Single Molecule Molecular Inversion Probes Research Articles

Development of a Genotype Assay for Age-Related Macular Degeneration: The EYE-RISK Consortium

To develop a genotype assay to assess associations with common and rare age-related macular degeneration (AMD) risk variants, to calculate an overall genetic risk score (GRS), and to identify potential misdiagnoses with inherited macular dystrophies that mimic AMD. Case-control study. Individuals (n= 4740) from 5 European cohorts. We designed single-molecule molecular inversion probes for target selection and used next generation sequencing to sequence 87 single nucleotide polymorphisms (SNPs), coding and splice-site regions of 10 AMD-(related) genes (ARMS2, C3, C9, CD46, CFB, CFH, CFI, HTRA1, TIMP3, and SLC16A8), and 3 genes that cause inherited macular dystrophies (ABCA4, CTNNA1, and PRPH2). Genetic risk scores for common AMD risk variants were calculated based on effect size and genotype of 52 AMD-associated variants. Frequency of rare variants was compared between late AMD patients and control individuals with logistic regression analysis. Genetic risk score, association of genetic variants with AMD, and genotype-phenotype correlations. We observed high concordance rates between our platform and other genotyping platforms for the 69 successfully genotyped SNPs (>96%) and for the rare variants (>99%). We observed a higher GRS for patients with late AMD compared with patients with early/intermediate AMD (P < 0.001) and individuals without AMD (P < 0.001). A higher proportion of pathogenic variants in the CFH (odds ratio [OR] = 2.88; P= 0.006), CFI (OR = 4.45; P= 0.005), and C3 (OR = 6.56; P= 0.0003) genes was observed in late AMD patients compared with control individuals. In 9 patients, we identified pathogenic variants in the PRPH2, ABCA4, and CTNNA1 genes, which allowed reclassification of these patients as having inherited macular dystrophy. This study reports a genotype assay for common and rare AMD genetic variants, which can identify individuals at intermediate to high genetic risk of late AMD and enables differential diagnosis of AMD-mimicking dystrophies. Our study supports sequencing of CFH, CFI, and C3 genes because they harbor rare high-risk variants. Carriers of these variants could be amendable for new treatments for AMD that currently are under development.

Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics

PurposeMissing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. MethodsSequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. ResultsIn 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. ConclusionDeep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.

THU0026 CLONAL HAEMATOPOIESIS ASSOCIATED SOMATIC MUTATIONS IN RHEUMATOID ARTHRITIS

Background:Clonal haematopoiesis of indeterminate potential (CHIP) occurs when somatic mutations arise in myeloid neoplasia driver genes of haematopoietic progenitor cells, in the absence of overt cytopenia or dysplasia. The prevalence of CHIP increases with age. The most common genes affected by CHIP mutations in unselected populations are DNMT3A, ASXL1, and TET2. The presence of CHIP is linked to increased basal level of inflammation and a high risk of cardiovascular disease and all-cause mortality. Rheumatoid arthritis (RA) is one of the most common and debilitating multi-system autoimmune disorders, affecting up to 1% of adults in developed countries. The role of somatic mutations in the pathogenesis of autoimmune diseases is an unexplored area; therefore, we aimed to test the hypothesis that clonal haematopoiesis (CH) is associated with the incidence and severity of RA.Objectives:To evaluate the association of CH somatic mutation with severity of RA.Methods:163 RA patients were recruited from the following cohorts: (i)Early RA/treatment naive (n=31), (ii)Refractory RA - non-responders to Disease-Modifying Anti-Rheumatic Drugs (DMARDs) and biologics (n=48), (ii)Flare (n=41) vsRemission patients (n=43) –patients treated with DMARDs and withdrawn from treatment on achieving remission. Six months later, 50% relapse and 50% sustain remission. Single molecule molecular inversion probes (smMIPs) were used to screen for somatic mutations in 40 loci known to carry clonal haematopoiesis driver mutations (CHDMs). Whole exome sequencing was also performed on Flare/Remission patients (n = 84) to screen for CHDMs and other somatic mutations. In-house bioinformatics pipelines were used to call mutations from both the datasets.Results:We identified CH in RA with an overall prevalence of 14%. Twenty-four unique variants with a variant allele frequency (VAF) of 2-35% were found in ten genes including ASXL1, CBL, DNMT3A, GNAS, GNB1, PTPN11, PTPN12, SF3B1, TET2, and TP53. The number of unique patients carrying mutations in these genes are follows:refractory: n=12/48, flare: n=6/41,remission: n=4/43 andearly RA: n=2/31. The majority of the mutations occurred in DNMT3A (n=6) followed by TP53 (N=4) and TET2 (n=3). Two variants with VAF of 15% were identified in two patients under the age of 30, both with clinically severe disease. In patients between the ages of 50-59 yrs., 60-69 yrs., and 70-79 yrs., CH was observed at 11% (4/35), 23% (11/46) and 17%(7/41), respectively.Conclusion:We here report the prevalence of CH in RA, affecting more patients with clinically advanced/refractory disease compared to those with early/less severe disease.Further study will be conducted to confirm the results.

