Arteriovenous malformation Map2k1 mutation affects vasculogenesis.

Somatic activating MAP2K1 mutations in endothelial cells (ECs) cause extracranial arteriovenous malformation (AVM). We previously reported the generation of a mouse line allowing inducible expression of constitutively active MAP2K1 (p.K57N) from the Rosa locus (R26GT-Map2k1-GFP/+) and showed, using Tg-Cdh5CreER, that EC expression of mutant MAP2K1 is sufficient for the development of vascular malformations in the brain, ear, and intestines. To gain further insight into the mechanism by which mutant MAP2K1 drives AVM development, we induced MAP2K1 (p.K57N) expression in ECs of postnatal-day-1 pups (P1) and investigated the changes in gene expression in P9 brain ECs by RNA-seq. We found that over-expression of MAP2K1 altered the transcript abundance of > 1600 genes. Several genes had > 20-fold changes between MAP2K1 expressing and wild-type ECs; the highest were Col15a1 (39-fold) and Itgb3 (24-fold). Increased expression of COL15A1 in R26GT-Map2k1-GFP/+; Tg-Cdh5CreER+/- brain ECs was validated by immunostaining. Ontology showed that differentially expressed genes were involved in processes important for vasculogenesis (e.g., cell migration, adhesion, extracellular matrix organization, tube formation, angiogenesis). Understanding how these genes and pathways contribute to AVM formation will help identify targets for therapeutic intervention.

Endothelial cell expression of mutant Map2k1 causes vascular malformations in mice.

Extracranial arteriovenous malformation (AVM) is a congenital vascular anomaly causing disfigurement, bleeding, ulceration, and pain. Most lesions are associated with somatic MAP2K1 activating mutations in endothelial cells (ECs). The purpose of this study was to determine if EC expression of mutant activated MAP2K1 is sufficient to produce vascular malformations in mice. We generated mice with a ROSA26 allele containing a lox-stop-lox gene trap (GT), Map2k1 cDNA with an activating p.K57N missense mutation, an internal ribosomal entry site, and green fluorescent protein cDNA (R26GT-Map2k1-GFP). We expressed mutant MAP2K1 and GFP in ECs of fetal and newborn mice using Tg-Cdh5Cre or Tg-Cdh5CreER alleles. Tg-Cdh5Cre+/-;R26GT-Map2k1-GFP/+ animals that express mutant MAP2K1 in ECs in utero developed diffuse vascular abnormalities and died by embryonic (E) day 16.5. Tg-Cdh5CreER+/-;R26GT-Map2k1-GFP/+ animals in which mutant MAP2K1 expression was induced in ECs by tamoxifen at postnatal (P) day 1 developed vascular malformations in the brain, ear, and intestines by P23. The lesions consisted of abnormal networks of blood vessels containing recombined and non-recombined ECs. In conclusion, expression of MAP2K1 p.K57N is sufficient to cause vascular malformations in mice. This model can be used to study the malformation process and for pre-clinical pharmacologic studies.

Exploring Medical Students' Perceptions of Organ Procurement: Need for a Formalized Medical Student Curriculum.

OBJECTIVE: We evaluated the medical student experience with a deceased-donor multiorgan procurement program at a single center. The program provided the opportunity to assist with organ procurement, but no formal curriculum was offered. DESIGN, SETTING, PARTICIPANTS: In 2018, medical students who registered for the program between 2014 and 2017 completed a voluntary survey about the experience and its impact on surgery interest and organ donation knowledge and advocacy. RESULTS: Of 139 respondents, 53.3% (N = 74) of students participated in at least one procurement. The experience was resoundingly positive: 81.7% (N = 58) believed it exceeded expectations, with less than one-third missing class and only 4.3% (N = 3) reporting a negative impact on academics. Although 60.6% (N = 43) students studied prior to procurement, 57.8% (N = 41) expressed the need for increased preparation. Preferred learning modalities included videos, discussion with the transplant fellows, and focused anatomy overview. Following participation, 53.5% (N = 38) of students had increased interest in pursuing an acting internship and career in surgery. However, participation was not associated with improved familiarity with organ donation concepts or advocacy. CONCLUSIONS: Adding a structured curriculum may turn medical students from passive observers into active learners, maximizing the educational value of procurement and better equipping future providers to promote organ donation.

Somatic PIK3CA mutations are present in multiple tissues of facial infiltrating lipomatosis.

