Enrichment Panel Research Articles

An enriched sugarcane diversity panel for utilization in genetic improvement of sugarcane

Sugarcane crop is important for both sugar and biofuels. A world collection of sugarcane and related grasses (WCSRG) maintained at Miami, FL contains > 1,200 non-redundant clones of different species and genera within the Saccharum complex. However, linkage of undesirable alleles with useful genes in wild species has hindered its efficient utilization in sugarcane breeding. A core collection developed previously with smaller number of clones representing WCSRG did not take into account > 120 wild/exotic clones maintained at the USDA-ARS Sugarcane Research Unit in Houma, Louisiana. Moreover, the genome complexity and sub-tropical to temperate growing climate of Louisiana warrant a region-specific core collection that can be used for base-broadening breeding aimed at efficient introgression of desirable alleles. Genetic diversity of 1,485 clones within WCSRG and Louisiana (commercials, wild/exotic) using 423 SSR alleles showed an average gene diversity (h) at 0.208 among all species groups where Erianthus-like Saccharum species (ELSS), Miscanthus spp., and S. spontaneum each formed a distinct cluster, Saccharum robustum, S. officinarum, hybrid cultivars, and S. edule grouped together in a major cluster, and Saccharum sinense and S. barberi formed distinct grouping. A 309-clone diversity panel (SDP1) was developed that captured the genetic diversity based on the combination of maximum length subtree and manual selection to maximize representation of Louisiana clones and minimize import of clones from Miami. SDP1 shared 324 alleles out of the 423 alleles in the entire population of 1,485 clones and captured the genetic diversity of the entire collection with an average gene diversity (h) at 0.163. The variation within (11–17%) and among (83–89%) the populations in SDP1 were comparable with the entire population of 1,485 clones (9–15% and 85–91%, respectively). The breadth of the genetic variation of SDP1 was exemplified by the intra- and inter-specific diversity of a 190-clone mini-core collection with markers derived from known cold-responsive genes. SDP1 will facilitate genome-wide association studies for identification of trait-specific markers for use in marker-assisted breeding in Louisiana and elsewhere.

Characteristics of genotype and phenotype in Chinese patients with Bardet-Biedl syndrome.

To investigate complex and different phenotypes in seven Chinese patients diagnosed with Bardet-Biedl syndrome (BBS) and carrying pathogenic mutations. Seven unrelated BBS patients were enrolled. Their medical and ophthalmic histories were reviewed, and comprehensive clinical examinations, such as fundus photography, optical coherence tomography, and medical imaging, were performed. A specific hereditary eye disease enrichment panel based on exome-capture technology was used to collect and amplify the protein-coding regions of 441 targeted hereditary eye disease genes, followed by high-throughput sequencing using the Illumina HiSeq platform. All patients exhibited the primary clinical phenotype of BBS. Seven BBS mutations were found in five patients (BBS7 in two patients, BBS10 in two patients, BBS12 in one patient), for a detection rate of 71% (5/7). The ratio of novel to known BBS mutations was 5:2. This study showed the phenotypic and genotypic spectrum of BBS patients from China, and the findings underscore the importance of obtaining comprehensive clinical observations and molecular analyses for ciliopathies.

Molecular correlation of the activity of evofosfamide (EVO) in combination with sunitinib (SUN) in pancreatic Neuroendocrine Tumors (pNETs) in the SUNEVO GETNE Trial.

e16706 Background: MEN1, DAXX, ATRX, and PI3K/AKT/mTOR pathway genes are frequently mutated in pNETs. Sunitinib is approved and widely used in the metastatic setting but unfortunately, no validated predictive biomarker has been identified to guide therapy yet. The SUNEVO phase II trial of the Spanish Task Force Group for Neuroendocrine and Endocrine Tumours (GETNE) trial combined the pro-drug of EVO under SUN-induced hypoxic conditions. Methods: We performed an exploratory analysis aimed to identify somatic mutations associated with the clinical benefit of pNET patients treated with SUN+EVO combination. Seventeen treatment-naïve pts with pNETS were included in the SUNEVO trial, and 4 (23.5%) achieved a partial response (by RECIST 1.1). Tumor DNA from 10 FFPE tumor samples were successfully sequenced with an enrichment panel (Nimblegen, Roche) including 42 cancer-related genes and TERT promoter region. Median coverage was 793x. Coding non-synonymous and loss-of-function variants were considered for the analysis. Final analysis included tumor samples from the 10 patients with NGS results. Results: Somatic mutations were found in MEN1 (70%), DAXX (30%), ATRX (20%), SETD2 (20%) and PTEN (10%). Mutations in the telomere maintenance genes DAXX and ATRX were concordant with mutual exclusivity. The loss of MEN1 was associated with a greater number of mutations (p = 0.019). Patients with complete and partial response showed heterogeneous genetic profiles. Conclusions: The molecular alterations of the patients in the SUNEVO trial were consistent with those previously described for metastatic pNETs. No clear association between molecular defects and treatment response was found. Clinical trial information: NCT02402062 .

