Letter | Published:

ARAF recurrent mutation causes central conducting lymphatic anomaly treatable with a MEK inhibitor

Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

WES data have been deposited in dbGaP with the accession number phs001802.v1.p1.

Ethics declarations

Competing interests

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.

Additional information

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.

References

  1. 1.

    Trenor, C. C. 3rd & Chaudry, G. Complex lymphatic anomalies. Semin. Pediatr. Surg. 23, 186–190 (2014).

  2. 2.

    Collins, F. S. & Varmus, H. A new initiative on precision medicine. N. Engl. J. Med. 372, 793–795 (2015).

  3. 3.

    Adams, D. M. et al. Efficacy and safety of sirolimus in the treatment of complicated vascular anomalies. Pediatrics 137, e20153257 (2016).

  4. 4.

    Hammill, A. M. et al. Sirolimus for the treatment of complicated vascular anomalies in children. Pediatr. Blood Cancer 57, 1018–1024 (2011).

  5. 5.

    McCormick, A., Rosenberg, S., Trier, K. & Balest, A. A case of a central conducting lymphatic anomaly responsive to sirolimus. Pediatrics 137, e20152694 (2016).

  6. 6.

    Hilliard, R. I., McKendry, J. B. & Phillips, M. J. Congenital abnormalities of the lymphatic system: a new clinical classification. Pediatrics 86, 988–994 (1990).

  7. 7.

    Levine, C. Primary disorders of the lymphatic vessels—a unified concept. J. Pediatr. Surg. 24, 233–240 (1989).

  8. 8.

    Smeltzer, D. M., Stickler, G. B. & Fleming, R. E. Primary lymphatic dysplasia in children: chylothorax, chylous ascites, and generalized lymphatic dysplasia. Eur. J. Pediatr. 145, 286–292 (1986).

  9. 9.

    Wassef, M. et al. Vascular anomalies classification: recommendations from the International Society for the Study of Vascular Anomalies. Pediatrics 136, e203–e214 (2015).

  10. 10.

    Chen, W., Adams, D., Patel, M., Gupta, A. & Dasgupta, R. Generalized lymphatic malformation with chylothorax: long-term management of a highly morbid condition in a pediatric patient. J. Pediatr. Surg. 48, e9–e12 (2013).

  11. 11.

    Lala, S. et al. Gorham–Stout disease and generalized lymphatic anomaly—clinical, radiologic, and histologic differentiation. Skeletal Radiol. 42, 917–924 (2013).

  12. 12.

    Clemens, R. K., Pfammatter, T., Meier, T. O., Alomari, A. I. & Amann-Vesti, B. R. Combined and complex vascular malformations. Vasa 44, 92–105 (2015).

  13. 13.

    Li, D. et al. Pathogenic variant in EPHB4 results in central conducting lymphatic anomaly. Hum. Mol. Genet. 27, 3233–3245 (2018).

  14. 14.

    Wellbrock, C., Karasarides, M. & Marais, R. The RAF proteins take centre stage. Nat. Rev. Mol. Cell Biol. 5, 875–885 (2004).

  15. 15.

    Lavoie, H. & Therrien, M. Regulation of RAF protein kinases in ERK signalling. Nat. Rev. Mol. Cell Biol. 16, 281–298 (2015).

  16. 16.

    Molzan, M. et al. Impaired binding of 14-3-3 to C-RAF in Noonan syndrome suggests new approaches in diseases with increased Ras signaling. Mol. Cell Biol. 30, 4698–4711 (2010).

  17. 17.

    Jung, H. M. et al. Development of the larval lymphatic system in zebrafish. Development 144, 2070–2081 (2017).

  18. 18.

    Karkkainen, M. J. et al. Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat. Immunol. 5, 74–80 (2004).

  19. 19.

    Carmeliet, P. & Jain, R. K. Molecular mechanisms and clinical applications of angiogenesis. Nature 473, 298–307 (2011).

