Abstract Aortopathy is a group of disorders that includes aneurysms, dilation, and tortuosity of the aorta. Although the FBN1 gene plays a major role in pathogenesis, several other genes are known to be involved. Because of the phenotypic overlap and genetic heterogeneity of these disorders, we developed a next generation sequencing (NGS) assay to detect mutations in ten genes that cause thoracic aortic aneurysms (ACTA2, COL3A1, FBN1, FBN2, MYH11, MYLK, SLC2A10, SMAD3, TGFBR1, and TGFBR2). We report the clinical and molecular findings in a series of 175 individuals submitted for aortopathy panel testing at ARUP Laboratories. Capture enrichment was used to target the ten aortopathies genes in the patient samples. Enriched samples were indexed, pooled, and sequenced. NGS results were analyzed using an internally developed program, and mutations were confirmed using Sanger sequencing. Of the 175 individuals evaluated, most pathogenic mutations were identified in the FBN1 gene with the clinical presentation of these individuals fitting the Marfan Syndrome diagnosis. In addition to the NGS assay, individuals were also evaluated for large deletions and duplications of the same aortopathy genes using a CGH-array. A few large structural variants were detected including a large duplication of SMAD3 and a single exon deletion of FBN1. Interestingly, a substantial number of individuals who had thoracic aortic aneurysms or other aortic involvement tested negative for a mutation in the aortopathy genes, suggesting that additional aortopathy genes exist. We anticipate that the clinical sensitivity (32.6%) will continue to rise as additional aortopathy genes are identified and included in the diagnostic panel. Importantly, the aortopathy clinical NGS panel assay aids in the timely molecular diagnosis of individuals with disorders featuring aortopathy and helps to guide their proper treatment. I. Subjects Overall clinical findings for 175 symptomatic, unrelated patients whose samples had been submitted for aortopathy panel testing at ARUP in the last year are summarized in Table 1. II. SureSelect capture enrichment All coding regions and exon-intron boundries of ten aortopathy genes (Table 2) were enriched using SureSelect capture baits. Enriched samples were indexed, pooled, and sequenced (HiSeq). NGS results were analyzed using an internally developed program, and mutations were confirmed using Sanger sequencing. III. Microarray A custom designed CGH microarray was used to identify large deletions and duplications in the ten aortopathy genes. 1. The aortopathy clinical panel is a cost effective diagnostic assay that facilitates a timely molecular diagnosis to guide patient therapy. 2. Pathogenic mutations or VUS were identified in 57/175 individuals in our cohort giving a mutation detection rate of 32.6%. Approximately 52% of these variants were novel. Of those evaluated using the array, only two had a large structural aberration (1.2%). 3. Seven additional genes were recently added to the aortopathy panel (CBS, COL5A1, COL5A2, PLOD1, SKI, SMAD4, and TGFB2). These and other newly discovered genes will continue to increase the clinical sensitivity of the aortopathy panel assay. Conclusions Results and Discussion Materials and Methods Table 3. Molecular & clinical findings for all 57/175 positive aortopathy cases. Figure 1. Aortopathy panel results for 3 cases. An FBN1 p.R2242C mutation detected by the NGS assay (257x, 51% variant) was confirmed by Sanger Sequencing. Array results are shown for the large FBN1 exon 30 deletion and SMAD3 duplication (bottom). SMAD3 exons 2-5 duplication Clinical utility of a next generation sequencing panel assay for Marfan and Marfan-like syndromes featuring aortopathy Whitney Wooderchak-Donahue 1,2 , Chad VanSant-Webb 1 , Tatiana Tvrdik 1 , Tracey Lewis 1 , Parker Plant 