Novel mutations of RPGRIP1 gene in a family with Leber congenital amaurosis

Objective To identify the pathogenic gene mutations in a family with Leber congenital amaurosis (LCA). Methods In October 2018, 1 patient and 3 normal family members from a LCA family was enrolled in this retrospective study. Detailed medical history of proband was obtained and fixation test, cycloplegic refraction, slit-lamp, fundus color photography and full-field ERG were performed. And other family members underwent BCVA, refraction slit-lamp, fundus biomicroscopy with the slit lamp, fundus color photography and full-field ERG. The family was investigated with a specific hereditary eye disease enrichment panel which contained 441 known pathogenic genes and based on targeted exome capture technology first to indentify the potential pathogenic genes and mutations. Then the potential pathogenic mutations were conformed by Sanger sequencing. Finally, the results were analyzed via bioinformatics analysis. Results The proband showed no trace object from childhood, but had obvious photophobia and nystagmus. No positive changes were found in the anterior segment, vitreous and retina in both eyes. Both cone and rod system function decreased significantly in full-field ERG in both eyes. Gene tests showed the proband carried both RPGRIP1 c.1635dupA and c.3565C> T, which composited a heterozygous mutation. Bioinformatics analysis showed RPGRIP1 c.1635dupA was a pathogenic mutation, and RPGRIP1 c.3565C> T which was a novel potential pathogenic mutation in LCA. Conclusion The compound heterozygous mutation, c.1635dupA and c.3565C> T in RPGRIP1 may be responsible for the pathogenesis in this Chinese Han LCA pedigree. Key words: Leber congenital amaurosis/etiology; Genes; Mutation; RPGRIP1 gene

Simultaneous expression of two pathogenic genes in four Chinese patients affected with inherited retinal dystrophy.

To describe the complex, overlapping phenotype of four Chinese patients with inherited retinal dystrophies (IRDs) who harbored two pathogenic genes simultaneously. This retrospective study included 4 patients affected with IRDs. Medical and ophthalmic histories were obtained, and clinical examinations were performed. A specific Hereditary Eye Disease Enrichment Panel (HEDEP) based on exome capture technology was used for genetic screening. Four patients were identified to harbor disease-causing variants in two different genes. Patient retinitis pigmentosa (RP) 01-II:1 exhibited both classical ABCA4-induced Stargardt disease (STGD) 1 and USH2A-associated RP, patient RP02-III:2 exhibited both classical ABCA4-induced STGD1 and CDH23-associated RP, patient RP03-II:1 exhibited both USH2A-induced autosomal recessive retinitis pigmentosa (arRP) syndrome and SNRNP200-induced autosomal dominant retinitis pigmentosa (adRP), and patient RP04-II:2 exhibited USH2A-induced arRP syndrome and EYS-induced arRP at the same time. Our study demonstrates that genotype-phenotype correlations and comprehensive genetic screening is crucial for diagnosing IRDs and helping family planning for patients suffering from the disease.

Clinical and Genetic Characterization of a Constitutional Delay of Growth and Puberty Cohort

Introduction: Constitutional delay of growth and puberty (CDGP) is the most prevalent cause of delayed puberty in both sexes. Family history of delayed puberty (2 or more affected members in a family) has been evidenced in 50–75% of patients with CDGP and the inheritance is often consistent with autosomal dominant pattern, with or without complete penetrance. However, the molecular basis of CDGP is not completely understood. Objective: To characterize the clinical and genetic features of a CDGP cohort. Methods: Fifty-nine patients with CDGP (48 boys and 11 girls) underwent careful and long-term clinical evaluation. Genetic analysis was performed using a custom DNA target enrichment panel designed to capture 36 known and candidate genes implicated with pubertal development. Results: All patients had spontaneous or induced pubertal development (transient hormonal therapy) prior to 18 years of age. The mean clinical follow-up time was 46 ± 28 months. Male predominance (81%), short stature (91%), and family history of delayed puberty (59%) were the main clinical features of this CDGP ­cohort. Genetic analyses revealed 15 rare heterozygous missense variants in 15 patients with CDGP (25%) in seven different genes (IGSF10, GHSR, CHD7, SPRY4, WDR11, SEMA3A,and IL17RD). IGSF10 and GHSR were the most prevalent affected genes in this group. Conclusions: Several rare dominant variants in genes implicated with GnRH migration and metabolism were identified in a quarter of the patients with familial or sporadic CDGP, suggesting genetic heterogeneity in this frequent pediatric condition.

