Novel EPHB4 mutation in capillary malformation-arteriovenous malformation syndrome 2 (CM-AVM2): the rst genetic study in Asians

Capillary malformation-arteriovenous malformation syndrome (CM-AVM), a rare vascular malformation, is autosomal dominant and characterized by hereditary capillary malformations (CMs) and potential fast-ow vascular malformation underlying CMs in around one-third of patients, affecting approximately 0.3-0.5% of newborns. CM-AVM was rst discovered in association with germline RASA-1 mutation and termed CM-AVM type 1 in 2003. Later, germline EPHB4 mutations were detected in RASA-1-negative CM-AVMs, also known as CM-AVM type 2. Although there have been recent genetic studies conrming the molecular basis of CM-AVM, few have focused on Asian populations, leading to the lack of comprehensive molecular proling. In this study, we report a female patient diagnosed as CM-AVM2 with a novel stop-gain mutation causing loss of function of the EPHB4 gene. As far as we know, this is the rst genetic report of germline EPHB4 loss-of-functional mutation related CM-AVM 2 in Asians, extending our understanding of CM-AVM.


Introduction
Capillary malformation-arteriovenous malformation (CM-AVM) syndrome, a vascular malformation, is known as a autosomal dominant disease that is present in 100,000 individuals 1 . CM-AVM is characterized with hereditary capillary malformations (CMs). Around one-third of affected individuals are associated with fast-ow vascular malformation lesions, including arteriovenous malformation (AVM) and arteriovenous stula (AVF). Capillary malformations (CMs) affected approximately 0.1-0.3% of newborns and are the most common type of vascular malformations 2 . CMs present as cutaneous, at, sporadic, unifocal, irregular, pink, red to purple patches anywhere in the body, particularly affecting the head and neck 3 . Although no proliferation was noted, CMs may gradually become darker and thicker with increasing age and do not regress spontaneously. In contrast, the atypical CM of CM-AVM is usually multiple, round to oral, small (1-2 cm) and pink to red 4  Although there have been recent genetic studies con rming the molecular basis of CM-AVM, few have focused on Asian populations. In this report, we described one female patient with CM-AVM 2 on the left arm and germline EPHB4 gene (c.1093C>T) mutation. As far as we know, this is the rst report of germline EPHB4 loss-of-functional mutation related CM-AVM 2 in Asians, extending our understanding of CM-AVM.

Methods
This study was authorized and approved by the Ethics Committee of Shanghai 9th People's Hospital A liated to Shanghai Jiaotong University School. We obtained oral and written informed consent from the patient's parents prior to the study. Detailed clinical data of the patient and her family were recorded.

Case Presentation
A three-year-old girl presented at our clinic with sporadic and multifocal erythema on her left arm, left shoulder and chest. According to her parents, the erythema is rstly apparent three month after birth on her left arm, and progressively extended to the left shoulder and chest area. The erythema was scattered with various shapes and sizes (maximum diameter was 3 cm) ( Figure 1a). Physical examination revealed higher skin temperature of the erythema than that of the adjacent normal skin. Besides, a mass with detectable palpation was found in the axillary lesion (Figure 1a, arrow). Family history disclosed that the proband's grandfather had a single patch of erythema on his right forearm that had not proliferated for several decades (Figure 1b). Neither erythema nor vascular disease was found in other family members of the proband.
Enhanced CT demonstrated multiple well-de ned nodular and lamellar masses in the axillary area. The masses were homogeneously enhanced after reinforcement. Besides, The vessels in the medial left arm were enlarged (Figure 1c,d). Considering the harm of anesthesia and radiation, digital subtraction angiography (DSA) examination was not performed on the patient.
To determine the genetic and histological alterations in this patient, we performed minimally invasive needle biopsy (D=2 mm, all layer biopsy) on the erythema lesion under local anesthesia (Penles and 1% xylocaine). One tissue specimen was stained with hematoxylin-eosin for histological analysis. Then, we performed targeted next-generation sequencing (NGS) in peripheral blood specimens as well as in tissue specimens.

Next-generation sequencing (NGS)
The targeted NGS gene panel was designed according to the ISSVA classi cation. 6 We designed a series of capture RNA baits in order to target the exons and the boundaries of the exons/introns of EPHB4. The SureSelect XT kit reagents (Agilent Technologies, Santa Clara, CA) were used for the Illumina adaptors.