Comprehensive routine diagnostic screening to identify predictive mutations, gene amplifications, and microsatellite instability in FFPE tumor material

BackgroundSensitive and reliable molecular diagnostics is needed to guide therapeutic decisions for cancer patients. Although less material becomes available for testing, genetic markers are rapidly expanding. Simultaneous detection of predictive markers, including mutations, gene amplifications and MSI, will save valuable material, time and costs.MethodsUsing a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach, we developed an NGS panel allowing detection of predictive mutations in 33 genes, gene amplifications of 13 genes and microsatellite instability (MSI) by the evaluation of 55 microsatellite markers. The panel was designed to target all clinically relevant single and multiple nucleotide mutations in routinely available lung cancer, colorectal cancer, melanoma, and gastro-intestinal stromal tumor samples, but is useful for a broader set of tumor types.ResultsThe smMIP-based NGS panel was successfully validated and cut-off values were established for reliable gene amplification analysis (i.e. relative coverage ≥3) and MSI detection (≥30% unstable loci). After validation, 728 routine diagnostic tumor samples including a broad range of tumor types were sequenced with sufficient sensitivity (2.4% drop-out), including samples with low DNA input (< 10 ng; 88% successful), low tumor purity (5–10%; 77% successful), and cytological material (90% successful). 75% of these tumor samples showed ≥1 (likely) pathogenic mutation, including targetable mutations (e.g. EGFR, BRAF, MET, ERBB2, KIT, PDGFRA). Amplifications were observed in 5.5% of the samples, comprising clinically relevant amplifications (e.g. MET, ERBB2, FGFR1). 1.5% of the tumor samples were classified as MSI-high, including both MSI-prone and non-MSI-prone tumors.ConclusionsWe developed a comprehensive workflow for predictive analysis of diagnostic tumor samples. The smMIP-based NGS analysis was shown suitable for limited amounts of histological and cytological material. As smMIP technology allows easy adaptation of panels, this approach can comply with the rapidly expanding molecular markers.

Single Molecule Molecular Inversion Probes for High Throughput Germline Screenings in Dystonia.

Background: This study's aim was to investigate a large cohort of dystonia patients for pathogenic and rare variants in the ATM gene, making use of a new, cost-efficient enrichment technology for NGS-based screening.Methods: Single molecule Molecular Inversion Probes (smMIPs) were used for targeted enrichment and sequencing of all protein coding exons and exon-intron boundaries of the ATM gene in 373 dystonia patients and six positive controls with known ATM variants. Additionally, a rare-variant association study was performed.Results: One patient (0.3%) was compound heterozygous and 21 others were carriers of variants of unknown significance (VUS) in the ATM gene. Although mutations in sporadic dystonia patients are not common, exclusion of pathogenic variants is crucial to recognize a potential tumor predisposition syndrome. SmMIPs produced similar results as routinely used NGS-based approaches.Conclusion: Our results underline the importance of implementing ATM in the routine genetic testing of dystonia patients and confirm the reliability of smMIPs and their usability for germline screenings in rare neurodegenerative conditions.

ACLY (ATP Citrate Lyase) Mediates Radioresistance in Head and Neck Squamous Cell Carcinomas and is a Novel Predictive Radiotherapy Biomarker.