1 , Jennifer Stocks 1 , Joshua Raney 1 , Tyler Wayman 1 , Brendan O’Fallon 1 , Alan F. Rope 3 , Angela T. Yetman 4 , David Bull 5 , Pinar Bayrak-Toydemir 1,2 1 ARUP Institute for Clinical and Experimental Pathology, 2 Dept of Pathology, University of Utah, 3 Dept of Pediatrics, Division of Medical Genetics, University of Utah, 4 Dept of Pediatrics, Division of Cardiology, University of Utah, 5 Dept of Surgery, Division of Cardiothoracic Surgery, University of Utah Gene Protein Disease(s) Proportion of disease attributed to a mutation in this gene Exons FBN1 Fibrillin 1 Marfan syndrome TAAD syndrome 93% Unknown 66 TGFΒR1 Transforming growth factor, beta receptor 1 Loeys‐Dietz syndrome TAAD Syndrome Marfan Syndrome Type II 25% 1% Unknown 9 TGFΒR2 Transforming growth factor, beta receptor 2 Loeys‐Dietz syndrome TAAD Syndrome Marfan Syndrome Type II 75 % 4% Unknown 7 COL3A1 Collagen type III alpha 1 Ehlers Danlos Type IV 95% 51 MYH11 Myosin heavy chain 11 TAAD a ‐patent ductus arteriosus ~ 1% 41 ACTA2 smooth muscle actin, alpha 2 TAAD syndrome 10 – 14 % 9 SLC2A10 solute carrier family 2 (facilitated glucose transporter), member 10 Arterial Tortuosity syndrome 100% 5 SMAD3 mothers against decapentaplegic homolog (SMAD) 3 TAAD Syndrome, Aneurysms‐osteoarthritis syndrome (AOS) 2% Unknown 9 FBN2 Fibrillin 2 CCA b 75% 71 MYLK myosin light chain kinase TAAD Syndrome ~1% 34 Table 2. Aortopathy Panel Genes. Table 1. Clinical findings of patient cohort. aThoracic aortic aneurysms and dissections (TAAD); bCongenital contractural arachnodactyly (CCA) Clinical Findings Number of cases (n=175) Aortic Dilation (Z ≥ 2 for Root) 87 (49.7%) Dissection 70 (40.0%) Rupture 60 (34.3%) Aneurism 8 (4.6%) Tortuosity 2 (1.1%) Cardiac Mitral Valve Prolapse 21 (12.0%) Bicuspid Valve 12 (6.9%) Valve (Other) 26 (14.9%) Cardiac (Other) 19 (10.9%) Ocular Myopia 25 (14.3%) Ectopia lentis 6 (3.4%) Other 15 (8.6%) Musculoskeletal Pectus Carinatum/Excavatum 43 (24.6%) Joint Hypermobility/Laxity 34 (19.4%) Increased Limb Length 16 (9.1%) Increased Height 27 (15.4%) Scoliosis 25 (14.3%) Thoracolumbar kyphosis 21 (12.0%) Pes Planus 22 (12.6%) Hindfoot Deformity 14 (8.0%) Decreased elbow extension 8 (4.6%) Wrist/Thumb signs 16 (9.1%) Dural ectasia 3 (1.7%) Arachnodactyly 12 (6.9%) Other 33 (18.9%) Craniofacial Hypertelorism 3 (1.7%) Bifid/broad uvula 6 (3.4%) Palate anomaly 21 (12.0%) Microretrognathia 15 (8.6%) Other 36 (20.6%) Skin Striae 22 (12.6%) Thin, velvety skin 12 (6.9%) Other 16 (9.1%) Gastrointestinal 19 (10.9%) Family History 73 (41.7%) SureSelect Capture: C. Case # Gene Nucleotide Change Protein Change Mutation Classification Age Sex Clinical Diagnosis Aortic Ocular Craniofacial Musculo‐ skeletal Cutaneous Family Hx 170 ACTA2 c.536G>A p.Arg179His Pathogenic 11 M Aortic dilation, ductus arteriosus X X no 171 ACTA2 c.536G>A p.Arg179His Pathogenic <1 F Aortopathy Aneurysmal ductus arteriosus X X unknown 138 FBN1 c.184C>T p.Arg62Cys Pathogenic 21 M MFS X no 141 FBN1 c.266G>T p.Cys89Phe Pathogenic 23 F MFS Aortic dilation X X yes 7 FBN1 c.1160_1161delA p.Lys387fs a Pathogenic 46 F MFS/LDS Aortic dilation/dissection X X X X no 140 FBN1 c.1633C>T p.Arg545Cys Pathogenic 24 M MFS X X X yes 52 FBN1 c.1878delG p.Arg627fs Pathogenic 26 M thoracic aneurysm/dissection yes 22 FBN1 c.3058A>G p.Thr1020Ala Suspected Path. 1 F MASS MVP/stenosis/atresia X X X no 5 FBN1 c.3919T>A p.Cys1307Ser a Suspected Path. 25 F MFS Aortic dilation (Z=3.7) X X yes 50 FBN1 c.4270C>C p.Pro1424Ala Pathogenic 8 M Aortic dilation/dissection X X X unknown 126 FBN1 c.4489T>G p.Cys1497Gly Suspected Path. 5 M MFS Aortic dilation (Z=3.2) X X X yes 49 FBN1 c.6181T>C p.Cys2061Arg Pathogenic 18 M LDS Aortic dilation/dissection, BAV X X X yes 146 FBN1 c.6418G>A p.Gly2140Arg Suspected Path. 15 F MFS Aortic dissection, CAD X no 89 FBN1 MYH11 c.6444delC c.3766_3768delAAG