Liquid Biopsy Is Useful to Identify the Genetic Profile of NHL-B at Diagnosis in Different Histological Subtypes

Introduction: Non-Hodgkin B lymphomas (NHL-B) are a large group of heterogeneous diseases, with well-defined, histological and clinical features. Currently, the lymph node biopsy is essential for diagnosis, often obtained from hard to reach areas. Our aims with this study was to analyze genetics variants at diagnosis trying to discriminate between different NHL-B histologic subtypes using formalin fixed paraffin embedded (FFPE) tissue sections and circulating tumor free DNA (ctDNA) samples. Material and Methods: This is a retrospective, cross-sectional, single-center study. Sixty patients were selected with a diagnosis of different NHL-B subtypes: Diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal lymphoma (ML), mantle cell lymphoma (MCL) and plasmablastic lymphoma (PL). Sixty FFPE tissue samples and 23 ctDNA specimens obtained at diagnosis were selected. We performed an enrichment panel of 54 genes recurrently mutated in lymphomas (Lymphoma Solution; Sophia Genetics) by next generation sequencing (NGS; NextSeq; Illumina). The depth of 90% of the readings was greater than 2600x. Quantitative variables were expressed as median and range. Categorical variables were expressed as frequency and percentage. The Fisher exact test was used to compare the distribution of categorical variables. The Mann-Whitney test was used to compare differences between quantitative variables. Statistical significance was set at p < 0.05. Results: Our study shows that 97% (58/60) of patients presented any variant. The most frequently mutated genes were KMT2D, EP300 and SOCS1. Exclusive variants were detected in FL in the genes CCND1, PAX5, CREBBP, BCL2 and REL genes. In the same way, in DLCBL the variants detected in the BRAF, TCF3, XPO1, PIM1, CHD2, ID3 and BCL6 genes were exclusive of this subtype. Furthermore, the genes BCL2 (p=0.0021), CREBBP (p=0.0004), NOTCH2 (p=0.03), PAX5 (p=0.01) and TNFRSF14 (p=0.04) are more frequently altered in patients with FL, ATM (p=0.02) gene in MCL; NFKBIE (p=0.03), CIITA (p=0.02), MYC (p=0.009) and PIM1 (p=0.04) in DLCBL, among which it was found that high-grade lymphomas are more frequently associated with mutations in CHD2 (p=0.02), MYC (p=0.03) and MYD88 (p=0.02; data not shown). In the subgroup of 23 patients with paired samples (FFPE/ctDNA), 95% (22/23) of patients had mutations in any of the genes on the panel in FFPE tissue samples and 82% (19/23) in ctDNA specimens. The number of variants detected was different depending on the type of sample analysed, 52% of the variants were detected in both specimens, 39 only in FFPE tissue and 9% in ctDNA; Figure 1). In variants detected in both samples, the average value of the VAF was higher that when is detected only in one of the samples (FFPE: 28.1 vs 5.8 (p<0.00001); ctDNA: 8.7 vs. 2.3 ((p<0.0009); Figure 2). Patients with MCL presented lower number of variants and primary mediastinal lymphoma presented higher number of variants compared with the others subtypes (Table 1). We did not find significant differences in the number of variants depending on the stage, classification, tumour burden or bulky mass (Table 1). Conclusion: In our study, we were able to identify genetic variables that could characterize different histological groups of NHL-B in FFPE tissue and ctDNA samples, by using a commercial gene panel of NGS. The mutational profile obtained from ctDNA and FFPE tissue is comparable in all histological subtypes, regardless tumour burden, aggressiveness or stage, throwing each one of them complemental information. These results support the hypothesis that could be possible to distinguish different histologies through non-invasive strategies, specially oriented to lesions where obtaining a tissue sample is difficult. We are working on the development of clinical-genetic algorithms that allow us to achieve our goal. Disclosures No relevant conflicts of interest to declare.

Novel mutations associated with autosomal-dominant congenital cataract identified in Chinese families.

