (reverse); VHL1-p.(Asn78His), 5′-GTCATCTTCTGCCATCGCAGTCCGC-3′ (forward) and 5′-GCGGACTGCGATGGCAGAAGATGAC-3′ (reverse); VHL2-c.341-1G>C, 5′-GCTTGTCCCGATACGTCACCTTTGGC-3′ (forward) and 5′-GCCAAAGGTGACGTATCGGGACAAGC-3′ (reverse); VHL3-p.(Tyr156Cys), 5′-GCCCTTCCAGTGTGTACTCTGAAAGAGC-3′ (forward) and 5′-GCTCTTTCAGAGTACACACTGGAAGGGC-3′ (reverse); VHL3-p.(Arg161*), 5′-GTATACTCTGAAAGAGTGATGCCTCCAGGTTG-3′ (forward) and 5′-CAACCTGGAGGCATCACTCTTTCAGAGTATAC-3′ (reverse); VHL3-p.(Arg161Gln), 5′-GTATACTCTGAAAGAGCAATGCCTCCAGGTTGTC-3′ (forward) and 5′-GACAACCTGGAGGCATTGCTCTTTCAGAGTATAC-3′ (reverse); and VHL3-p.(Arg167Gln), 5′-CTCCAGGTTGTCCAGAGCCTAGTCAAG-3′ (forward) and 5′-CTTGACTAGGCTCTGGACAACCTGGAG-3′ (reverse). The presence of each mutation was checked by Sanger sequencing. Each mutated plasmid was mixed with the wild-type one of the corresponding exon to obtain an allelic ratio between 1% and 2%, and then the wild-type plasmids of the two other exons were added. The starting amount of DNA had to be modified for library preparation with VHL amplicons cloned into plasmids. A total of 2 pg of insert was used for the three constructions (which corresponds to 0.66 pg/insert). Taking into account the ratio insert size/plasmid vector size, the insert amount (pg) was converted into plasmid amount (pg). Totals of 6.42 pg of pCR2.1-VHL exon 1 construct, 7.52 pg of pCR2.1-VHL exon 2 construct, and 6.64 pg of pCR2.1-VHL exon 3 construct were pooled. According to the mutation individually studied (unitary plasmid), the mutated plasmids were diluted and pooled with the corresponding wild-type construction, and the wild-type constructions for the two other exons were added. Then, an internal quality control (iQc) was generated that contained all of the VHL mutations generated at the level of 1% to 2%. Each mutated VHL construct was mixed with the wild-type corresponding construct to get a final level of mosaicism around 1% to 2% for each studied position. SNaPshot Multiplex System technology (Thermo Fisher Scientific) was used as previously described.14Coppin L. Grutzmacher C. Crépin M. Destailleur E. Giraud S. Cardot-Bauters C. Porchet N. Pigny P. VHL mosaicism can be detected by clinical next-generation sequencing and is not restricted to patients with a mild phenotype.Eur J Hum Genet. 2014; 22: 1149-1152Crossref PubMed Scopus (21) Google Scholar Primers used for VHL mutation detection were as follows: VHL-1-65, 5′-CGCGGCCCGTGCTGCGCT-3′ (forward) and 5′-AGGGCTCGCGCGAGTTCACC-3′ (reverse); VHL-1-76, WT 5′-*N*NAGCCCTCCCAGGTCATCTT-3′ (forward), MUT 5′-*N*NCCCTCCCAGGTCATCTGC-3′ (forward), and VHL-1-76 5′-*N*NGCGGACTGCGATTGCAGA-3′ (reverse); VHL-1-78, 5′-*N*N*N*CCTCCCAGGTCATCTTCTGC-3′ (forward) and 5′-*N*N*N*CACGACGCGCGGACTGCGAT-3′ (reverse); VHL-3-156, 5′-*N*N*N*NTTGGTTTTTGCCCTTCCAGTGT-3′ (forward) and 5′-*N*N*N*NGAGGCATCGCTCTTTCAGAGTA-3′ (reverse); VHL-1-c.341, 5′-AACCTTTGCTTGTCCCGATA-3′ (forward) and 5′-TCTGAAGAGCCAAAGGTGAC-3′ (reverse); VHL-1-161Gln, 5′-*NAGTGTATACTCTGAAAGAGC-3′ (forward) and 5′-*NTCCGGACAACCTGGAGGCAT-3′ (reverse); VHL-1-167Gln, 5′-*N*N*NGATGCCTCCAGGTTGTCC-3′ (forward) and 5′-*N*N*NTCTCAGGCTTGACTAGGCTC-3′ (reverse); and VHL-3-161*, 5′-*N*N*N*NCCAGTGTATACTCTGAAAGAG-3′ (forward) and 5′-*N*N*N*NCGGACAACCTGGAGGCATC-3′ (reverse). For VHL c.226_228del, p.