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Show 172 THE CLINICAL UTILITY OF NEXT GENERATION SEQUENCING IN THE DIAGNOSIS OF PATIENTS WITH MARFAN AND MARFAN LIKE SYNDROMES Parker Plant (Whitney Wooderchak- Donahue, Pinar Bayrak-Toydemir, FACMG Department of Pathology, ARUP University of Utah RESEARCH POSTERS ON THE HILL SPRING 2012 II. SureSelect target enrichment of aortapathy genes (Table 2): III. Sequencing and Data Analysis Samples were barcoded/indexed using PCR, pooled accordingly based on qPCR results, and were sequenced using 2x100 PE reads on a HiSeq2000 instrument (Illumina). Samples were aligned using BWA and GaTK was used to call variants. Annovar was used to annotate the variants, and variants from dbSNP were filtered out. The IGV viewer (Broad Institute) was used to view the results. Average exon coverage between the enrichment methods was compared among all 294 exons in the panel. Aortapathy is a group of disorders that includes aneurysms, dilation, and tuortosity of the aorta. Ten aortapathy genes have been described. Marfan syndrome (MFS), the most common aortapathy, is caused by mutations in fibrillin-1 (FBN1). Loeys-Dietz syndrome (LDS) and Ehlers-Danlos syndrome (EDS) type IV overlap clinically with MFS and are caused by mutations in the transcription growth factor beta receptor (TGFBR1/ TGFBR2) and collagen 3 (COL3A1), respectively. Purpose: Because of the phenotypic overlap and genetic heterogeneity of these disorders, we developed a next generation sequencing assay to detect mutations in these four genes and five others that cause thoracic aortic aneurysms. This assay will allow physicians to test patients for many genetic disorders that manifest symptoms suggestive of an aortapathy disorder in a single test at a lower cost. Methods: SureSelect hybridization capture enrichment was used to enrich for nine aortapathies genes in nine symptomatic patient samples. Six of these patient samples had previously undergone DNA testing and were negative for Marfan Syndrome and Loeys-Dietz Syndrome. Enriched samples were then barcoded/indexed, pooled, and sequenced (Illumina HiSeq2000). Results were analyzed, and overall coverage distribution was evaluated for each sample. Results: The hybridization capture enrichment and subsequent DNA sequencing accurately identified sequencing variants in two of the eight patient samples. Overall coverage was comparable across the 294 exons. Analysis of the sequence variants identified is currently underway for remaining samples. These results may reveal the disease-causative mutation in these patients and facilitate proper treatment. Conclusion: Our panel assay will aid physicians in a clinical setting in the timely diagnosis of patients with aortapathy disorders. The clinical utility of next generation sequencing in the diagnosis of patients with Marfan and Marfan like Syndromes. Parker Plant1, Whitney Wooderchak-Donahue1, Larissa Furtado1, Brendan O'Fallon1, Pinar Bayrak-Toydemir1,2 1ARUP Institute for Clinical and Experimental Pathology, University of Utah 2Department of Pathology, University of Utah Table 3. Aortapathy panel results Parker Plant Pinar Bayrak- Toydemir ABSTRACT MATERIALS AND METHODS Table 2. Aortapathy panel genes Gene Protein Disease Exons FBN1 (chr. 15) Fibrillin Marfan Syndrome 66 TGFBR1 (chr. 9) Transforming growth factor, beta receptor 1 Loeys-Dietz syndrome 9 TGFBR2 (chr. 3) Transforming growth factor, beta receptor 2 Loeys-Dietz syndrome 7 COL3A1 (chr. 2) Collagen, type III, alpha 1 Ehlers Danlos Type IV 51 MYH11 (chr. 16) Myosin, heavy chain 11, smooth muscle TAAD*-patent ductus arteriosus 41 ACTA2 (chr. 10) Actin, alpha 2, smooth muscle, aorta TAAD4 9 SLC2A10 (chr. 20) Solute carrier family 2 (facilitated glucose transporter), member 10 Arterial Tortuosity syndrome 5 NOTCH1 (chr. 9) Notch homolog 1, translocation-associated BAV-TAA syndrome 34 FBN2 (chr. 5) Fibrillin 2 BAV-TAA syndrome 71 I. Patient Samples We studied ten unrelated patients (Table I) who met clinical diagnostic criteria for Marfan syndrome and Loeys-Dietz syndrome. Barcode here! RESULTS AND DISCUSSION 0 100 200 300 400 500 600 700 800 900 1000 COL3A1 FBN2 TGFBR2 TGFBR1 NOTCH1 ACTA2 FBN1 MYH11 SLC2A10 Exon Average Coverage Gene CONCLUSIONS SureSelect capture: Craniofacial Uvula / Palate Cardiac / Aortic Other cardiac Other vascular Skeletal Skin Ocular Other General Family Hx Patient 1* + + + Patient 2 + ? Patient 3* + + + Patient 4 + + + Patient 5* + + + + + Patient 6* + + + Patient 7* + + + + Patient 8 + + + + Patient 9 + + + Table 1. Patient Phenotypes