As the leading cause of impaired vision, congenital cataracts, particularly autosomal dominant congenital cataract (ADCC), have been considered as a hereditary disease. The present study aimed to identify genetic defects in Chinese pedigrees with ADCC. A total of 6 Chinese families with ADCC were included, comprising 103 members and 27 patients assessed in total. Genomic DNA samples were extracted from the peripheral blood of probands; mutations were determined using a specific eye disease enrichment panel with next-generation sequencing. Following pathogenicity prediction, sites with notable pathogenicity were screened for further validation. Sanger sequencing was performed in the remaining individuals of the families and 100 normal controls. The pathogenic effects of the mutations, including amino acid substitutions, as well as structural and functional alterations of proteins linked to ADCC, were investigated via bioinformatics analysis. A total of seven mutations in six candidate genes associated with ADCC were identified in the 6 families: Myosin heavy chain 9 (MYH9) c.4150G>C, β-crystallin A4 (CRYBA4) c.169T>C, RPGR-interacting protein 1 (RPGRRIP1) c.2669G>A, wolframin (WFS1) c.1235T>C, CRYBA4 c.26C>T, Ephrin receptor subfamily 2 (EPHA2) c.2663+1G>A and paired box 6 (PAX6) c.11-2A>G. The seven mutations were only detected in affected individuals. Among them, there were three novel mutations (MYH9: c.4150G>C; CRYBA4: c.169T>C; RPGRRIP1: c.2669G>A) and four previously reported ones. Mutations in RPGRIP1 (c.2669G>A) and CRYBA4 (c.26C>T) were predicted to be benign according to bioinformatics analysis. Conversely, other mutations in EPHA2, PAX6, MYH9, CRYBA4 (c.169T>C) and WFS1 were determined to be pathogenic. The present study reported two novel heterozygous mutations (MYH9 c.4150G>C and CRYBA4 c.169T>C) identified by analyzing 6 Chinese families with ADCC, supporting their important roles in the development of the disease.

PS942 CD19 MUTATION FREQUENCY AFTER BLINATUMOMAB TREATMENT IS LOWER THAN AFTER CAR T‐CELLS TREATMENT

Background:A significant antileukemic activity of CD19‐directed T‐cell immunotherapies, namely chimeric antigen receptor (CAR) T‐cells and Blinatumomab (Blin), has been demonstrated in advanced B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL). However, despite high response rate, up to 30% of patients develop relapse with loss of CD19 antigen on leukemic cells (Ruella, JCI, 2016). Due to the novelty of this escape mechanism, only few studies investigating its genetic cause have been published so far.Aims:Our aim was to identify somatic mutations acquired at the time of CD19‐negative relapse in patients after Blin treatment, employing exome sequencing of remission (rem), screening (scr) and relapse (rel) samples.Methods:Among patients with relapse/refractory or minimal residual disease (MRD)‐positive BCP‐ALL treated within MT103–203, MT103–206, MT103–211, and TOWER trials, 17 patients suffering a CD19‐negative relapse with sufficient DNA available were identified. Rem, scr and rel samples (median infiltration at scr 40%, range 6–95%; at rel 63%, 6–95%) from these patients were analysed with Nextera Exome Enrichment or IDT xGEN Exome Panel on an Illumina HiSeq 4000 sequencer with 2x 75 bp reads and 6 samples per sequencing lane, resulting in 80X‐90X on‐target average coverage. Two scr and five rel samples with IG/TR‐based MRD below 10% were sequenced with double coverage. Somatic variant calling was done by the bcbio‐nextgen software. Variants with abundance over 1/5 of the sample infiltration were marked as dominant (dom), otherwise marked as subclonal (sub).Results:Mutations of the CD19 gene affecting the final protein were identified in only 5/17 (29.4%) patients (Table 1). Two of the mutations were frameshift variants producing a premature termination codon, 2 were nonsense variants, and 1 of them was located in the splicing region. In 4/5 patients loss of heterozygosity was present in the CD19 region, leaving no functional allele. The remaining patient showed inconclusive results, likely due to a very low leukemic infiltration (9%).We further assessed all somatic variants present in the rel sample but not in the scr sample (i.e. when <5 alternative reads). On average, 10 (range 1–40) dom mutations and 7 (range 0–29) sub mutations were identified per patient. The difference between samples with and without a CD19 mutation was not significant. No mutation was shared among different patients and only ABCE1 and KMT2D genes were recurrently mutated – each of them in 2 different patients without a CD19 mutation.Summary/Conclusion:Mutations with an effect on CD19 protein were identified in less than 1/3 of patients in our cohort. In contrast to our findings, a recent study identified truncating CD19 mutations in 12/12 patients with a CD19‐negative relapse after CAR T‐cell treatment (Orlando, Nature Medicine, 2018). This difference could be attributed to a stronger selection pressure of CAR T‐cell treatment compared to Blin, where also lower incidence of CD19‐negative relapses has been reported (Ruella, Comput Struct Biotechnol J., 2016).imageA genetic explanation for the loss of CD19 in the remaining patients (n = 12) of our cohort is currently lacking. Clustering of all mutations into pathways, as well as analysis of a control cohort of patients with relapse on standard of care chemotherapy are ongoing with the aim to identify other molecular pathways responsible for the CD19 loss. RNA is not available for this retrospective cohort of patients, therefore studying alternative splicing via transcriptome analysis is not possible.

