Cellular and Molecular Mechanisms of Lymphatic Disorders
- Sarah E. Sheppard, MD, PhD, MST, Head, Unit on Vascular Malformations
- Scott Paulissen, PhD, Research Specialist IV
- Luciana Daniela Garlisi Torales, MD, Postdoctoral Fellow
- Georgia Krikorian, BA, Postbaccalaureate Fellow
- Dhyanam Shukla, BA, Postbaccalaureate Fellow
- Kristina Woodis, BS, Postbaccalaureate Fellow
- Christopher Marshall, BA, Research Specialist II
The primary goal of our translational research group is to develop efficacious therapies for patients with complex lymphatic anomalies. To do this, we seek to understand the molecular etiologies of these complex lymphatic malformations, how the molecular etiologies alter molecular signaling, and how this affects the cellular mechanisms regulating normal lymphatic development. Ultimately, these answers will allow us to develop novel therapies.
Complex lymphatic anomaly is a term that encompasses four different complex lymphatic malformations: central conducting lymphatic anomaly (CCLA); generalized lymphatic anomaly (GLA); Kaposiform lymphangiomatosis (KLA); and Gorham Stout disease (GSD). Patients suffer from symptoms such as pleural effusions, pericardial effusions, ascites, and bone lesions, which can cause significant morbidity and even death. Currently, there is only one medication approved for patients with complex lymphatic anomalies caused by PIK3CA, a gene mutation known known to cause lymphatic malformations. Similar, precision-medicine approaches are needed for patients with other complex lymphatic anomalies.
Research in our lab will combine patient studies and genomics with the zebrafish model to identify novel therapies. The zebrafish model allows for us to manipulate the genetics rapidly to create patient-based models, image the developing vasculature, understand cellular dynamics in vivo, and perform drug screening.
Figure 1. A bedside-to-bench precision medicine program for lymphatic anomalies
Click image to view.
The figure shows a circle with the major components of the research in the lab: a child with abnormal lymphatics, a DNA molecule, a representation of a zebrafish model and organoid model, and a pill bottle. This represents a bedside-to-bench-to-bedside program using organoid and zebrafish to model patient's lymphatic anomalies and develop therapies that will be translated back to the patients.
Natural history study of lymphatic disorders
Our group launched a prospective natural history study for individuals with lymphatic anomalies to systematically evaluate the disease phenotypes and long-term outcomes. This will allow us to provide improved prognostication to families, establish screening/monitoring guidelines, determine best practices for genetic diagnosis, and explore fertility outcomes for those on long-term medication management. The study will allow us to identify novel end-points for future clinical trials.
Genotype-phenotype correlations in central conducting lymphatic anomaly
Central conducting lymphatic anomalies (CCLA) occur when there is a disruption of central lymphatic flow, resulting in complications such as non-immune fetal hydrops, chylothorax, chylous ascites, protein-losing enteropathy, other effusions, or lymphedema. The heterogeneity of CCLA complicates diagnosis, treatment, and prognostication. Understanding the molecular etiology of a patient’s disease can improve medical care, including novel treatment strategies. However, few genetic causes have been identified for CCLA. Clinical geneticists use distinct facial features to assist in diagnosis of rare disorders. Given the recent advances in lymphatic imaging, we sought to understand whether we could use features identified by dynamic contrast magnetic resonance imaging for diagnosing CCLA. We discovered that only about a quarter of patients with CCLA have an underlying genetic diagnosis that can be identified by routine clinical evaluation. We also demonstrated that germline RASopathies (group of genetic syndromes caused by germline mutations in genes that encode components or regulators of the Ras/mitogen-activated protein kinase pathway), mosaic KRASopathies (a disorder caused by a somatic pathogenic variant in KRAS), PIEZO1 (a gene that encodes a mechanically activated ion channel that links mechanical forces to biological signal)–related lymphatic dysplasia, and Trisomy 21 have distinct central lymphatic flow phenotypes. In recent published work [Reference 2], we developed novel techniques such as the use of cfDNA and identified novel causes for CCLA and other complex lymphatic anomalies. We also collaborated on the spectrum of complex lymphatic anomalies caused by PIK3CA.