Radiotherapy is an important treatment modality of head and neck squamous cell carcinomas (HNSCC). Multiple links have been described between the metabolic activity of tumors and their clinical outcome. Here we test the hypothesis that metabolic features determine radiosensitivity, explaining the relationship between metabolism and clinical outcome. Radiosensitivity of 14 human HNSCC cell lines was determined using colony forming assays and the expression profile of approximately 200 metabolic and cancer-related genes was generated using targeted RNA sequencing by single molecule molecular inversion probes. Results: Correlation between radiosensitivity data and expression profiles yielded 18 genes associated with radiosensitivity or radioresistance, of which adenosine triphosphate (ATP) citrate lyase (ACLY) was of particular interest. Pharmacological inhibition of ACLY caused an impairment of DNA damage repair, specifically homologous recombination, and lead to radiosensitization in HNSCC cell lines. Examination of a The Cancer Genome Atlas (TCGA) cohort of HNSCC patients revealed that high expression of ACLY was predictive for radiotherapy failure, as it was only associated with poor overall survival in patients who received radiotherapy (hazard ratio of 2.00, 95% CI: 1.12–3.55; p = 0.0184). These data were further validated in an independent cohort of HNSCC patients treated with chemoradiation. Furthermore, patients with poor locoregional control after radiotherapy have significantly higher nuclear ACLY protein levels. Together, we here show that ACLY affects DNA damage repair, and is a predictive factor for radiotherapy outcome in HNSCC.

Genomic Landscape of Juvenile Myelomonocytic Leukemia: A Real World Context

Introduction: Juvenile myelomonocytic leukaemia (JMML) is a clonal haematopoietic disorder occurring in pediatric age group usually characterized by uncontrolled granulocytic and monocytic lineage proliferation and a poor outcome. JMML is characterised by aberrant signal transduction of the RAS signalling pathways, usually manifesting in molecular alteration of one of the five cardinal genes: NF1, NRAS, KRAS, PTPN11 and CBL. Recent high-throughput studies have started to push the horizon of JMML associated mutations wider and have proposed molecular-based risk algorithms. We comprehensively evaluated the genomic profile of JMML that were referred to our hospital for diagnosis and treatment. Methods: We developed a 51 gene (103 kB) low-cost targeted sequencing myeloid panel based on single molecule molecular inversion probes. This focussed panel interrogated sets of genes implicated in pathogenesis of myeloid malignancies. A total of 50 children with clinical and pathological features of JMML were sequenced at high coverage using this assay on an Illumina MiSeq. Results:The median age of our cohort was 2 years, with a male preponderance. Among the 50 patients, 43 (86%) harboured mutations in one of the RAS/MAPK pathway genes. The most frequently mutated gene in our cohort was PTPN11 (14, 28%), and NRAS (14, 28%), followed by NF1 mutation in 22% (11) cases (Figure 1). Interestingly, 20% (10) of children had more than one mutation, with 5 cases harbouring two RAS pathway mutations. Of these 5 patients, one patient harboured two coexistent mutations in the NF1 gene, three patients had coexistent PTPN11 and NF1 mutations and one patient had a coexistent NF1 and NRAS mutations. Additionally, two patients also had an ASXL1 mutation, coexistent with NF1 and NRAS mutations respectively. Other non-RAS pathway mutations involved ABL1, ATRX, SETBP1, SH2B3 and ZRSR2 genes. The median variant allele frequency of RAS pathway mutations detected was 40.75%. Monosomy 7 was detected in 32% (16) patients, and five of these did not harbour any RAS pathway mutations. The follow up data revealed that 37 (74%) of these patients have succumbed to the disease. In our cohort, only five patients received an allogenic stem cell transplant, owing to economic constraints; two of these patients have succumbed to the disease. The median time period from diagnosis to death was 7.2 months in the cohort. On survival analysis, patients with PTPN11 mutations showed a trend to shorter overall survival, compared to their wildtype counterparts (p=0.7, median OS 10.6 months vs 38.1 months). Additionally, patients harbouring more than one mutation also showed a trend to shorter OS (p=0.64, median OS 10.3 months vs 38.1 months). Among the thirteen patients who are still on regular follow-up, two patients have relapsed with disease progression to acute myeloid leukemia and T/myeloid mixed phenotypic acute leukemia respectively. Conclusion: Our study represents the largest cohort of JMML from a single centre in the Indian subcontinent. This study depicted that almost 90% cases of JMML harbor at least one mutation with 86% harbouring at least one RAS pathway mutation. Presence of PTPN11 mutations and co-existence of more than one mutation may be the major determinants of outcome in JMML. Figure 1 Disclosures No relevant conflicts of interest to declare.