A customized nuclear target enrichment approach for developing a phylogenomic baseline for Dioscorea yams (Dioscoreaceae).

PremiseWe developed a target enrichment panel for phylogenomic studies of Dioscorea, an economically important genus with incompletely resolved relationships.MethodsOur bait panel comprises 260 low‐ to single‐copy nuclear genes targeted to work in Dioscorea, assessed here using a preliminary taxon sampling that includes both distantly and closely related taxa, including several yam crops and potential crop wild relatives. We applied coalescent‐based and maximum likelihood phylogenomic inference approaches to the pilot taxon set, incorporating new and published transcriptome data from additional species.ResultsThe custom panel retrieved ~94% of targets and >80% of full gene length from 88% and 68% of samples, respectively. It has minimal gene overlap with existing panels designed for angiosperm‐wide studies and generally recovers longer and more variable targets. Pilot phylogenomic analyses consistently resolve most deep and recent relationships with strong support across analyses and point to revised relationships between the crop species D. alata and candidate crop wild relatives.DiscussionOur customized panel reliably retrieves targeted loci from Dioscorea, is informative for resolving relationships in denser samplings, and is suitable for refining our understanding of the independent origins of cultivated yam species; the panel likely has broader promise for phylogenomic studies across Dioscoreales.

OR17-5 Clinical and Genetic Features of Constitutional Delay of Growth and Puberty

Constitutional delay of growth and puberty (CDGP) is the most prevalent cause of delayed puberty in both sexes. It has a strong family history, however, its genetic basis is still poorly understood. Objective: To characterize the clinical and genetic features of a CDGP cohort. Methods: 59 unrelated probands with CDGP were selected. They underwent careful and long-term clinical evaluation. Genetic analysis was performed using a custom DNA target enrichment panel (NextSeq 500 next-generation sequencing, Illumina Inc) designed to capture 36 known and candidate genes implicated with delayed puberty. Results: All CDGP patients had spontaneous pubertal development or it was induced by a transient sexual steroid replacement. Male predominance (81%), short stature (91%) associated with delayed bone age and positive family history (59%) were the major clinical features of this cohort. Genetic analyses revealed 16 rare heterozygous predicted to be deleterious variants in 15 CDGP patients (25%) in 8 distinct genes (HS6ST1, IGSF10, GHSR, CHD7, SEMA3A, IL17RD, SPRY4 and WDR11). IGSF10 and GHSR were the most prevalent affected genes in this group (3 variants in 4 patients each one). The vast majority of variants were missense. We identified a stop-gain variant, likely pathogenic, in HS6ST1 gene (p.Cys27X) in a male proband with no family history of delayed puberty. 5% of the subjects had oligogenicity (variants in more than one gene). Conclusions: The classic phenotype of CDGP has as clinical features short stature in the prepubertal period, higher prevalence in males and more than a half of the individuals had a positive family history. Genetic heterogeneity was demonstrated in typical CDGP phenotype.

Comprehensive data analysis for development of custom qRT-PCR miRNA assay for glioblastoma: a prevalidation study.

Glioblastoma (GB) is one notable example of miRNA-modulated neoplasms. Given its unique expression signature, proper miRNA profiling can help discriminate between GB and other types of brain tumors. The current work aimed to develop a more GB-specific and applicable custom designed quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) miRNA assay. A comprehensive data analysis of bioinformatics databases, previous literature and commercially available pre-designed miRNA PCR arrays within the market. A highly enriched panel of 84 deregulated and GB-specific miRNAs has been developed. After validation of this newly developed array, it can not only save the researcher's time and effort, but can also have a potential diagnostic and/or prognostic role in GB, paving the road toward personalized medicine.

Slip-stimulated grain boundary sliding in Ti-6Al-4 V at room temperature

Grain boundary sliding has a substantial contribution to high temperature deformation of α/β titanium alloys. Despite the low homologous temperature, the operation of grain boundary sliding is presently reported at room temperature using a pre-deformed Ti-6Al-4 V specimen. Scanning electron microscopy and atomic force microscopy observations were combined with electron back-scattered diffraction characterization in order to clarify the underlying mechanisms. Intragranular prismatic slip seemingly triggers grain boundary sliding. The analysis of activated slip systems while accounting for crystallographic aspects unveiled several highly influential parameters. Notably, strain partitioning, as a result of plastic anisotropy and microstructural heterogeneities, promotes grain boundary sliding. The orientation of the incident slip band relative to the boundary plane was also found correlated with the occurrence of grain boundary sliding. These findings lay the foundations for identification of conditions favoring grain boundary sliding in titanium alloys and highlight an enriched panel of possible interactions between slip bands and grain boundaries that is likely to play a critical role in crack initiation processes.