Cellular and molecular mechanisms of lymphatic disorders
We published work [Reference 1] demonstrating that activating variants in KRAS can drive lymphatic malformations in the zebrafish, which can be treated with MEK (kinase that phosphorylates mitogen-activated protein kinase) inhibitors. We identified several genetic causes of CCLA. We are evaluating these novel potential causes to understand their effect on the cellular and molecular mechanisms driving lymphatic development and identify new therapies.
Natural history study of lymphatic disorders
We are developing a prospective natural history study for patients with lymphatic anomalies to systematically evaluate the disease phenotypes and long-term outcomes. This will allow us to provide improved prognostication to families, establish screening/monitoring guidelines, determine best practices for genetic diagnosis, and explore family opinions and fertility for those on long-term medication management. The study will allow us to identify novel end-points for future clinical trials.
Genotype-phenotype correlations in central conducting lymphatic anomaly
Central conducting lymphatic anomalies (CCLA) occur when there is a disruption of central lymphatic flow resulting in complications such as non-immune fetal hydrops, chylothorax, chylous ascites, protein-losing enteropathy, other effusions, or lymphedema. The heterogeneity of CCLA complicates diagnosis, treatment, and prognostication. Understanding the molecular etiology of a patient’s disease can drive medical care, including novel treatment strategies. However, few genetic causes have been identified for CCLA. Clinical geneticists use distinct facial features to assist in diagnosis of rare disorders. Given the recent advances in lymphatic imaging, we sought to understand whether we could use features identified by dynamic contrast magnetic resonance imaging for diagnosing CCLA. We discovered that only about a quarter of patients with CCLA have an underlying genetic diagnosis that can be identified by routine clinical evaluation. We also demonstrated that germline RASopathies, mosaic KRASopathies, PIEZO1–related lymphatic dysplasia, and Trisomy 21 have distinct central lymphatic flow phenotypes.
Figure 2. Clinical imaging of lymphatic anomalies according to genotype
Click image to view.
T2 space and dynamic contrast MR lymphangiography (DCMRL) from seven different genotypes, illustrating lymphatic conduction abnormalities.
A. Mosaic BRAF (p.Val600Glu): T2 space shows significant edema in the intercostal, mesentery, and liver lymphatics (left panel) (arrows) that correlates with abnormal perfusion patterns on intrahepatic DCMRL (right). Also note the abnormal lymphatic thoracic vessels with absence of a normal thoracic duct (BRAF encodes a serine/threonine kinase involved in cell growth and survival).
B. Mosaic KRAS (p.Gly12Asp): There is edema on T2 space within the mediastinum and lungs (arrows). Patient also with cystic right kidney (asterisk). Intrahepatic DCMRL demonstrates correlation with mediastinal, pulmonary, and supraclavicular edema, with perfusion of dilated lymphatic structures. Of note, this patient has a central thoracic duct (arrow heads), but it was not patent to the venous circulation on ultrasound contrast imaging.
C. Noonan syndrome (PTPN11 p.Gln510His): T2 space imaging demonstrating mediastinal and intercostal edema predominately. With intranodal DCMRL, these areas correlate with abnormal perfusion (arrows). Again, note there is no central thoracic duct, but persistent pulmonary and intercostal perfusion.
D. Trisomy 21: T2 space imaging with edema in the supraclavicular and superior mediastinal lymphatics (arrows). On intrahepatic DCMRL, there is retrograde flow into retroperitoneal lymphatics, intercostal, mediastinal, pulmonary, and supraclavicular perfusion (arrows). There is a patent thoracic duct that courses to the left venous angle (arrowhead).
E. PIEZO1: T2 space shows bilateral pleural effusions and pulmonary and retroperitoneal edema (arrows). Intrahepatic DCMRL shows extensive flow to the hepatic capsular lymphatics, with extension into the mediastinum and pulmonary lymphatics (arrows). There is also retrograde flow into the retroperitoneal lumbar and mesenteric lymphatics. A small thoracic duct can be seen coursing to the left venous angle (arrow head), patent on follow-up imaging.