A Novel Machine Learning Derived Genomics-Based Scoring System Is Highly Predictive of Outcome in Core Binding Factor Acute Myeloid Leukemia

Introduction: Core binding factor acute myeloid leukemia (CBF-AML) is one of the commonest subtypes of AML characterized presence of t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22). It is characterised by a high frequency of somatic mutations especially in RAS and tyrosine kinase signalling pathways. Here we investigated the feasibility of improving risk prediction of CBF-AML using machine learning algorithms. Methods: We developed a next generation sequencing panel that targeted 50 genes implicated in the pathogenesis of myeloid malignancies using single molecule molecular inversion probes. This panel was used to sequence 106 patients of CBF-AML accrued over a six year period (March 2012 - December 2018) treated with conventional "3 + 7" chemotherapy. Post data analysis, we devised a supervised machine learning (ML) approach for identification of mutations most likely to predict for favorable outcome in CBF-AML. We included somatic mutations in genes occurring in CBF-AML at a frequency of &gt;5%. A total of 11 variables were included for feature selection to predict for favorable outcome (including mutations in ASXL2, CSF3R,FLT3, KIT, NF1, NRAS, RAD21, TET2 and WT1 genes as well as mutation burden). Approaches for supervised ML were naïve bayes, generalized linear model, logistic regression, deep learning and random forest methods. Based on the ML results top 6 selected variables were allotted an individual score. A final score for that case was devised as a sum total of the individual scores. These sum were used to generate a genetic risk for a patient. Overall survival (OS) was calculated from date of diagnosis to time of last follow up or death. Relapse free survival (RFS) was calculated from date of CR till time to relapse or death or last follow up if in CR. Results of the genetic risk were analyzed for their impact on OS and RFS using log rank test. Multivariate analysis was performed using cox proportional hazards regression model. Results: The median follow up of the cohort was 27.6 months. A total of 181 somatic mutations were identified in this subset of AML with 86.7% harbouring at least one somatic mutation (median = 2). Based on ML data, a genetic score was formulated that incorporated mutations in RAD21, FLT3, KIT D816, ASXL2, NRAS genes as well as high mutation burden (≥2) into two genetic risk classes (favorable risk and poor ML derived genetic genetic risk). Patients classified as poor genetic risk had a significantly lower OS [median OS: 34.8 months; 95% confidence interval (CI) (14.2-34.8); p=0.0086] and RFS [median RFS: 17.9 months; 95%CI (12.7-33.6); p=0.0043] as compared to patients with favorable genetic risk (median OS and RFS not reached). These results can be seen in Figure 1. On multivariate analysis poor genetic risk was the most important independent risk factor that predicted for inferior OS [hazard ratio(HR), 2.7; 95% CI 1.3 to 5.7] and RFS (HR, 2.6; 95% CI:1.3 to 5.1). Conclusions In a proof of concept, we describe a novel ML derived genomics scoring model that provides a mechanism to risk stratify CBF-AML, a seemingly homogeneous disease entity. This study, to the best of our knowledge represents a novel application of ML to CBF mutated AML. Our data indicates that this scoring system will be useful in identifying CBF mutated AML patients who are at higher risk of relapse and distinguishes them from patients who are truly good risk. Figure 1 Disclosures No relevant conflicts of interest to declare.

Open-Sourced CIViC Annotation Pipeline to Identify and Annotate Clinically Relevant Variants Using Single-Molecule Molecular Inversion Probes.

PURPOSEClinical targeted sequencing panels are important for identifying actionable variants for patients with cancer; however, existing approaches do not provide transparent and rationally designed clinical panels to accommodate the rapidly growing knowledge within oncology.MATERIALS AND METHODSWe used the Clinical Interpretations of Variants in Cancer (CIViC) database to develop an Open-Sourced CIViC Annotation Pipeline (OpenCAP). OpenCAP provides methods to identify variants within the CIViC database, build probes for variant capture, use probes on prospective samples, and link somatic variants to CIViC clinical relevance statements. OpenCAP was tested using a single-molecule molecular inversion probe (smMIP) capture design on 27 cancer samples from 5 tumor types. In total, 2,027 smMIPs were designed to target 111 eligible CIViC variants (61.5 kb of genomic space).RESULTSWhen compared with orthogonal sequencing, CIViC smMIP sequencing demonstrated a 95% sensitivity for variant detection (n = 61 of 64 variants). Variant allele frequencies for variants identified on both sequencing platforms were highly concordant (Pearson’s r = 0.885; n = 61 variants). Moreover, for individuals with paired tumor and normal samples (n = 12), 182 clinically relevant variants missed by orthogonal sequencing were discovered by CIViC smMIP sequencing.CONCLUSIONThe OpenCAP design paradigm demonstrates the utility of an open-source and open-access database built on attendant community contributions with peer-reviewed interpretations. Use of a public repository for variant identification, probe development, and variant interpretation provides a transparent approach to build dynamic next-generation sequencing–based oncology panels.