BackgroundFacial infiltrating lipomatosis (FIL) is a congenital disorder that causes overgrowth of one side of the face. The purpose of this study was to determine whether PIK3CA mutations are present in tissues outside of the subcutaneous adipose.MethodsFIL tissues from three patients were dissected to enrich for cells from skin, subcutaneous tissue, orbicularis oris muscle, buccal fat, zygomatic bone, and mucosal neuroma. Endothelial cells within the affected tissue also were enriched using CD31 microbeads. Laser capture microdissection on formalin-fixed paraffin-embedded histologic sections was performed to collect specific cell types. DNA was extracted from each tissue and cell type, and measured for the abundance of mutant PIK3CA alleles using droplet digital PCR.ResultsWe detected mutant PIK3CA alleles in every tissue and cell type tested from each overgrown face; frequencies ranged from 1.5 to 53%. There were fewer mutant endothelial cells compared with nonendothelial cells, and the stromal cell compartment had the highest frequency of mutant cells in each tissue.ConclusionsPIK3CA mutations are not restricted to a single tissue or cell type in FIL. Overgrowth in this condition is likely due to the mutation arising in a cell that contributes to several different facial structures during embryogenesis.

Endothelial Cells from Capillary Malformations Are Enriched for Somatic GNAQ Mutations.

BACKGROUND: A somatic mutation in GNAQ (c.548G>A; p.R183Q), encoding Gαq, has been found in syndromic and sporadic capillary malformation tissue. However, the specific cell type containing the mutation is unknown. The purpose of this study was to determine which cells in capillary malformations have the GNAQ mutation. METHODS: Human capillary malformation tissue was obtained from 13 patients during a clinically indicated procedure. Droplet digital polymerase chain reaction, capable of detecting mutant allelic frequencies as low as 0.1 percent, was used to quantify the abundance of GNAQ mutant cells in capillary malformation tissue. Six specimens were fractionated by fluorescence-activated cell sorting into hematopoietic, endothelial, perivascular, and stromal cells. The frequency of GNAQ mutant cells in these populations was quantified by droplet digital polymerase chain reaction. RESULTS: Eight capillary malformations contained GNAQ p.R183Q mutant cells, two lesions had novel GNAQ mutations (p.R183L and p.R183G), and three capillary malformations did not have a detectable GNAQ p.R183 mutation. Mutant allelic frequencies ranged from 2 to 11 percent. Following fluorescence-activated cell sorting, the GNAQ mutation was found in the endothelial but not the platelet-derived growth factor receptor-ß-positive cell population; mutant allelic frequencies were 3 to 43 percent. CONCLUSION: Endothelial cells in capillary malformations are enriched for GNAQ mutations and are likely responsible for the pathophysiology underlying capillary malformation.

A somatic MAP3K3 mutation is associated with verrucous venous malformation.

Verrucous venous malformation (VVM), also called "verrucous hemangioma," is a non-hereditary, congenital, vascular anomaly comprised of aberrant clusters of malformed dermal venule-like channels underlying hyperkeratotic skin. We tested the hypothesis that VVM lesions arise as a consequence of a somatic mutation. We performed whole-exome sequencing (WES) on VVM tissue from six unrelated individuals and looked for somatic mutations affecting the same gene in specimens from multiple persons. We observed mosaicism for a missense mutation (NM_002401.3, c.1323C>G; NP_002392, p.Iso441Met) in mitogen-activated protein kinase kinase kinase 3 (MAP3K3) in three of six individuals. We confirmed the presence of this mutation via droplet digital PCR (ddPCR) in the three subjects and found the mutation in three additional specimens from another four participants. Mutant allele frequencies ranged from 6% to 19% in affected tissue. We did not observe this mutant allele in unaffected tissue or in affected tissue from individuals with other types of vascular anomalies. Studies using global and conditional Map3k3 knockout mice have previously implicated MAP3K3 in vascular development. MAP3K3 dysfunction probably causes VVM in humans.

Lymphatic and other vascular malformative/overgrowth disorders are caused by somatic mutations in PIK3CA.

OBJECTIVES: To test the hypothesis that somatic phosphatidylinositol-4,5-bisphospate 3-kinase, catalytic subunit alpha (PIK3CA) mutations would be found in patients with more common disorders including isolated lymphatic malformation (LM) and Klippel-Trenaunay syndrome (KTS). STUDY DESIGN: We used next generation sequencing, droplet digital polymerase chain reaction, and single molecule molecular inversion probes to search for somatic PIK3CA mutations in affected tissue from patients seen at Boston Children's Hospital who had an isolated LM (n = 17), KTS (n = 21), fibro-adipose vascular anomaly (n = 8), or congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (n = 33), the disorder for which we first identified somatic PIK3CA mutations. We also screened 5 of the more common PIK3CA mutations in a second cohort of patients with LM (n = 31) from Seattle Children's Hospital. RESULTS: Most individuals from Boston Children's Hospital who had isolated LM (16/17) or LM as part of a syndrome, such as KTS (19/21), fibro-adipose vascular anomaly (5/8), and congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (31/33) were somatic mosaic for PIK3CA mutations, with 5 specific PIK3CA mutations accounting for ∼ 80% of cases. Seventy-four percent of patients with LM from Seattle Children's Hospital also were somatic mosaic for 1 of 5 specific PIK3CA mutations. Many affected tissue specimens from both cohorts contained fewer than 10% mutant cells. CONCLUSIONS: Somatic PIK3CA mutations are the most common cause of isolated LMs and disorders in which LM is a component feature. Five PIK3CA mutations account for most cases. The search for causal mutations requires sampling of affected tissues and techniques that are capable of detecting low-level somatic mosaicism because the abundance of mutant cells in a malformed tissue can be low.