  20. 20.

    Karaman, S., Leppanen, V. M. & Alitalo, K. Vascular endothelial growth factor signaling in development and disease. Development 145, dev151019 (2018).

  21. 21.

    Potente, M. & Makinen, T. Vascular heterogeneity and specialization in development and disease. Nat. Rev. Mol. Cell Biol. 18, 477–494 (2017).

  22. 22.

    Coso, S., Bovay, E. & Petrova, T. V. Pressing the right buttons: signaling in lymphangiogenesis. Blood 123, 2614–2624 (2014).

  23. 23.

    Brouillard, P., Boon, L. & Vikkula, M. Genetics of lymphatic anomalies. J. Clin. Invest. 124, 898–904 (2014).

  24. 24.

    Bulow, L. et al. Hydrops, fetal pleural effusions and chylothorax in three patients with CBL mutations. Am. J. Med. Genet. A 167A, 394–399 (2015).

  25. 25.

    Gargano, G. et al. Hydrops fetalis in a preterm newborn heterozygous for the c.4A>G SHOC2 mutation. Am. J. Med. Genet. A 164A, 1015–1020 (2014).

  26. 26.

    Gos, M. et al. Contribution of RIT1 mutations to the pathogenesis of Noonan syndrome: four new cases and further evidence of heterogeneity. Am. J. Med. Genet. A 164A, 2310–2316 (2014).

  27. 27.

    Hanson, H. L. et al. Germline CBL mutation associated with a Noonan-like syndrome with primary lymphedema and teratoma associated with acquired uniparental isodisomy of chromosome 11q23. Am. J. Med. Genet. A 164A, 1003–1009 (2014).

  28. 28.

    Milosavljevic, D. et al. Two cases of RIT1 associated Noonan syndrome: further delineation of the clinical phenotype and review of the literature. Am. J. Med. Genet. A 170, 1874–1880 (2016).

  29. 29.

    Koenighofer, M. et al. Mutations in RIT1 cause Noonan syndrome - additional functional evidence and expanding the clinical phenotype. Clin. Genet. 89, 359–366 (2016).

  30. 30.

    Lee, K. A. et al. PTPN11 analysis for the prenatal diagnosis of Noonan syndrome in fetuses with abnormal ultrasound findings. Clin. Genet. 75, 190–194 (2009).

  31. 31.

    Croonen, E. A. et al. Prenatal diagnostic testing of the Noonan syndrome genes in fetuses with abnormal ultrasound findings. Eur. J. Hum. Genet. 21, 936–942 (2013).

  32. 32.

    Joyce, S. et al. The lymphatic phenotype in Noonan and cardiofaciocutaneous syndrome. Eur. J. Hum. Genet. 24, 690–696 (2016).

  33. 33.

    Yaoita, M. et al. Spectrum of mutations and genotype–phenotype analysis in Noonan syndrome patients with RIT1 mutations. Hum. Genet. 135, 209–222 (2016).

  34. 34.

    Lo, I. F. et al. Severe neonatal manifestations of Costello syndrome. J. Med. Genet. 45, 167–171 (2008).

  35. 35.

    Ebrahimi-Fakhari, D. et al. Congenital chylothorax as the initial presentation of PTPN11-associated Noonan syndrome. J. Pediatr. 185, 248–248.e1 (2017).

  36. 36.

    Morcaldi, G. et al. Lymphodysplasia and Kras mutation: a case report and literature review. Lymphology 48, 121–127 (2015).

  37. 37.

    Manevitz-Mendelson, E. et al. Somatic NRAS mutation in patient with generalized lymphatic anomaly. Angiogenesis 21, 287–298 (2018).

  38. 38.

    Barclay S. F. et al. A somatic activating NRAS variant associated with kaposiform lymphangiomatosis. Genet. Med. https://doi.org/10.1038/s41436-018-0390-0 (2018).

  39. 39.