Sequencing of RAS/RAF pathway genes in primary colorectal cancer and matched liver and lung metastases

BackgroundMutations in the RAS/RAF pathway predict resistance to anti-epidermal growth factor receptor antibodies in colorectal cancer (CRC), and may be targets for future therapies. This study investigates concordance of BRAF, HRAS, KRAS, NRAS and PIK3CA mutation status in primary CRC with matched liver (n = 274), lung (n = 114) or combined liver and lung metastases (n = 14).MethodsNext generation sequencing was performed on DNA from formalin-fixed paraffin embedded CRC and matched liver and/or lung metastases, for recurrent mutations in BRAF, HRAS, KRAS, NRAS and PIK3CA and using the single-molecule molecular inversion probe method.ResultsPaired sequencing results on all five genes were reached in 249 of the 402 cases (62%). The obtained number of unique reads was not always sufficient to confidently call the absence or presence of mutations for all regions of interest. The mutational status of matched pairs was highly concordant; 91.1% concordance for all five genes, 95.5% for KRAS, 99.1% for NRAS. Lung metastases more often harboured RAS mutations compared to liver metastases (71% vs. 48%, p < 0.001).ConclusionsIn this large series of CRC we show that both primary tumors and corresponding metastases can be used to determine the mutational status for targeted therapy, given the high concordance rates. Next generation sequencing including a single molecule tags is feasible, however in combination with archival formalin-fixed paraffin embedded material is limited by coverage depth.

Sequencing-based microsatellite instability testing using as few as six markers for high-throughput clinical diagnostics.

Microsatellite instability (MSI) testing of colorectal cancers (CRCs) is used to screen for Lynch syndrome (LS), a hereditary cancer‐predisposition, and can be used to predict response to immunotherapy. Here, we present a single‐molecule molecular inversion probe and sequencing‐based MSI assay and demonstrate its clinical validity according to existing guidelines. We amplified 24 microsatellites in multiplex and trained a classifier using 98 CRCs, which accommodates marker specific sensitivities to MSI. Sample classification achieved 100% concordance with the MSI Analysis System v1.2 (Promega) in three independent cohorts, totaling 220 CRCs. Backward–forward stepwise selection was used to identify a 6‐marker subset of equal accuracy to the 24‐marker panel. Assessment of assay detection limits showed that the 24‐marker panel is marginally more robust to sample variables than the 6‐marker subset, detecting as little as 3% high levels of MSI DNA in sample mixtures, and requiring a minimum of 10 template molecules to be sequenced per marker for >95% accuracy. BRAF c.1799 mutation analysis was also included to streamline LS testing, with all c.1799T>A variants being correctly identified. The assay, therefore, provides a cheap, robust, automatable, and scalable MSI test with internal quality controls, suitable for clinical cancer diagnostics.

Gene Mosaicism Screening Using Single-Molecule Molecular Inversion Probes in Routine Diagnostics for Systemic Autoinflammatory Diseases

Diagnosis of systemic autoinflammatory diseases (SAIDs) is often difficult to achieve and can delay the start of proper treatments and result in irreversible organ damage. In several patients with dominantly inherited SAID, postzygotic mutations have been detected as the disease-causing gene defects. Mutations with allele frequencies <5% have been detected, even in patients with severe phenotypes. Next-generation sequencing techniques are currently used to detect mutations in SAID-associated genes. However, even if the genomic region is highly covered, this approach is usually not able to distinguish low-grade postzygotic variants from background noise. We, therefore, developed a sensitive deep sequencing assay for mosaicism detection in SAID-associated genes using single-molecule molecular inversion probes. Our results show the accurate detection of postzygotic variants with allele frequencies as low as 1%. The probability of calling mutations with allele frequencies ≥3% exceeds 99.9%. To date, we have detected three patients with mosaicism, two carrying likely pathogenic NLRP3 variants and one carrying a likely pathogenic TNFRSF1A variant with an allele frequency of 1.3%, confirming the relevance of the technology. The assay shown herein is a flexible, robust, fast, cost-effective, and highly reliable method for mosaicism detection; therefore, it is well suited for routine diagnostics.

Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease.

Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation-scanning methods. We aimed to develop a cost-effective sequencing method for ABCA4 exons and regions carrying known causal deep-intronic variants. Fifty exons and 12 regions containing 14 deep-intronic variants of ABCA4 were sequenced using double-tiled single molecule Molecular Inversion Probe (smMIP)-based next-generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. Thirty-four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep-intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep-intronic variant (c.4539+2065C>G) resulted in a 170-nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). smMIPs-based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost-effective mutation detection in STGD1 cases in previously unsolved cases.

SUN-245 Heterozygous GH1 p.R209H Variant within a Large Pedigree May Mimic a Growth Hormone Insensitivity Pattern

Isolated growth hormone deficiency (IGHD) is most frequently caused by mutations in the GH1 gene. Pathogenic mutations in the GHRHR and GHSR have also been reported to cause IGHD. Individuals with IGHD type II present with variable clinical phenotype. Autosomal dominant GH1 p.R209H variant impairs GH secretion despite normal GH synthesis. The aim of this work is to expand the clinical and biochemical phenotype of heterozygous GH1 p.R209H missense mutation (previously known as p.R183H) in a large Argentinean pedigree. We report a non-consanguineous 4 generation pedigree. Two affected members showed classical IGHD phenotype: short stature (height -4.13 and -2.6 SD), low GH peak after arginine/clonidine provocative tests (3.6 and 2.6 ng/ml, cut-off value 4.7 ng/ml, GH IS 98/574, Immulite), non-detectable IGF-1, and low IGFBP-3 (-2.83 and -2.28 SD). Brain MRI showed anterior pituitary gland of 3.3 mm and 2.5 mm, respectively. The use of a customized panel for congenital hypopituitarism using single molecule molecular inversion probes sequencing (smMIPS) revealed a c.626G>A transition in GH1 gene, predicted to result in p.R209H mutation. The variant was subsequently confirmed by Sanger sequencing. Parents affected with the same variant presented either short (-1.91 SDS) or normal stature (-0.72 SDS). The patients’ response to rhGH treatment was adequate (Δ heights were 2.49 and 1.37 SDS in 2.4 and 1.0 year of treatment, respectively). Additionally, 4/9 siblings within this pedigree had short stature (mean height -2.48 ± 0.62 SD) with mild or no clinical phenotype of GHD, normal GH response to provocative pharmacologic tests (maximum GH peaks 5.46 to 10.9 ng/ml), associated to low serum IGF-1 (mean -3.27 ± 0.89 SD) and IGFBP3 (mean -2.61 ± 0.42 SD), resembling a pattern of growth hormone insensitivity (GHI). IGF-1 generation tests (GH dose 33 µg/kg.day) showed a mean increment in IGF-1 of 3.25 times over basal, confirming adequate GH sensitivity. Despite the absence of complete GHD phenotype, data from the extended pedigree suggested GH1 as the initial candidate gene, identifying the same pathogenic GH1 variant in these four siblings. Brain MRI showed normal height of the anterior pituitary gland (range 3.9 to 4.6 mm). Their affected father had normal stature (-1.69 SDS) with low serum IGF-1 (64 ng/mL). We have shown that members of a large pedigree affected with the p.R209H GH1 variant showed either a classical clinical and biochemical pattern of GHD or an initial biochemical pattern resembling GHI. This variability could lead in certain cases to an elusive diagnosis of GHD when the classical phenotype is absent. Therefore, these results highlight the need to explore GH1 gene in children with short stature associated to low IGF-1 and IGFBP-3 serum levels even when GH response to pharmacological tests is normal.

Molecular Profiling of Druggable Targets in Clear Cell Renal Cell Carcinoma Through Targeted RNA Sequencing.