(Phe76del) mutation, specific forward primers targeting the wild-type and the mutated event were designed. ddPCR was performed based on water–oil emulsion droplet technology. This technology requires a preliminary optimization step to determine the best annealing temperature. Temperatures ranging from 52°C to 60°C were tested and it was found that 56°C could be used for all of our assays. Sample partitioning was performed using the QX200 Droplet Generator (Bio-Rad, Marnes-la-Coquette, France), PCR amplification with the C1000 Thermal Cycler (Bio-Rad; 95°C for 10 minutes; 94°C for 30 seconds and 56°C for 60 seconds, for 40 cycles; and 98°C for 10 minutes) and droplet reading with the QX 200 Droplet Reader (Bio-Rad), which provides absolute quantification in digital form. TaqMan probe–based assays were used (Bio-Rad). Each mutated event was detected with a primer labeled with FAM fluorophore. The corresponding wild-type allele was detected with a primer labeled with HEX fluorophore. References of ddPCR Mutation Assay (BioRad) were the following: p.(Ser65Trp), dHsaMDS389884862; p.(Phe76del), dHsaMDS544200506; p.(Asn78His), dHsaMDS8356884801; c.341-1G>C, dHsaMDS618563711; p.(Tyr156Cys), dHsaMDS456108094; p.(Arg161*), dHsalS2503094; p.(Arg161Gln), dHsaMDS649820635; and p.(Arg167Gln), dHsalS2504838. As mentioned, in silico primers were designed to perform sequencing of the three exons of the VHL gene with the PGM platform using Ion AmpliSeq Designer software version 2.2.1 (Thermo Fisher Scientific). The three exons of VHL were covered by a total of five amplicons [three in exon 1 (Supplemental Figure S1), one in exon 2, and one in exon 3]. A preliminary NGS run allowed identification of amplicons with a depth of coverage below 700 reads that would not allow mosaic detection at the level of 1% (1× concentration) (Supplemental Table S1). This was the case for two of the three amplicons covering VHL exon 1 (1710843567 and 1809669404). These two amplicons are longer (213 and 198 bp, respectively) than amplicon 1512083324 (114 bp), which was also located in exon 1 and well covered. Amplicons in VHL exon 2 and 3 were well covered. To obtain a higher depth of coverage for these two VHL-exon 1 amplicons, the primer mixes provided by the manufacturer were enriched with individual primers available separately in a 384-well plate (+1.25× and +2.5× enrichment). The same DNA samples were then reanalyzed in a new library prepared with these enriched mixes. According to the results (Supplemental Table S1), +1.25X and +2.5X enrichments successfully enabled to reach a minimal coverage of 700× for amplicons 1809669404 and 1710843567, respectively. The results generated by the 454 and PGM platforms for the two positive control samples were compared using the same settings in the SeqPilot software with the SeqNext module (JSI Medical Systems GmbH, Ettenheim, Germany). The mosaic mutations were properly identified in both samples regardless of the platform used, with a concordant allelic ratio of mutated alleles; in patient 1, the ratio of mutated alleles was 2.1% with the PGM platform versus 1.7% with 454 (Supplemental Table S2) and in patient 2, the ratio was 7.4% with PGM versus 5.7% with 454 (Supplemental Table S2). Mosaic events detected by PGM were equilibrated on both strands. These results demonstrate that PGM is able to detect VHL mosaic mutations as well as 454 platform. Mosaic mutations at other positions on the VHL gene were next detected. Eight plasmids, each harboring a specific VHL mosaic mutation at levels between 1% and 2%, were generated and analyzed on the PGM platform. These eight mutations were distributed on the whole VHL gene. Individual mosaic mutation in one exon was systematically analyzed in the presence of the two other plasmids corresponding to the wild-type form of the remaining exons in order to be closer to the conditions of VHL patient analysis in clinical practice. Results presented in Table 1 (unitary plasmid) and Table 2 show that mosaic mutations were found at levels corresponding to the theoretical dilution.Table 1Proportion of Mutated VHL Event Detected by NGS on the PGM Platform for Each Individual Mutant Plasmid and for the Mixed iQc (Dilution, 1% to 2%)Eventc.194C>G p.