    Gao, J. et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci. Signal. 6, pl1 (2013).

  40. 40.

    Imielinski, M. et al. Oncogenic and sorafenib-sensitive ARAF mutations in lung adenocarcinoma. J. Clin. Invest. 124, 1582–1586 (2014).

  41. 41.

    Johannessen, C. M. et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468, 968–972 (2010).

  42. 42.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

  43. 43.

    Grzegorski, S. J., Chiari, E. F., Robbins, A., Kish, P. E. & Kahana, A. Natural variability of Kozak sequences correlates with function in a zebrafish model. PLoS One 9, e108475 (2014).

  44. 44.

    Kwan, K. M. et al. The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev. Dyn. 236, 3088–3099 (2007).

  45. 45.

    Villefranc, J. A., Amigo, J. & Lawson, N. D. Gateway compatible vectors for analysis of gene function in the zebrafish. Dev. Dyn. 236, 3077–3087 (2007).

  46. 46.

    Kawakami, K. & Shima, A. Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene 240, 239–244 (1999).

Download references

Acknowledgements

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.

Author information

H.H. designed and supervised all aspects of the study. D.L. conducted the analysis and writing of the study. D.L., L.T., T.W., C.N.K., P.M.A.S. and H.H. arranged and performed genomic testing/analysis. M.E.M., A.G.-U., C.K., C.S., L.S.M. and M.R.B contributed the functional investigations. E.P., E.J.B., T.L.W., J.A.P., M.A.L., P.J.H., J.S., J.B.B., Y.D. and H.H. contributed the clinical phenotyping and treatment. B.M.W., M.S. and H.M.J. provided the zebrafish line. N.R. and R.C. coordinated research study subject enrollment. D.L., M.E.M., A.G.-U., C.S., C.N.K, R.C., J.A.P., M.A.L., P.M.A.S., Y.D. and H.H. read, edited and approved of the manuscript, along with all other authors.

Competing interests

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.

Correspondence to Hakon Hakonarson.

Extended Data

Extended Data Fig. 1 Western blot analysis of ARAF overexpression in HeLa cells.

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

Extended Data Fig. 2 Cell morphology study in primary HDLECs.

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

Extended Data Fig. 4 Examples of dilated lymphatic vessels induced by ARAF-S214P.

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.

Extended Data Fig. 5 ARAF-S214P expression in zebrafish induces phosphorylation of ERK.

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.

Supplementary information

Reporting Summary

Source data

Source Data Fig. 2

Unprocessed/uncropped Western Blots.

Source Data Fig. 2

Statistical Source Data.

Source Data Extended Data Fig. 1

Unprocessed/uncropped Western Blots.

Source Data Extended Data Fig. 1

Statistical Source Data.

Source Data Extended Data Fig. 3

Source Data for a single proliferation experiment.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark
Fig. 1: Clinical images in the lead proband with lymphatic anomaly and molecular analysis.
Fig. 2: The ARAF-S214P mutation increases ERK1/2 activity, enhances lymphangiogenic capacity and alters actin skeleton and VE-cadherin junctions in HDLECs, and results in dilation of the thoracic duct (TD) in zebrafish that is reversed by cobimetinib.
Fig. 3: Pulmonary function tests and clinical images in the lead proband before and after MEK inhibitor therapy.
Extended Data Fig. 1: Western blot analysis of ARAF overexpression in HeLa cells.
Extended Data Fig. 2: Cell morphology study in primary HDLECs.
Extended Data Fig. 3: MTT assay demonstrating that the ARAF-S214P mutation does not enhance proliferation.
Extended Data Fig. 4: Examples of dilated lymphatic vessels induced by ARAF-S214P.
Extended Data Fig. 5: ARAF-S214P expression in zebrafish induces phosphorylation of ERK.
Extended Data Fig. 6: WT larvae show good tolerance following treatment with 1 μM cobimetinib from 3 to 7 dpf.