Clear cell renal cell carcinoma (ccRCC) comprises more than 80% of all renal cancers and when metastasized leads to a 5-year survival rate of only 10%. The high rate of therapy failure and resistance development calls for reliable methods that provide information on the actionable biological pathways and predict optimal treatment protocols for individual patients. We here applied targeted RNA sequencing (t/RNA-NGS) using single molecule Molecular Inversion Probes on tumor nephrectomy samples of five ccRCC patients, comparing tumor with healthy kidney tissues. Transcriptome profiling focused on expression of genes with involvement in ccRCC biology that can be targeted with clinically available drugs. Results confirm high expression of vascular endothelial growth factor-A (VEGF-A) in tumor tissue relative to healthy-appearing kidney, in line with the angiogenic nature of ccRCC. PDGFRα and KIT, targets of the multi-kinase inhibitor sunitinib which is one of the current choices of first-line drug in metastasized ccRCC patients, were expressed at relatively low levels in tumor tissues, whereas significantly increased in normal kidney. Of all measured druggable tyrosine kinases, MET, AXL, or EGFR were expressed at higher levels in tumors than in normal kidney tissues, although intertumor differences were observed. Using cancer cell lines we show that t/RNA-NGS gene expression profiles can be used to predict in vitro sensitivity to targeted drugs. In conclusion, t/RNA-NGS analysis may provide insights into the (druggable) molecular make-up of individual renal cancers, and may guide personalized therapy of renal cell cancers.

Identification of novel GNAS mutations in intramuscular myxoma using next-generation sequencing with single-molecule tagged molecular inversion probes

BackgroundIntramuscular myxoma (IM) is a hypocellular benign soft tissue neoplasm characterized by abundant myxoid stroma and occasional hypercellular areas. These tumors can, especially on biopsy material, be difficult to distinguish from low-grade fibromyxoid sarcoma or low-grade myxofibrosarcoma. GNAS mutations are frequently involved in IM, in contrast to these other malignant tumors. Therefore, sensitive molecular techniques for detection of GNAS aberrations in IM, which frequently yield low amounts of DNA due to poor cellularity, will be beneficial for differential diagnosis.MethodsIn our study, a total of 34 IM samples from 33 patients were analyzed for the presence of GNAS mutations, of which 29 samples were analyzed using a gene-specific TaqMan genotyping assay for the detection of GNAS hotspot mutations c.601C > T and c602G > A in IM, and 32 samples using a novel next generation sequencing (NGS)-based approach employing single-molecule tagged molecular inversion probes (smMIP) to identify mutations in exon 8 and 9 of GNAS. Results between the two assays were compared for their ability to detect GNAS mutations with high confidence.ResultsIn total, 23 of 34 samples were successfully analyzed with both techniques showing GNAS mutations in 12 out of 23 (52%) samples. The remaining 11 samples were analyzed with either TaqMan assay or smMIP assay only. The TaqMan assay revealed GNAS mutations in 16 out of 29 samples (55%), with six samples c.601C > T (p.R201C; 38%) and ten samples c.602G > A (p.R201H; 62%) missense mutations. The smMIP assay identified mutations in 16 out of 28 samples (57%), with five samples c.601C > T (p.R201C; 31%) and seven samples c.602G > A (p.R201H; 44%) missense mutations. In addition, four samples (25%) revealed novel IM-associated mutations, including c.601C > A (p.R201S), c.602G > T (p.R201L), c.602G > C (p.R201P) and c.680A > G (p.Q227R). Combining the results of both tests, 23 out of 34 sporadic IM samples (68%) showed a GNAS mutation.ConclusionsBoth the TaqMan and the smMIP assay a show a high degree of concordance in detecting GNAS hotspot mutations in IM with comparable sensitivity. However, since the NGS-based smMIP assay permits mutation detection in whole exons of GNAS, a broader range of GNAS mutations can be identified by the smMIP approach.

Assessment of parental mosaicism in SCN1A-related epilepsy by single-molecule molecular inversion probes and next-generation sequencing

BackgroundDravet syndrome is a severe genetic encephalopathy, caused by pathogenic variants in SCN1A. Low-grade parental mosaicism occurs in a substantial proportion of families (7%–13%) and has important implications for recurrence...

SA27 - EXOME SEQUENCING OF MULTIPLY AFFECTED BIPOLAR DISORDER FAMILIES AND FOLLOW-UP RESEQUENCING IMPLICATE RARE VARIANTS IN NEURONAL GENES CONTRIBUTING TO DISEASE ETIOLOGY