(ser65Trp)c.226_228del p.(Phe76del)c.232A>C p.(Asn78His)c.341-1G>Cc.467A>G p.(Tyr156Cys)c.481C>T p.(Arg161*)c.482G>A p.(Arg161Gln)c.500G>A p.(Arg167Gln)Proportion of mutated event (unitary plasmid), %1.341.11.351.031.311.721.682.39P5.69 × 10−181.20 × 10−171.09 × 10−206.06 × 10−154.73 × 10−234.11 × 10−231.05 × 10−232.81 × 10−48Proportion of mutated event [mixed plasmids (iQc)], %1.412.31.541.411.851.731.121.02P2.79 × 10−178.63 × 10−171.99 × 10−109.21 × 10−186.07 × 10−332.30 × 10−234.21 × 10−164.10 × 10−15“Unitary plasmid” corresponds to plasmid individually analyzed (each had a different barcode); “mixed plasmids” corresponds to all plasmid mixed all together in the same sample (all had the same barcode).iQc, internal quality control; NGS, next-generation sequencing; PGM, Personal Genome Machine. Open table in a new tab Table 2SeqNext Analyses of Mutant Unitary PlasmidsEventTotal read number (forward + reverse) at the position of the eventNumber of reads for each nucleotide (forward + reverse) at the position of the eventProportion of the event, %ATCGc.194C>G p.(Ser65Trp)10,848581410,6311451.34c.226_228del p.(Phe76del)15,744180 reads with the deletion1.1c.232A>C p.(Asn78His)17,28617,0213233291.35c.341-1G>C6101366360291.03c.467A>G p.(Tyr156Cys)18,34818,107012401.31c.481C>T p.(Arg161*)72131124708711.72c.482G>A p.(Arg161Gln)66821121656901.68c.500G>A p.(Arg167Gln)21,84852331921,3032.39At each mutated position, the number of reads for each nucleotide is shown in the table. Open table in a new tab “Unitary plasmid” corresponds to plasmid individually analyzed (each had a different barcode); “mixed plasmids” corresponds to all plasmid mixed all together in the same sample (all had the same barcode). iQc, internal quality control; NGS, next-generation sequencing; PGM, Personal Genome Machine. At each mutated position, the number of reads for each nucleotide is shown in the table. Next, by mixing the eight mutated plasmids with the three wild-type plasmids, a 1% mosaic mutation–positive sample was generated, which simultaneously harbored the eight VHL mutations previously studied, each at a level between 1% and 2%. Results for mixed plasmids obtained on the PGM platform are shown in Table 1. The P values reflect the probability that the VHL event detected was significantly different from background noise. All mutations were detected in the sample at the expected levels with significant P values. This positive sample, after evaluation, could be used as an iQc during NGS runs to validate the assay, allowing for the assessment of both the detection and the detection level of each VHL mosaic mutation. Twelve patients were analyzed for VHL mosaic mutations using the PGM platform. Taking into account the 35 patients previously analyzed with the 454 platform, a total of 47 patients were analyzed for VHL mosaic mutations and tested negative for heterozygous VHL germline mutations (Table 3 and Supplemental Table S3). Twenty-three patients had suggestive VHL disease (one tumor), and 24 presented with typical VHL disease (two or more tumors, or one tumor with a familial context). Their clinical characteristics are described in Table 3 and Supplemental Table S3. Forty-six patients had sporadic VHL disease, and one patient had an affected offspring. A VHL mosaic mutation was identified in four patients (4/47, 8.5%). Patients with typical VHL disease corresponded to a VHL mosaicism frequency of 16.6% (4/24). The identified events were c.500G>A p.