Cellular and Molecular Mechanisms of Vascular Malformations

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

A prospective natural history study of lymphatic anomalies We developed a prospective natural history study for patients with lymphatic anomalies to systematically evaluate the disease phenotypes and long-term outcomes. This study will allow us to provide improved prognostication to families, establish screening/monitoring guidelines, determine best practices for genetic diagnosis, and explore family opinions and explore fertility for those on long term medication management. This study will allow us to identify novel end points for future clinical trials. This study is currently recruiting and has enrolled 81 total participants (as of August 2025). Previously, we partnered with a family advocacy organization to create a prenatal support guide to help families with rare disease in the prenatal period. This is available at https://www.cmtc.nl/en/parents-support-guide/ A virtual support session intervention for caregivers of family members with rare disease inspires mothers to create their own support group Parents and caregivers of children with rare genetic conditions face multiple challenges during their journey to manage the medical and psychosocial needs of their children. A pilot project designed to assess the efficacy of virtual support sessions for caregivers of children with rare conditions was led by Dr. Andrea Bowling, DNP, in my group. Participants provided informed consent to participate in the study “Impact of brief group session on anxiety and coping in primary caregivers of those with rare disorders.” Demographic information, practical and clinical significance of the sessions, and the most discussed topics of the session were analyzed quantitatively and qualitatively using questionnaires. The State portion of the State-Trait Anxiety Inventory measured anxiety and a subscale of the Coping Health Inventory for Parents (maintaining social support, self-esteem, and psychological stability) measured coping before and after three support sessions led by licensed facilitators. Although 11 parents/caregivers indicated interested, only four mothers of children with either Smith Lemli Opitz Syndrome or Complex Lymphatic Anomaly completed the study. Although the sample was too small for quantitative statistical analysis, the clinical impact was evident, as two of the participants created their own virtual support group which has grown to 91 participants. Further work is needed to systematically evaluate the need for virtual support groups for this population. Genotype-phenotype associations in vascular malformations My group has also contributed to the expansion of the phenotype of capillary malformation-arteriovenous malformation 2 syndrome, lymphatic anomalies caused by mosaic variants in BRAF, and verrucous venous malformation. Development of liquid biopsy for vascular anomalies Vascular anomalies are malformations or tumors of the blood or lymphatic vasculature and can be life-threatening. Although molecularly targeted therapies can be life-saving, identification of the molecular etiology is often impeded by lack of accessibility to affected tissue samples, mosaicism or insufficient sequencing depth. In a cohort of 356 participants with vascular anomalies, including 104 with primary complex lymphatic anomalies (pCLAs), DNA from CD31+ cells isolated from lymphatic fluid or cell-free DNA from lymphatic fluid or plasma underwent ultra-deep sequencing thereby uncovering pathogenic somatic variants down to a variant allele fraction of 0.15%. A molecular diagnosis, including previously undescribed genetic causes, was obtained in 41% of participants with pCLAs and 72% of participants with other vascular malformations, leading to a new medical therapy for 63% (43/69) of participants and resulting in improvement in 63% (35/55) of participants on therapy. Taken together, these data support the development of liquid biopsy-based diagnostic techniques to identify previously undescribed genotype-phenotype associations and guide medical therapy in individuals with vascular anomalies. We are currently using this research technology to evaluate individuals in the natural history study. Therapies for KRAS–related lymphatic disorders Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. Previous work in the lab showed that pathogenic, mosaic variants in KRAS cause central conducting lymphatic anomaly and found that for the KRAS p.G12D MEK inhibitors were efficacious (Sheppard et al, JCI Insight 2023). We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS. To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2-dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants. However, issues regarding therapies for KRAS–related lymphatic anomalies still remain. First, different variants in KRAS lead to up-regulation of MAPK signaling (a central signaling pathway) by different mechanisms. Second, the patients have different phenotypes. Third, previous patients with KRAS–related lymphatic anomalies have mixed responses to MEK inhibitors. Additional individuals with KRAS-related lymphatic anomalies were identified through the natural history study. Therefore, we developed three new zebrafish lymphatic anomaly models for three different KRAS–related variants which showed abnormalities in the lateral facial lymphatic and the thoracic duct. There are ongoing studies in the laboratory to evaluate the efficacy of additional therapeutics. Cellular and molecular mechanism of lymphatic disorders Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. Novel candidate genes and known genetic causes associated with disease must be validated to ensure that they truly cause disease. Mechanisms of disease must be elucidated for known causes of lymphatic disease as a first step toward identifying a precision-based treatment. Previously, we identified pathogenic variants in the RIT1 gene (RIT1 belongs to the Ras family of GTPases) as a novel cause of CCLA. However, the mechanisms that drive disease are unknown. Therefore, we created a mosaic zebrafish model of RIT1 lymphatic anomalies which in preliminary studies recapitulates the human phenotype with pericardial edema, cyst formation and interruption of blood flow. Studies are ongoing to determine the effect of this variant on the cellular and molecular mechanisms driving the lymphatic malformations, and, in the future will be used to identify therapies. SHOC2 contributes to the pathogenesis of central conducting lymphatic anomaly SHOC2 is a scaffolding protein that regulates RAS-MAPK signaling but has not yet been described CCLA. A patient with complex congenital heart disease presented with post-operative refractory chylothorax. Lymphatic imaging demonstrated abnormal intercostal lymphatic perfusion consistent with CCLA and suggestive of a RASopathy, a group of genetic disorders characterized by dysregulated RAS-MAPK signaling. Genetic testing showed a variant of uncertain significance (p.Ala308Val) in SHOC2. Trametinib was trialed without response. We generated transient transgenic zebrafish expressing the SHOC2 VUS in the developing lymphatic endothelium. Transgenic zebrafish expressing SHOC2 p.Ala308Val in the lymphovenous endothelium developed lymphatic malformations, confirming this variant contributed to CCLA. In concordance with the lack of clinical response to trametinib, immunoblot and immunostaining indicate that SHOC2 p.Ala308Val may be inducing lymphatic malformations through non-canonical mTOR signaling rather dysregulated RAS-MAPK signaling. In summary, SHOC2 is a novel cause of CCLA through non-canonical mTOR signaling and these data provide support for investigating the utility of additional pharmaceuticals that may inhibit mTOR signaling.