Application of Whole Exome and Targeted Panel Sequencing in the Clinical Molecular Diagnosis of 319 Chinese Families with Inherited Retinal Dystrophy and Comparison Study.

Inherited retinal dystrophies (IRDs) are a group of clinically and genetically heterogeneous diseases involving more than 280 genes and no less than 20 different clinical phenotypes. In this study, our aims were to identify the disease-causing gene variants of 319 Chinese patients with IRD, and compare the pros and cons of targeted panel sequencing and whole exome sequencing (WES). Patients were assigned for analysis with a hereditary eye disease enrichment panel (HEDEP) or WES examination based on time of recruitment. This HEDEP was able to capture 441 hereditary eye disease genes, which included 291 genes related to IRD. As RPGR ORF15 was difficult to capture, all samples were subjected to Sanger sequencing for this region. Among the 163 disease-causing variants identified in this study, 73 had been previously reported, and the other 90 were novel. Genes most commonly implicated in different inheritances of IRDs in this cohort were presented. HEDEP and WES achieved diagnostic yield with 41.2% and 33.0%, respectively. In addition, nine patients were found to carry pathogenic mutations in the RPGR ORF15 region with Sanger sequencing. Our study demonstrates that HEDEP can be used as a first-tier test for patients with IRDs.

Two rare loss-of-function variants in the STAG3 gene leading to primary ovarian insufficiency

Background/AimPrimary ovarian insufficiency (POI) is characterized by primary or secondary amenorrhea, infertility, low estradiol levels, and increased gonadotropin levels. Most cases of POI remain unsolved even after exhaustive investigation. Here, we performed a targeted massively parallel sequencing to identify the genetic diagnosis of primary ovarian insufficiency (POI) in a Brazilian patient. Patient and methodsAn adopted 21-year-old Brazilian woman with isolated POI was selected. A custom SureSelectXT DNA target enrichment panel was designed and sequenced on an Illumina NextSeq 500 sequencer. The variants were confirmed using Sanger sequencing. ResultsTwo rare heterozygous pathogenic variants in the STAG3 gene were identified in our patient. An unpublished 1-bp duplication c.291dupC (p.Asn98Glnfs*2) and one stop codon variant c.1950C > A (p.Tyr650*) were identified in the STAG3 gene. Both undescribed heterozygous variants were absent in the public databases [1000Genomes, Exome Aggregation Consortium (ExAC), National Heart, Lung, and Blood Institute Exome Variant Server (NHLBI/EVS), database of Single Nucleotide Polymorphisms (dbSNP), Genome Aggregation Database (gnomAD)], and Online Archive of Brazilian Mutations (ABraOM) databases. Moreover, neither heterozygous variants were found in 400 alleles from fertile Brazilian women screened by Sanger sequencing. The parents' DNA was not available to segregate these variants. ConclusionOur results suggested that POI is caused by pathogenic compound heterozygous variants in the STAG3 gene, supporting the key role of the STAG3 gene in the etiology of primary ovarian insufficiency.

Abstract 580: A method for estimating mutation load from tumor research samples using a targeted next-generation sequencing panel