(Arg167Gln) (allelic ratio, 5.7%), c.481C>T p.(Arg161*) in two unrelated patients (same allelic ratio, 1.7%) and c.490C>T p.(Gln164*) (3.3%). One of them corresponds to the familial case of the cohort, and his daughter was heterozygous for the same mutation. No VHL mosaic mutation was identified in the other patients at the detection level of 1%. The four patients harboring a VHL mosaic mutation had a clinical VHL disease with a median number of lesions of 2.5, which is significantly higher than in patients without a mosaic mutation (median 1.0, P = 0.0058, U-test). Of interest, all patients with a unique hemangioblastoma were negative for VHL mosaic mutation.Table 3Clinical Characteristics of Patients Harboring a VHL Mosaic MutationSexDate of birthAge of first clinical manifestation, yearsFamilial historyPhenotypeNumber of tumorsClinical diagnosis of VHLEvent% Of mutated allele in blood identified by NGS% Of mutated allele in blood identified by ddPCR% Of mutated allele in tumor identified by ddPCRM199016NoHb, pheochromocytoma, pancreatic endocrine tumor, left adrenal nodule3Yesc.500G>A p.(Arg167Gln)5.75.534.6 (pheochromocytoma)M199416UnknownSeveral retinal Hb, renal cysts2Yesc.481C>T p.(Arg161*)1.71.9Tissue not availableM193948Yes†An affected daughter.Bilateral RCC2Yesc.481C>T p.(Arg161*)1.71.811.1 (right RCC)M198434NoCentral nervous system Hb, two RCC3Yesc.490C>T p.(Gln164*)3.33.5Tissue not availableM, male; ddPCR, droplet digital PCR; Hb, hemangioblastoma; RCC, renal clear cell carcinoma.† An affected daughter. Open table in a new tab M, male; ddPCR, droplet digital PCR; Hb, hemangioblastoma; RCC, renal clear cell carcinoma. In molecular diagnostics practice, the presence of a germline mutation should be validated before the result is reported to the physician who prescribed the molecular test. The confirmation of a mosaic event detected by NGS is possible either with another NGS technology or with another highly sensitive technique, such as SNaPshot or ddPCR. To evaluate these two techniques for the detection of mosaic mutation, each mutant plasmid was used to determine the limit of detection. SNaPshot was able to detect mutations until a threshold set at 5% (Table 4 and Figure 1A). The ddPCR technique was able to detect an event with a threshold of 1% (Table 4 and Figure 1B). For c.194C>G, p.(Ser65Trp) VHL mutation, no signal was detected by ddPCR with the wild-type probe, possibly due to technical issues during probe production.Table 4Proportion of VHL Mutated Event Detected by SNaPshot and ddPCR for Each Individual Plasmid MutantEventSNaPshotddPCRTheoretical proportion of mutated event, %10511051Observed proportion of mutated event (forward + reverse), %ThresholdThresholdc.194C>G p.(Ser65Trp)17.78.43ND5NDNDNDNDc.226_228del p.(Phe76del)7.83.37ND56.382.950.691c.232A>C p.(Asn78His)20.012.95.5<54.712.040.0531c.341-1G>C25.818.21ND513.616.81.011c.467A>G p.(Tyr156Cys)12.17.02ND521.297.991.191c.481C>T p.(Arg161*)13.2NDND>524.659.982.89" @default.
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• W2912454862 title "Optimization of Next-Generation Sequencing Technologies for von Hippel Lindau (VHL) Mosaic Mutation Detection and Development of Confirmation Methods" @default.
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