Expression of follicle-stimulating hormone receptor in vascular anomalies.

BACKGROUND: The mechanism for the growth of infantile hemangioma and vascular malformations is unknown. Follicle-stimulating hormone secretion mirrors the life cycle of infantile hemangioma and increases during adolescence, when vascular malformations often progress. The purpose of this study was to determine whether vascular anomalies express the receptor for follicle-stimulating hormone. METHODS: Human vascular tumors (i.e., infantile hemangioma, congenital hemangioma, kaposiform hemangioendothelioma, and pyogenic granuloma) and vascular malformations (i.e., capillary, lymphatic, venous, and arteriovenous) were subjected to immunofluorescence for follicle-stimulating hormone receptor. Control specimens included normal skin/subcutis, mucosa, liver, spleen, Crohn disease, granulation, pancreatitis, rheumatoid arthritis, and synovitis. Receptor and microvessel density were quantified using imaging software. RESULTS: Follicle-stimulating hormone receptor was found in the endothelium of all vascular anomalies but was not present in control specimens. Expression was greater in proliferating infantile hemangioma (6.0 percent) compared with other vascular tumors (congenital hemangioma, 0.61 percent; kaposiform hemangioendothelioma, 0.55 percent; pyogenic granuloma, 0.56 percent; p < 0.0001), despite similar microvessel density (p = 0.1). Follicle-stimulating hormone receptor was elevated in arteriovenous malformations (2.65 percent) compared with other types of vascular malformations (capillary, 1.02 percent; lymphatic, 0.38 percent; venous, 0.76 percent; p < 0.0001). CONCLUSIONS: Vascular anomalies express follicle-stimulating hormone receptor on their endothelium, in contrast to vascular control tissues. Vascular anomalies are the only benign, pathologic tissue known to express this receptor. Because the secretion of follicle-stimulating hormone correlates with the growth pattern of infantile hemangioma and vascular malformations, follicle-stimulating hormone might be involved in the pathogenesis of these lesions.

PIK3CA activating mutations in facial infiltrating lipomatosis.

BACKGROUND: Facial infiltrating lipomatosis is a nonheritable disorder characterized by hemifacial soft-tissue and skeletal overgrowth, precocious dental development, macrodontia, hemimacroglossia, and mucosal neuromas. The authors tested the hypothesis that this condition is caused by a somatic mutation in the phosphatidylinositide-3 kinase (PI3K) signaling pathway, which has been indicted in other anomalies with overgrowth. METHODS: The authors extracted DNA from abnormal tissue in six individuals, generated sequencing libraries, enriched the libraries for 26 genes involved in the PI3K pathway, and designed and applied a sequential filtering strategy to analyze the sequence data for mosaic mutations. RESULTS: Unfiltered sequence data contained variant reads affecting ~12 percent of basepairs in the targeted genes. Filtering reduced the fraction of targeted basepairs containing variant reads to ~0.008 percent, allowing the authors to identify causal missense mutations in PIK3CA (p.E453K, p.E542K, p.H1047R, or p.H1047L) in each affected tissue sample. CONCLUSIONS: Affected tissue from individuals with facial infiltrating lipomatosis contains PIK3CA mutations that have previously been reported in cancers and in affected tissue from other nonheritable, overgrowth disorders, including congenital lipomatous overgrowth, vascular, epidermal, and skeletal anomalies syndrome, Klippel-Trenaunay syndrome, hemimegalencephaly, fibroadipose overgrowth, and macrodactyly. Because PIK3CA encodes a catalytic subunit of PI3K, and in vitro studies have shown that the overgrowth-associated mutations increase this enzyme's activity, PI3K inhibitors currently in clinical trials for patients with cancer may have a therapeutic role in patients with facial infiltrating lipomatosis. The strategy used to identify somatic mutations in patients with facial infiltrating lipomatosis is applicable to other somatic mosaic disorders that have allelic heterogeneity.