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ARAF Recurrent Mutation Causes Central Conducting Lymphatic Anomaly Treatable with a MEK Inhibitor

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ARAF Recurrent Mutation Causes Central Conducting Lymphatic Anomaly Treatable with a MEK Inhibitor LETTERS https://doi.org/10.1038/s41591-019-0479-2 ARAF recurrent mutation causes central conducting lymphatic anomaly treatable with a MEK inhibitor Dong Li1, Michael E. March1, Alvaro Gutierrez-Uzquiza1,12, Charlly Kao1, Christoph Seiler2, Erin Pinto3, Leticia S. Matsuoka1, Mark R. Battig1, Elizabeth J. Bhoj1, Tara L. Wenger4, Lifeng Tian1, Nora Robinson1, Tiancheng Wang1, Yichuan Liu1, Brant M. Weinstein5, Matthew Swift6, Hyun Min Jung5, Courtney N. Kaminski1, Rosetta Chiavacci1, Jonathan A. Perkins7, Michael A. Levine8,9, Patrick M. A. Sleiman1,9, Patricia J. Hicks9, Janet T. Strausbaugh9, Jean B. Belasco9,10, Yoav Dori3,9 and Hakon Hakonarson 1,9,11* The treatment of lymphatic anomaly, a rare devastating dis- rarity and overlapping of diagnostic criteria, has hampered the ease spectrum of mostly unknown etiologies, depends on development of innovative therapies6–9. GLA is defined as multifo- the patient manifestations1. Identifying the causal genes will cal lymphatic anomaly that has multiple areas of micro/macrocystic allow for developing affordable therapies in keeping with pre- lymphatic malformation and often involves bone destruction9–11. cision medicine implementation2. Here we identified a recur- CCLA, on the other hand, describes dysfunction of the thoracic rent gain-of-function ARAF mutation (c.640T>C:p.S214P) duct (TD) or cisterna chyli, leading to a retrograde flux of lymphatic in a 12-year-old boy with advanced anomalous lymphatic fluid or abnormal drainage of lymphatic fluid1,12,13. Both conditions disease unresponsive to conventional sirolimus therapy and can manifest with chylothorax, effusions, chylous ascites or lymph- in another, unrelated, adult patient. The mutation led to loss edema. The overlap of these apparently disparate disorders suggests of a conserved phosphorylation site. Cells transduced with that a common pathway rather than a common gene is responsible ARAF-S214P showed elevated ERK1/2 activity, enhanced for the various clinical syndromes, and implies that the distinction lymphangiogenic capacity, and disassembly of actin skeleton between entities may be artificial. Here we report the use of whole- and VE-cadherin junctions, which were rescued using the MEK exome sequencing (WES) to identify a recurrent missense muta- inhibitor trametinib. The functional relevance of the mutation tion in ARAF as the basis for a severely advanced lymphatic disease was also validated by recreating a lymphatic phenotype in characterized by a complex lymphatic anomaly in two unrelated a zebrafish model, with rescue of the anomalous phenotype patients. Our results provide a representative demonstration of how using a MEK inhibitor. Subsequent therapy of the lead pro- genetic classification presents a way to categorize complex medical band with a MEK inhibitor led to dramatic clinical improve- disorders, thereby guiding biologically based medical treatments, ment, with remodeling of the patient’s lymphatic system with which in our instance was life-saving. resolution of the lymphatic edema, marked improvement in The first tier of WES analyses of the known lymphatic anom- his pulmonary function tests, cessation of supplemental oxy- aly-associated genes was unrevealing, including mutation analysis gen requirements and near normalization of daily activities. of AKT1, PIK3CA, KRAS, HRAS, NRAS, BRAF, RAF1, PTPN11, Our results provide a representative demonstration of how SHOC2, CBL, RIT1 and SOS1. Subsequent gene prioritization knowledge of genetic classification and mechanistic under- revealed a novel X chromosomal ARAF mutation, c.640T>C:p. standing guides biologically based medical treatments, which S214P, in both patient P1, a male with CCLA (Fig. 1a,c–e; see in our instance was life-saving. Methods for a detailed clinical description), and patient P2, a female Although recent studies have demonstrated the benefit of siro- diagnosed with lymphangiomatosis in 2012 before the establish- limus in the treatment of generalized lymphatic anomaly (GLA) ment of the 2015 International Society for the Study of Vascular and central conducting lymphatic anomaly (CCLA)3–5, the absence Anomalies classification. The mutation affects a conserved phos- of clear clinical distinctions between these entities, due to their phorylation site, which putatively resulted in a gain-of-function 1Center for Applied Genomics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 2Zebrafish Core Facility, The Children’s Hospital of Philadelphia Research