Sample Mutation x coverage (%Variant frequency) Results Patient 1 None Reported allelic changes: SLC2A10: p.A206T, p. A385G. NOTCH1: IVS16-4C>CT. Patient 2 COL3A1: IVS9-7T>TC, splice site? 891x (45%) Reported allelic changes: COL3A1: p.A698T. FBN2: p.V965I. Patient 9 FBN1: c.1585C>CT, p.R529X 1214x (36%) FBN1 disease causiative mutation identified. COL3A1: c.198A>AG, p.I66M, a novel change. B. Next gen. data *These individuals had previously undergone DNA testing and were negative for Marfan Syndrome and/or Loeys-Dietz Syndrome. Three samples were run initially to assess the performance of the aortapathy assay (Table 3). An FBN1 mutation detected in patient 9 (Figure 1A) confirmed the accuracy of the assay. This mutation (p.R529X ) was also detected using Sanger Sequencing (Figure 1B). Figure 1. Patient 9 FBN1 mutation. Sanger A. *Results are pending for patient samples 3-8. Figure 2. Average coverage for the aortapathy genes was obtained using the custom panel (in A) and exome sequencing (in B). High enough coverage is obtained from each method to detect mutations in the known aortapathy genes. Exome sequencing is useful for samples that test negative for the aortapathy genes because mutations in unknown genes that may cause the patient's syndrome can be identified. *NOTCH1 will be removed from the custom panel based on the lower overall coverage obtained due to gene homology. Figure 2. Average coverage of aortapathy genes. A. Custom Panel* 1. Our results show the clinical utility of next generation sequencing in a clinical diagnostic setting for the diagnosis of patients with aortapathy disorders. Such cost effective, rapid testing will facilitate a timely diagnosis and therapy for these patients. 2. Of the patient samples tested using the aortapathy panel assay, Patient 9 had a pathogenic mutation in FBN1 (Figure 1). 3. Patient samples 1 and 2 tested negative for a mutation in the 9 aortapathy genes (Table 3). 4. High enough coverage to correctly identify variants was obtained for the aortapathy genes using exome sequencing (Figure 2). 5. Exome sequencing may be useful for patients who test negative for the aortapathy gene panel (Patients 1 and 2). Exome sequencing may lead to gene discovery in these patients and ultimately increase the mutation detection rate for aortapathies. Future Plans: Results for patient samples 3-8 will be interpreted in an effort to identify the disease causing mutation in the affected individual. Exome sequencing of patients who test negative for a mutation in one of the nine aortapathy genes may identify new aortapathy causative genes. 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 FBN1 TGFBR1 TGFBR2 COL3A1 MYH11 ACTA2 SLC2A10 FBN2 Average read depth Gene Average Exon 1 B. Exome Aortapathy is a group of disorders that includes aneurysms, dilation, and tuortosity of the aorta. Ten aortapathy genes have been described. Mar-fan syndrome (MFS), the most common aortapathy, is caused by mutations in brillin-1 (FBN1). Loeys-Di-etz syndrome (LDS) and Ehlers-Danlos syndrome (EDS) type IV overlap clinically with MFS and are caused by mutations in the transcription growth factor beta receptor (TGFBR1/ TGFBR2) and collagen 3 (COL3A1), respectively. Purpose: Because of the phenotypic overlap and genetic heterogeneity of these disorders, we developed a next generation sequencing assay to detect mutations in these four genes and six others that cause thoracic aortic aneurysms. This assay will allow physicians to test patients for many genetic disorders that manifest symptoms suggestive of an aortapathy disorder in a single test at a lower cost. Methods: SureSelect hybridization capture enrichment was used to enrich for ten aortapathies genes in eight symptomatic patient samples. Six of these patient samples had previously undergone DNA testing and were negative for Marfan Syndrome and Loeys-Dietz Syndrome. Enriched samples were then bar-coded/ indexed, pooled, and sequenced (Illumina HiSeq2000). Results were analyzed, and overall coverage distribution was evaluated for each sample. Results: The hybridization capture enrichment and subsequent DNA sequencing accurately identi ed sequencing variants in all eight patient samples. Overall coverage was comparable across the 294 exons. Analysis of the sequence variants identi ed is currently underway. These results may reveal the disease-causative mutation in these patients and facilitate proper treatment. Conclusion: Our panel assay will aid physicians in a clinical setting in the timely diagnosis of patients with aortapathy disorders. |