Background Bipolar Disorder (BD) is a complex neuropsychiatric disorder characterized by recurrent episodes of mania and depression. It is a severe disorder of mood with a lifetime prevalence of about 1% and a high heritability of about 70%. Models of illness are most consistent with a polygenic contribution of common and rare variants to disease susceptibility. As the cumulative impact of common alleles with small effect may only explain around 25–38% of the phenotypic variance for BD, rare variants of high penetrance have been suggested to contribute to BD susceptibility. One way to evaluate this hypothesis is to investigate large pedigrees densely affected with BD, in which the existence of a genetic variant of strong effect inherited from a common ancestor may be more likely. Methods In the present study, we investigated 226 individuals of 68 large multiply affected families of European origin using Whole Exome Sequencing (WES). In each family, two to five affected individuals with BD or recurrent major depression were selected for sequencing. WES was performed on the Illumina HiSeq. 2500 platform and the Varbank pipeline of the Cologne Center for Genomics was used for data analysis. All identified variants shared within each family were filtered for a minor allele frequency Results We identified a total of 1214 rare, segregating and functional variants implicating 1122 different genes, of which 903 were brain expressed. Subsequently, we applied the Residual Variation Intolerance Scores (Petrovski et al., 2013) and identified 294 genes that were ranked among the 20% most “intolerant” genes in the genome. Gene enrichment analysis of these genes showed a significant enrichment for 18 pathways (p We prioritized genes that were (i) found in at least two unrelated families in the present study, (ii) previously reported in next generation sequencing or GWAS studies of BD, or (iii) predominantly driving the significant pathways in our gene enrichment analysis. The different approaches of prioritization yielded 42 promising candidate genes including CDH22 which encodes a calcium-dependent cell adhesion protein that plays an important role in the morphogenesis of neural cells during brain development. Discussion Our preliminary results strongly suggest that rare and highly-penetrant variants in neuronal and cell adhesion genes contribute to BD etiology. The 42 prioritized candidate genes are currently being followed up by resequencing in cohorts of about 2500 independent BD cases and 2500 controls of European ancestry. For resequencing, we use the single molecule molecular inversion probes (smMIPs) technology that enables multiplex targeted resequencing in large cohorts. The smMIPs sequences have been designed with the empirically trained design algorithm MIPgen (Boyle et al., 2014) and sequencing is currently performed on the Illumina HiSeq. 2500 platform. The resequencing results will provide further insights into the particular genes and pathways which are involved in BD development.

Inherited and multiple de novo mutations in autism/developmental delay risk genes suggest a multifactorial model

BackgroundWe previously performed targeted sequencing of autism risk genes in probands from the Autism Clinical and Genetic Resources in China (ACGC) (phase I). Here, we expand this analysis to a larger cohort of patients (ACGC phase II) to better understand the prevalence, inheritance, and genotype–phenotype correlations of likely gene-disrupting (LGD) mutations for autism candidate genes originally identified in cohorts of European descent.MethodsWe sequenced 187 autism candidate genes in an additional 784 probands and 85 genes in 599 probands using single-molecule molecular inversion probes. We tested the inheritance of potentially pathogenic mutations, performed a meta-analysis of phase I and phase II data and combined our results with existing exome sequence data to investigate the phenotypes of carrier parents and patients with multiple hits in different autism risk genes.ResultsWe validated recurrent, LGD, de novo mutations (DNMs) in 13 genes. We identified a potential novel risk gene (ZNF292), one novel gene with recurrent LGD DNMs (RALGAPB), as well as genes associated with macrocephaly (GIGYF2 and WDFY3). We identified the transmission of private LGD mutations in genes predominantly associated with DNMs and showed that parental carriers tended to share milder autism-related phenotypes. Patients that carried DNMs in two or more candidate genes show more severe phenotypes.ConclusionsWe identify new risk genes and transmission of deleterious mutations in genes primarily associated with DNMs. The fact that parental carriers show milder phenotypes and patients with multiple hits are more severe supports a multifactorial model of risk.

Somatic variants in autosomal dominant genes are a rare cause of sporadic Alzheimer's disease

IntroductionA minority of patients with sporadic early-onset Alzheimer's disease (AD) exhibit de novo germ line mutations in the autosomal dominant genes such as APP, PSEN1, or PSEN2. We hypothesized that negatively screened patients may harbor somatic variants in these genes. MethodsWe applied an ultrasensitive approach based on single-molecule molecular inversion probes followed by deep next generation sequencing of 11 genes to 100 brain and 355 blood samples from 445 sporadic patients with AD (>80% exhibited an early onset, <66 years). ResultsWe identified and confirmed nine somatic variants (allele fractions: 0.2%–10.8%): two APP, five SORL1, one NCSTN, and one MARK4 variants by independent amplicon-based deep sequencing. DiscussionTwo of the SORL1 variant might have contributed to the disease, the two APP variants were interpreted as likely benign and the other variants remained of unknown significance. Somatic variants in the autosomal dominant AD genes may not be a common cause of sporadic AD, including early onset cases.