Abstract Introduction: Tumor mutation load predicts durable benefit from immune checkpoint inhibitors in several cancer types. Existing methods to estimate tumor mutation load have large input DNA and extensive infrastructure requirements and are associated with delays due to shipping precious biopsy samples to central laboratories. Herein, we demonstrate the ability of a targeted panel with fast turn-around time to estimate mutation load from tumor research samples using low input. Methods: We developed a single sample analysis workflow to estimate mutation load (mutation count per megabase) from FFPE and fresh frozen tumor research samples. The assay utilizes a PCR-based target enrichment panel that covers 409 genes and 1.7 Mb of genomic regions. The workflow requires only 10 ng of input DNA, and enables a 2.5-day turn-around time from sample to the final report. The workflow enables < 60 minutes of hands-on time for automated library preparation and templating on a batch of 8 samples. Sequencing is performed on high throughput semiconductor sequencing platform to achieve sufficient depth (~500x coverage) and accuracy. Our analysis pipeline calls variants at ≥5% allelic frequency with parameters optimized for high accuracy. The workflow is tumor sample only, with no matched normal sample required, and germ-line variants, along with background noise, are removed through application of filters based on variants prevalent in population databases. Results: In-silico analysis demonstrated that the predicted mutation counts associated with the covered regions of the targeted panel could effectively stratify responders and non-responders to immune checkpoint inhibitors in 3 separate cohorts with high statistical significance. The assay was applied to colorectal specimens previously typed for microsatellite instability (MSI) and distinguished samples that were MSI-H (median mutation count 94.5) and MSS (median mutation count 17.8). Matched tumor-normal analyses on 7 colorectal tumor samples showed that the single tumor analysis workflow detected somatic mutations with strong correlation (r = 0.97) with tumor-normal analysis. To assess reproducibility, we compared somatic mutation count in 6 library replicates of breast cancer cell line HCC1143 and observed low relative variability (CV=9.57%) with 19.17 average mutation count. The analysis report characterizes substitution type and context of somatic mutations, and highlights mutation signatures consistent with UV damage and spontaneous deamination of 5-methyl-cytosine, as well as FFPE deamination. Conclusions: We developed a simple analysis workflow on the Ion Torrent sequencing platform with an AmpliSeq panel to estimate mutation load from FFPE and fresh frozen tumor research samples. This solution will advance research in immuno-oncology. Citation Format: Ruchi Chaudhary, Dinesh Cyanam, Vinay Mittal, Warren Tom, Janice Au-Young, Seth Sadis, Fiona Hyland. A method for estimating mutation load from tumor research samples using a targeted next-generation sequencing panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 580.

Abstract 1421: Flexible tools for the development and performance verification of customized target enrichment panels

Abstract Implementation of next generation sequencing for variant identification and discovery presents difficulties both in the selection of genomic loci for inclusion in a panel, as well as the ability to effectively verify the performance of the assay based on the content selected. Balancing the relevance of genomic content with the practical hurdles of developing specific assays necessitates new approaches for the application of next generation sequencing toward precision medicine that are aimed at providing the flexibility of content to efficiently provide data capable of testing fundamental biological hypotheses. NEBNext Direct Custom Ready Panels overcome these obstacles by enabling the rapid development and deployment of customized target enrichment panels. This offering allows researchers to select from a large library of genes for which baits have been developed and optimized to produce sequencing data with extremely high specificity and coverage uniformity. These panels are effective in targeting a wide range of content, from highly focused, single-gene panels, to comprehensive panels comprised of hundreds of genes. In a similar approach for the verification and validation of new panels, SeraCare has developed the VariantFlex™ system, which is comprised of a broad library of mutations to develop custom controls and reference materials for NGS-based assays. These biosynthetic constructs contain variants such as SNVs, indel, frameshift and fusion variants that can be used to formulate controls across a range of variant allele frequencies, providing the necessary content and flexibility to verify assay performance. Here we will present data demonstrating the combined utility of these offerings to assess target enrichment panel performance with a range of genomic content, panel sizes and variant allele frequencies. Citation Format: Cynthia L. Hendrickson, Evan Mauceli, Andrew Barry, Kruti Patel, Catherine Huang, Scott Adams, Bharathi Anekella. Flexible tools for the development and performance verification of customized target enrichment panels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1421.

Estimating mutation load at 5% LOD from FFPE samples using a targeted next-generation sequencing assay.

28 Background: High tumor mutation load is a biomarker which positively correlates with response to immune checkpoint inhibitors. Current methods to estimate tumor mutation load often require large amounts of DNA. Herein, we demonstrate the ability of a targeted panel to estimate mutation load from FFPE samples using low input amount of DNA. Methods: We developed a single-sample analysis workflow to estimate mutation load (somatic mutation count per Megabase (Mb)) from FFPE and fresh frozen tumor research samples. The assay utilizes a PCR-based target enrichment panel that covers ~1.7 Mb. Our workflow requires only 10 ng of input DNA, and enables a 2.5-day turn-around time from sample to the final report. The workflow enables < 60 minutes of hands-on time for automated library preparation and templating on a batch of 8 samples. Sequencing is performed using a high throughput semiconductor sequencing platform to achieve sufficient depth (~500x coverage) and accuracy. Our analysis pipeline calls variants with optimized parameters on the tumor sample only, with no matched normal sample required, and applies filters to remove germ-line. Results: An in-silico analysis using 21,000 exomes from COSMIC demonstrated the panel can achieve high sensitivity (>=93%) and specificity (>99%) necessary to stratify high and low mutation burden samples. Matched tumor-normal analyses on 14 lung and colorectal samples showed that our single tumor analysis workflow detects mutation load with strong correlation (r=0.8699) with tumor-normal analysis. Through dilution experiments on engineered control we learned that the workflow provides accurate estimates of mutation load, predicting ± 7% of expected mutation load on >=20% tumor content. To assess reproducibility, we compared mutation load in library replicates for a cohort of 8 samples (FFPE and fresh frozen tumors, engineered control, and NIST cell-lines) and observed high correlation (r=0.9906) among replicates. Conclusions: We developed a simple workflow on the Ion Torrent sequencing platform to estimate mutation load from FFPE and fresh frozen tumor research samples. This solution will advance research in immuno-oncology.