p.Tyr2149fs p.Lys1256del a Pathogenic VUS 18 M MFS Aortic dilation/dissection X no 82 FBN1 c.6724C>T p.Arg2242Cys Pathogenic 10 M MFS Aortic dilation (Z=2.51) X X yes 175 FBN1 MYH11 c.7254dupT c.2496G>C p.Val2419fsa p.Trp832Cysa Pathogenic VUS 35 F MFS MVP X yes 133 FBN1 Large deletion Exon 30 del a Pathogenic <1 F Beals‐Hecht Aortic dilation X X no 166 SMAD3 c.369dupT p.Pro124fs Pathogenic 51 F Aortic root dilation yes 65 SMAD3 c.977G>A p.Trp326X Pathogenic 56 M Aortic dilation/dissection unknown 109 TGFBR1 c.722C>T P.Ser241Leu Pathogenic 15 M MFS/CCA Aortic dilation/dissection X X unknown 21 ACTA2 c.569A>C p.Tyr190Ser VUS b 6 F Aortic dilation/dissection X X no 164 COL3A1 c.560C>T p.Thr187Ile a VUS 18 M MFS pneumomediastinum X X X no 41 COL3A1 c.1804C>A p.Pro602Thr VUS 27 M MFS X X X X no 106 FBN1 c.1052A>G p.Gln351Arg a VUS 17 M pneumothorax X X X yes 1 FBN1 c.2927G>A p.Arg976His VUS 28 M MFS X unknown 91 FBN1 c.3712+9G>A a Splicing VUS 7 M LDS Aortic dilation (Z>3.0) X X yes 167 FBN1 c.5463C>T p.Asn1821Asn a VUS 13 M Aortic involvement yes 144 FBN1 c.6560G>A p.Gly2187Asp a VUS 47 M Aortic aneurysm X X yes 163 FBN1 c.6601A>G p.Met2201Val VUS 45 M unknown 40 FBN1 c.7852G>A p.Gly2618Arg VUS 15 M unknown 79 FBN2 c.2260G>A p.Gly754Ser VUS 72 F TAAD chest pain, dyspnea yes 8 FBN2 c.3302A>G p.Asn1101Ser a VUS 66 M MFS Aortic root aneurysm (Z=4.6) X no 162 FBN2 SMAD3 c.3793G>A c.317T>C p.Glu1265Lys a p.Met106Thr VUS VUS 30 F MVP, pneumothorax X yes 43 FBN2 c.4454A>G p.Asp1485Gly a VUS 56 M Vascular dissections unknown 174 FBN2 c.4657C>T p. Arg1553Cysa VUS 79 M MFS/LDS Aortic dilation/dissection X no 86 FBN2 c.5627G>C p.Cys1876Ser a VUS 14 M MFS X X X no 119 MYH11 c.2195C>T p.Ala732Val a VUS 55 F Aortic involvement X X yes 123 MYH11 c.3766_3768delAAG p.Lys1256del a VUS 48 M Aortopathy Ascending aortic aneurysm yes 63 MYH11 c.3856C>T p.Gln1286X VUS c 14 F Aortic dilation/dissection, BAV X X yes 95 MYH11 c.3928G>A p.Val1310Met a VUS 40 F Ascending aortic dilation yes 118 MYH11 MYLK c.4235C>T c.4165T>G p.Val1412Val a p.Phe1389Val a VUS VUS 12 M MFS Aortic dilation, assymetric chest X X yes 44 MYLK c.1991A>G p.Gln664Arg VUS 5 M Aortic dilation/dissection no 112 MYLK c.2113C>T p.Arg705Cys a VUS 32 F MFS Aortic dilation, MVP X yes 148 MYLK c.2777G>A p.Arg926His a VUS 36 M TAAD Aortic dilation/dissection unknown 127 MYLK c.3641T>G p.Leu1214Arg a VUS 14 M MFS Aortic dilation/dissection X no 66 MYLK c.3987T>G p.Asp1329Glu VUS 18 M MFS X X no 134 SLC2A10 c.515C>T p.Thr172Ile a VUS <1 M Aortic dilation/dissection no 92 SLC2A10 c.671C>G p.Ala224Gly a VUS 48 M Aortic dilation/dissection X unknown 156 SMAD3 c.268C>T p.Arg90Cys VUS 32 M Aortic involvement unknown 107 SMAD3 c.284C>T p.Pro95Leu a VUS 29 M Aortic dilation/dissection no 42 SMAD3 c.803G>A p.Arg268His a VUS 28 M MFS Aortic dilation/dissection X no 36 SMAD3 c.1118G>A p.Arg373His a VUS 47 M Aortic dilation/dissection, yes 74 SMAD3 c.1168A>G p.Thr390Pro a VUS 18 F LDS/EDS/TAAD Aortic dissection (Type B) X X no 120 SMAD3 Large duplication Exons 2‐5 dup a VUS 41 M Aortic dissection (Type A), X yes 88 TGFBR1 c.868G>T p.Asp290Tyr a VUS 28 M EDS Aortic involvement X X unknown 116 TGFBR2 c.95‐3C>A a Splicing VUS 58 M unknown 110 TGFBR2 c.106A>G p.Met36Val a VUS 21 M MFS Aortic dilation (Z=2.5) X X X X no aIndicates a novel mutation; bVariant of uncertain significance; cVariant reclassified to VUS after family tracking studies. Abbreviations: Bicuspid aortic valve (BAV), Carotid artery dissection (CAD), Contractural Arachnodactyly (CCA), Ehlers Danlos Syndrome (EDS), Loeys-Dietz Syndrome (LDS), Marfan Syndrome (MFS), Mitral valve prolapse (MVP), Thoracic aortic aneurysms and dissections (TAAD). Figure I. Aortopathy panel results. Sanger confirmed FBN1 exon 30 deletion (Case 133) SMAD3 exons 2-5 duplication (Case 120) NGS data FBN1 p.R2242C (Case 82)