F. Gaucher’s disease Type III: T2 space notable for ascites. Intrahepatic DCMRL shows retrograde perfusion to retroperitoneal lumbar lymphatics and mesentery (arrows). The thoracic duct is mildly dilated and tortuous as it courses to the left venous angle (arrowhead).
G. Andersen's disease: T2 space imaging with significant ascites, pleural effusions, and anasarca (arrows). With intranodal DCMRL, there is extensive dermal perfusion and dilated retroperitoneal lymphatics. A thoracic duct is present and mildly dilated and tortuous (arrowhead). Figure from Liu M, Smith CL, Biko DM et al, Eur J Hum Genet 2022;30:1022.
Cellular and molecular mechanism of lymphatic disorders
Previously, I demonstrated that activating variants in KRAS can drive lymphatic malformations in the zebrafish, which can be treated with MEK inhibitors. We identified several genetic causes of CCLA. We will evaluate these novel potential causes to understand their effect on the cellular and molecular mechanisms driving lymphatic development and identify new therapies.
Additional Funding
- NIH Distinguished Scholars Program
- Uplifting Athletes/LGDA
Publications
- Sheppard SE, March ME, Seiler C, Matsuoka LS, Kim SE, Kao C, Rubin AI, Battig MR, Khalek N, Schindewolf E, O'Connor N, Pinto E, Priestley JR, Sanders VR, Niazi R, Ganguly A, Hou C, Slater D, Frieden IJ, Huynh T, Shieh JT, Krantz ID, Guerrero JC, Surrey LF, Biko DM, Laje P, Castelo-Soccio L, Nakano TA, Snyder K, Smith CL, Li D, Dori Y, Hakonarson H. Lymphatic disorders caused by mosaic, activating KRAS variants respond to MEK inhibition. JCI Insight 2023 8(9):e155888.
- Li D, Sheppard SE, March ME, Battig MR, Surrey LF, Srinivasan AS, Matsuoka LS, Tian L, Wang F, Seiler C, Dayneka J, Borst AJ, Matos MC, Paulissen SM, Krishnamurthy G, Nriagu B, Sikder T, Casey M, Williams L, Rangu S, O'Connor N, Thomas A, Pinto E, Hou C, Nguyen K, Pellegrino da Silva R, Chehimi SN, Kao C, Biroc L, Britt AD, Queenan M, Reid JR, Napoli JA, Low DM, Vatsky S, Treat J, Smith CL, Cahill AM, Snyder KM, Adams DM, Dori Y, Hakonarson H. Genomic profiling informs diagnoses and treatment in vascular anomalies. Nat Med 2023 29(6):1530–1539.
- Nriagu BN, Williams LS, Brewer N, Surrey LF, Srinivasan AS, Li D, Britt A, Treat J, Crowley TB, O'Connor N, Ganguly A, Low D, Queenan M, Drivas TG, Zackai EH, Adams DM, Hakonarson H, Snyder KM, Sheppard SE. Microcystic lymphatic malformations in Turner syndrome are due to somatic mosaicism of PIK3CA. Am J Med Genet A 2023 doi: 10.1002/ajmg.a.63385.
- Grenier JM, Borst AJ, Sheppard SE, Snyder KM, Li D, Surrey LF, Al-Ibraheemi A, Weber DR, Treat JR, Smith CL, Laje P, Dori Y, Adams DM, Acord M, Srinivasan AS. Pathogenic variants in PIK3CA are associated with clinical phenotypes of kaposiform lymphangiomatosis, generalized lymphatic anomaly, and central conducting lymphatic anomaly. Pediatr Blood Cancer 2023 e30419.
- Zarowin D, Heymann WR, Yan AC, Treat J, Sheppard SE. Segmental vasoconstricted patches with a border of telangiectasia. Pediatr Dermatol 2023 40(3):565–567.
- Bolli A, Nriagu B, Britt AD, Toole AD, Treat J, Srinivasan A, Sheppard SE. Mosaic pathogenic variants in AKT3 cause capillary malformation and undergrowth. Am J Med Genet A 2023 191(5):1442–1446.
Contact
For more information, email sarah.sheppard@nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/sheppard.