Identification of the first homozygous 1‐bp deletion in GDF9 gene leading to primary ovarian insufficiency by using targeted massively parallel sequencing

Targeted massively parallel sequencing (TMPS) has been used in genetic diagnosis for Mendelian disorders. In the past few years, the TMPS has identified new and already described genes associated with primary ovarian insufficiency (POI) phenotype. Here, we performed a targeted gene sequencing to find a genetic diagnosis in idiopathic cases of Brazilian POI cohort. A custom SureSelectXT DNA target enrichment panel was designed and the sequencing was performed on Illumina NextSeq sequencer. We identified 1 homozygous 1-bp deletion variant (c.783delC) in the GDF9 gene in 1 patient with POI. The variant was confirmed and segregated using Sanger sequencing. The c.783delC GDF9 variant changed an amino acid creating a premature termination codon (p.Ser262Hisfs*2). This variant was not present in all public databases (ExAC/gnomAD, NHLBI/EVS and 1000Genomes). Moreover, it was absent in 400 alleles from fertile Brazilian women screened by Sanger sequencing. The patient's mother and her unaffected sister carried the c.783delC variant in a heterozygous state, as expected for an autosomal recessive inheritance. Here, the TMPS identified the first homozygous 1-bp deletion variant in GDF9. This finding reveals a novel inheritance pattern of pathogenic variant in GDF9 associated with POI, thus improving the genetic diagnosis of this disorder.

Abstract 21281: Cardiosphere-Derived Cell and Mesenchymal Stem Cell Extracellular Vesicles Contain Distinct RNA Cargo

Introduction: Cardiosphere-derived cells (CDCs) possess regenerative, immunomodulatory and cardioprotective characteristics when delivered to the heart post-myocardial infarction (MI). CDCs derived from different donors possess variable regenerative potency in a MI model. We previously demonstrated that human CDC-derived extracellular vesicles (CDC-EVs) recapitulate the effects of CDCs in acute and chronic in vivo MI models. We are testing the hypothesis that a distinct cargo profile defines the functional efficacy between CDC-EVs and mesenchymal stem cell-derived EVs (MSC-EVs). Also, we assessed the immunomodulatory role of CDC-EVs derived from potent and non-potent CDCs in comparison with MSC-EVs. Methods: CDCs or MSCs were cultured in serum-free medium for 15 days and the conditioned media was concentrated by ultrafiltration to isolate EVs. The miRNeasy Serum/Plasma kit was used to extract total RNA from EVs followed by small RNA sequencing (NextSeq 500, Illumina) (CDC-EVs, n =12; MSC-EVs, n =4). We performed an in vitro and in vivo macrophage assay to assess the immunomodulatory role of EVs. Results: RNA sequencing analysis of CDC-EVs and MSC-EVs revealed a greater overall abundance of Y-RNA fragments and miRNA in CDC-EVs. Examining the specific Y-RNA classes, we found that MSC-EVs contained an expression profile with lower Y4 (p<0.05) but higher Y5 (p<0.05) Y-RNA fragments. Four miRNAs (miR-146a, miR-151, miR-409, miR-423) were highly enriched in CDC-EVs while miR-10a and miR-4792 were more abundant in MSC-EVs. EVs from potent CDCs showed a stronger upregulation of anti-inflammatory genes in activated macrophages ( in vitro ) and prevented accumulation of macrophages ( in vivo ) when compared with EVs obtained from non-potent CDCs or MSCs. Conclusions: We demonstrated that CDC-EVs contain a unique RNA cargo set that can differentiate CDC-EVs from MSC-EVs. The higher presence of Y-RNA in CDC-EVs as compared to MSC-EVs could be responsible for the ability of CDCs to regulate stem cell activation and tissue regeneration. The highly enriched panel of miRNAs observed in CDC-EVs and the ability to reduce the inflammatory reaction by macrophages, in contrast to MSC-EVs, suggest that they may support some of the functional benefits observed post-MI.