Results
Histological study showed capillary hyperplasia in the and dermis (Fig.1e, HE, 100x).
Molecular analysis showed that a germline stop-gain mutation (c.1093C>T, p.Arg365*) in the EPHB4 gene mutation was identi ed in the samples from peripheral blood of the proband. The same mutation was also detected in the tissue sample of the proband. According to family history of erythema, physical examination of potential fast-ow lesions, CT results and genetic study revealing germline EPHB4 mutation, the patient was diagnosed with capillary malformation-arteriovenous malformation syndrome type 2 (CM-AVM2).

Discussion
Our proband had multifocal, progressive, pink, irregular erythema on the left arm left shoulder and chest. The erythema was consistent with atypical CMs in CM-AVM. The suspicion of CM-AVM further deepened according to physical examination that revealed the higher skin temperature in the erythema and detectable palpation of the mass located in the axillary lesion, and family history of inherited erythema. Enhancement CT demonstrated potential invasion of fast-ow lesions underlying the erythema. Moreover, the suspicion of CM-AVM was then con rmed with de nite diagnosis at the molecular level based on detection of germline loss-of-functional mutation of EPHB4.
EPHB4 belongs to the Eph transmembrane receptor tyrosine kinase family, and mainly expressed in the vein endothelial cell membrane and forms a bidirectional signaling pathway with its ligand, EFNB2, which usually expressed in the arterial endothelial cell membrane 7 . Bidirectional signaling plays an essential role in primary angiogenesis and arteriovenous differentiation [8][9][10] . Elena Groppa et al. discovered that activation of EPHB4 reduced the phosphorylation of ERK1/2 and the proliferation of endothelial cells, while blockade of EPHB4 caused activation of ERK1/2 and sprouting angiogenesis, which was regulated by ephrinB2 reverse signaling 11 . Thus, the stop-gain mutation of EPHB4 results in an increase in ERK1/2 phosphorylation and spouting angiogenesis. In this case, the mutation of EPHB4 was stopped, resulting in loss-of-function mutation of EPHB4 gene. The MAPK/ERK pathway plays an essential role in various processes, including proliferation, differentiation, migration, and apoptosis in a variety of cells 12 .
Furthermore, RASA1 is the downstream effector of EPHB4 in endothelial cells 13 . Together, both mutations of RASA1 and EPHB4 lead to similar atypical erythema and high-ow vascular malformations. In addition, the "two-hit" hypothesis is that the occurrence of a disease requires the inactivation of two alleles, which was proposed to explain the AVM present in 30% of CM-AVM caused by germline mutation from the parent and second mutation in somatic cells 4,5,14,15 . However, we did not nd any additional mutations in EPHB4 in this study to support this hypothesis.
EPHB4 mutation related CM-AVM2 was rstly reported and named in 2017 5 . According to the literature, CM-AVM usually manifests as atypical CMs that are cutaneous, multifocal, pink to purple in color, round to oval in shape, 1-3 cm in diameter, and are surrounded by pale halos 4,16,17 . Although the CMs of CM-AVM2 have similar characteristics compared with those of CM-AVM1, they are slightly different. Telangiectasia and bier spots usually presented in CM-AVM2 but not in CM-AVM1, and the pale central region can present in large CM in CM-AVM2 5 . In addition, AVM was discovered in the armpit of our patient, and fast-ow vascular malformation can occur in CM-AVM2 and CM-AVM1, but the risk of anomalies in the central system is lower than that in CM-AVM1 5 . Molecular diagnosis when necessary, combined with detailed clinical history as well as physical examination, is important for differential diagnosis of CM-AVM, risk assessment of fast-ow lesions, as well as monitoring of complications including heart failure, seizures, and life-threating hemorrhage.
Molecular analysis provides new management strategies beyond precautions preventing AVM formation and complication with bleomycin injection 18  In conclusion, we report, to the best of our knowledge, the rst case of CM-AVM type 2 with a novel germline EPHB4 mutation in East Asia. The molecular mechanism of CM-AVM2 may be that the mutation results in the loss of EPHB4 function, raising the reversal of the EPHB4/ephrinB2 bidirectional signaling pathway and inducing angiogenesis. Moreover, the occurrence of AVM requires a second mutation to cause a complete loss of EPHB4 function.

Conclusion
In this case, we report one female patient with a novel germline EPHB4 loss-of-function mutation as well as multiple CMs and AVMs, consistent with the diagnosis of CM-AVM2. According to our knowledge, this is the rst description of CM-AVM2 with a germline EPHB4 mutation in Asians. This nding would extending our understanding of the angiogenesis of CM-AVM type 2, especially in Asian populations.