Institute, Philadelphia, PA, USA. 3Center for Lymphatic Imaging and Interventions, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 4Division of Craniofacial Medicine, Seattle Children’s Hospital, Seattle, WA, USA. 5Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA. 6Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC, USA. 7Division of Otolaryngology–Head and Neck Surgery, Seattle Children’s Hospital, Seattle, WA, USA. 8Division of Endocrinology and Diabetes, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 9Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 10Division of Oncology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 11Divisions of Human Genetics and Pulmonary Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 12Present address: Department of Biochemistry and Molecular Biology II, School of Pharmacy, Complutense University, Madrid, Spain. *e-mail: [email protected] 1116 Nature MEDICINE | VOL 25 | JULY 2019 | 1116–1122 | www.nature.com/naturemedicine NATURE MEDICINE LETTERS a bc f Patient P1 Patient P2 Mother of patient P2 Daughter of patient P2 de ARAF c.640T>C ARAF c.640T>C (p.S214P) (p.S214P) in patient P2 g ARAF c.640T>C (p.S214P) CR1 CR2 CR3 Human Rhesus Mouse Dog Elephant Xenopus tropicalis Zebrafish B-RAF C-RAF Fig. 1 | Clinical images in the lead proband with lymphatic anomaly and molecular analysis. a, The coronal slice of a T2-weighted non-contrast lymphangiogram, demonstrating a large pericardial effusion (arrow). b, The maximal intensity projection of a dynamic contrast-enhanced magnetic resonance lymphangiogram in a healthy control person, showing a normal TD coursing towards the left innominate vein. c, The maximal intensity projection of a dynamic contrast-enhanced magnetic resonance lymphangiogram in P1, showing dilated lumber lymphatic networks with retrograde liver hilar flow (arrowhead) and a dilated and tortuous TD (arrow) coursing towards the innominate vein on the left and also supplying retrograde perfusion to the mediastinum and pericardium (box). d, The contrast lymphangiogram of the boxed region in c, demonstrating dilated and tortuous distal TD with retrograde flow towards the mediastinum, pericardium and lungs through dilated lymphatic networks originating at the distal TD (arrows). e, The coronal maximal intensity projection of the pelvis and genitalia, demonstrating multiple dilated ducts (arrowhead) originating in bilateral groin lymph nodes and supplying retrograde flow into the penis and scrotum (arrow). f, The pedigrees and genotypes of a recurrent mutation, c.640T>C (p.S214P), in ARAF identified in unrelated kindreds. g, The schematic topology of the ARAF protein, where the asterisk indicates the position of the p.S214P mutation in CR2. The Ser 214 residue is highly conserved across vertebrate species and all RAF isoforms. (GoF) effect as the residue Ser 214 is a paralogous regulatory site significantly greater activation of ERK1/2, as measured by increased in its homologous protein C-RAF (also known as RAF1) for inhibi- phosphorylation, compared with HEK293T cells expressing wild- tion by 14-3-3 proteins. This missense mutation was absent from type (WT) ARAF (Fig. 2a,b). Phosphorylation of AKT, p70S6K, 1000 Genomes Project, ESP6500SI, ExAC v0.3, gnomAD v2.1 or mTOR and p38 (another family of MAP kinases) was not altered by additional exome-sequencing data from more than 5,000 samples ARAF-S214P (Fig. 2b). Similar results were obtained in HeLa cells that we had in our in-house database. Sanger sequencing of blood- (Extended Data Fig. 1) and in primary human dermal lymphatic derived DNA from P1 and both parents confirmed that this X-linked endothelial cells (HDLECs) (Fig. 2c). This marked overactivation ARAF mutation occurred as a somatic heterozygous event as shown was also present even in the absence of cytokines or growth factors in the male patient (Fig. 1f). Sanger sequencing of the ARAF muta- (Extended Data Fig. 1). tion in P2, her unaffected daughter and mother confirmed the HDLECs expressing ARAF-S214P manifest enhanced lymphan- mutation was present only in P2 (Fig. 1f). The father was unavail- giogenic capacity compared with HDLECs expressing ARAF-WT, able for sequencing; however, as her father had no reported respira- as measured by the number of sprouts and the sprout length in the tory symptoms it remains likely that the ARAF mutation arose as a three-dimensional lymphatic spheroid sprouting assay conducted de novo or somatic mutation in P2. Patient P2 was lost to follow-up in the absence of vascular endothelial growth factor C (VEGFC) and we were informed later that she died in 2017 from complica- (Fig. 2d). The MEK inhibitor trametinib rescued the increased tions of her lymphatic disease, five years after her diagnosis.
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