The treatment of lymphatic anomaly, a rare devastating disease spectrum of mostly unknown etiologies, depends on the patient manifestations1. Identifying the causal genes will allow for developing affordable therapies in keeping with precision medicine implementation2. Here we identified a recurrent gain-of-function ARAF mutation (c.640T>C:p.S214P) in a 12-year-old boy with advanced anomalous lymphatic disease unresponsive to conventional sirolimus therapy and in another, unrelated, adult patient. The mutation led to loss of a conserved phosphorylation site. Cells transduced with ARAF-S214P showed elevated ERK1/2 activity, enhanced lymphangiogenic capacity, and disassembly of actin skeleton and VE-cadherin junctions, which were rescued using the MEK inhibitor trametinib. The functional relevance of the mutation was also validated by recreating a lymphatic phenotype in a zebrafish model, with rescue of the anomalous phenotype using a MEK inhibitor. Subsequent therapy of the lead proband with a MEK inhibitor led to dramatic clinical improvement, with remodeling of the patient’s lymphatic system with resolution of the lymphatic edema, marked improvement in his pulmonary function tests, cessation of supplemental oxygen requirements and near normalization of daily activities. Our results provide a representative demonstration of how knowledge of genetic classification and mechanistic understanding guides biologically based medical treatments, which in our instance was life-saving.
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WES data have been deposited in dbGaP with the accession number phs001802.v1.p1.
H.H. is a scientific advisor to Aevi Genomic Medicine Inc. and he and CHOP own shares in the company. The other authors declare no competing interests.
Peer review information: Kate Gao and Brett Benedetti were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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We thank all of the families involved in this study for their participation. We gratefully acknowledge L. Klepper, T. Ferry and J. Kelly, who helped with the collection of the DNA samples and clinical data on patient P2. Research reported in this publication was supported in part by the Roberts Collaborative Functional Genomics Rapid Grant (to D.L.) from CHOP, Institutional Development Funds (to H.H.) from CHOP, CHOP’s Endowed Chair in Genomic Research (H.H) and donation from the Adele and Daniel Kubert family (to H.H. and CAG). The study was also funded in part through a sponsored research agreement from Aevi Genomic Medicine Inc., funding discovery and translation of rare and orphan disease genes at the CAG.
It demonstrates the phosphorylation status of ERK1/2, AKT, p70S6K, mTOR and p38 in HeLa cells after serum deprivation or serum deprivation followed by a short stimulation with 10% fetal bovine serum (FBS). Normalized ratios are illustrated by the panel on the bottom. The data are shown as the mean ± s.e.m. (error bars) of five independent experiments. Two-tailed unpaired t-test with 8 df. *P = 014; ****P = 7.7 × 10−5. The images were cropped for better presentation. Source data
Primary HDLECs transduced with ARAF-WT or ARAF-S214P were plated in the presence or absence of 100 nM trametinib. Cells were fixed and stained for VE-cadherin and actin. Here are the full ×10 magnification fields. a, VE-cadherin staining. b, Actin staining of the same ×10 field shown in a. c, Zoomed-in views of the regions with the red boxes in b. Zoomed-in views are presented for improved visualization of the actin filaments.
Extended Data Fig. 3 MTT assay demonstrating that the ARAF-S214P mutation does not enhance proliferation.
There is no increase in proliferation observed in transduced HDLECs from an independent experiment. The contents of triplicate wells (n = 3 independent wells) were collected at the indicated times as described in the Methods. The trend lines connect the means for each transductant at each time point, and the points show the measured values for all data points. This experiment is the second of two representative experiments, as described in Fig. 2f. Source data
a, The construct used for transgene expression. b,c, ARAF-S214P expression (red) leads to dilation of the intersegmental lymphatic vessel (ISLV, b) and the parachordal line (PL, c). Scale bar, 200 μm.
Staining with p-ERK antibody is labeled green, and transgene expression is labeled red; the bottom panel shows an overlay of the staining. a, For ARAF-WT the area of the TD is outlined by dotted lines; that of the PCV is outlined by dashed lines. Red ARAF-WT transgenic cells (arrows in a) and cells without transgene expression do not show significantly different p-ERK levels. b, Expression of ARAF-S214P causes expansion and fusion of the TD and PCV (outlined by dotted lines). (We observed comparable findings in n = 10 larvae.) Scale bar, 100 μm.
Extended Data Fig. 6 WT larvae show good tolerance following treatment with 1 μM cobimetinib from 3 to 7 dpf.
a,b, The treatment does not affect the overall morphology and development (b) in WT zebrafish compared to control (DMSO)-treated larvae (a). The morphologies of the TD at 7 dpf (dotted outline) and the PCV are also comparable in cobimetinib-treated (d) and DMSO-treated larvae (c). The boxes indicate the area investigated in c and d. Scale bar, 100 μm.
Unprocessed/uncropped Western Blots.
Statistical Source Data.
Unprocessed/uncropped Western Blots.
Statistical Source Data.
Source Data for a single proliferation experiment.