Background Capillary malformation-arteriovenous malformation is an autosomal dominant disorder, characterised by capillary malformations and increased risk of fast-flow vascular malformations, caused by loss-of-function mutations in the RASA1 or EPHB4 genes. Around 25% of the patients do not seem to carry a germline mutation in either one of these two genes. Even if other genes could be involved, some individuals may have mutations in the known genes that escaped detection by less sensitive techniques. We tested the hypothesis that mosaic mutations could explain some of previously negative cases. Methods DNA was extracted from peripheral blood lymphocytes, saliva or vascular malformation tissues from four patients. RASA1 and EPHB4 coding regions and exon/intron boundaries were analysed by targeted custom gene panel sequencing. A second panel and/or Sanger sequencing were used to confirm the identified mutations. Results Four distinct mosaic RASA1 mutations, with an allele frequency ranging from 3% to 25%, were identified in four index patients with classical capillary malformation-arteriovenous malformation phenotype. Three mutations were known, one was novel. In one patient, a somatic second hit was also identified. One index case had three affected children, illustrating that the mosaicism was also present in the germline. Conclusion This study shows that RASA1 mosaic mutations can cause capillary malformation-arteriovenous malformation. Thus, highly sensitive sequencing techniques should be considered as diagnostic tools, especially for patients with no family history. Even low-level mosaicism can cause the classical phenotype and increased risk for offspring. In addition, our study further supports the second-hit pathophysiological mechanism to explain the multifocality of vascular lesions in this disorder.

Primary lymphedema (PLE) is phenotypically heterogeneous and partially explained by mutations in 28 genes. In Nonne-Milroy disease (OMIM 153100), PLE appears typically at birth and is autosomal dominant with incomplete penetrance. Several mutations have been discovered in VEGFR3, but only 2 in its ligand VEGFC (c.571_572insTT; p.Pro191Leufs*10 and c.628C>T; p.Arg210*; Figure 1G), causing Milroy-like disease (OMIM 615907). VEGFC regulates lymphangiogenesis upon proper activation by ADAMTS3 and CCBE1, mutations in which cause Hennekam lymphangiectasia-lymphedema syndrome . We sequenced VEGFC (NM_005429.4) in 542 index patients with any kind of PLE using Ion Torrent technology. All participants gave their informed consent, as approved by the Medical Faculty ethical committee at University of Louvain, Brussels, Belgium or local committees of collaborators. With Highlander (http://sites.uclouvain.be/highlander/), we selected variants ≤0.0014 in ExAC, not in dbSNP, and predicted to affect splicing or being damaged by at least 5 softwares (Sift (http://sift. jcvi.org), Mutation Taster (http://www.mutationtaster.org), PolyPhen2_hdiv, PolyPhen2_hvar (http://genetics.bwh.harvard.edu/pph2/), Lrt (http://www.genetics.wustl.edu/jflab/lrt_query.html) and Mutation Assessor (http://mutationassessor.org/r3/)). We identified 2 heterozygous mutations, chr4:177650901delTG (c.148-3_148-2delCA) and chr4:177648932C>T (c.552G>A), which are rare in gnomAD (7/211120 alleles and 1/242972, respectively, both predicted to alter mRNA splicing (c.552G>A is the last nucleotide of exon 3). Ten other loss-offunction VEGFC variants are reported in gnomAD. Some of these could undergo gene rescue, whereas lymphedema might be undiagnosed in other individuals. c.148-3_148-2delCA was found in a 22 years old male (LE-627III-1, Figure 1A) with no familial history of PLE. Diagnosed at 7 years of age with left lower limb swelling, he presented, 1 year later, with bilateral lower extremity lymphedema and regular episodes of lymphangitis (Figure 1B,C). At 9 years of age, he developed hydrocele, which was operated. He follows intensive physiotherapy and wears elastic garments. Lymphoscintigraphy revealed absence of lymph nodes in the right groin, suggestive of aplasia/hypoplasia, and hypoplastic lymphatics in the left limb, with backflow or rerouting in both legs, more marked on the left (Figure 1D). This had not been seen in the previously reported families. The mutation was also detected in the unaffected father, but neither in the healthy grandmother nor in the aunt who had varicose veins. Thus, penetrance was 50% in this family. c.552G>A was discovered in LE-445-V-1, 6 other affected persons and unaffected younger sister (Figure 1E). Inheritance was autosomal dominant with 87.5% penetrance. Male members were more severely affected. At birth, LE-445-V-1 had lymphedema on the dorsum of right foot. Edema of left foot appeared after a fracture. Lymphedema progressed to legs but below the knees (Figure 1F). Congenital lymphedema of right foot of his brother progressed to bilateral below knees. The granduncle, his son, and an aunt also developed lymphedema of feet and ankles. In contrast, mother and grandmother had only swollen ankles during pregnancies or warm weather. The first recorded case was the great-grandmother with lymphedema in one leg since 12 years of age, progressing to bilateral below knees. Lymphocytic mRNA of LE-627-III-1 showed a smaller amplicon (Figure 1H), corresponding to skipping of exons 2 and 3 (Figure 1I), and deletion of 135 of the 419 aa. This removes most of the propeptide and the VEGF-homology domain (VHD), namely the core for VEGFC activity after cleavage (Figure 1G). As exon 3 deletion was unexpected, we excluded a genomic deletion in the region by detecting 3 heterozygous single-nucleotide polymorphisms (Figure 1G and not shown). In LE-445V-1 RNA (Figure 1H), exon 3 was missing (Figure 1J), leading to frameshift and premature stop-codon. This truncation alters the VHD similar to previous mutations (Figure 1G). If these mutant proteins are not unstable, they would still be nonfunctional. Our novel mutations underscore VEGFC haploinsufficiency as the pathophysiological mechanism. Variable expressivity and reduced penetrance were observed in both families, albeit LE-445V-3 could develop PLE with age. Similarly, 2 individuals of the first reported family had a mutation but no clinical signs of leg edema, albeit prominent veins, or hydrocele. Thus, variants in other components of the VEGFC-VEGFR3 axis, such as ADAMTS3, or environmental factors may act as modifiers. VEGFC accounts for ~0.4% of 542 patients screened and VEGFR3 for ~10%. In several PLE genes, only few mutations have been found, underscoring pathophysiological heterogeneity. VEGFC screening should be considered for precise diagnosis, management, and counseling of patients with PLE, as well as to refine the phenotypic spectrum linked to VEGFC mutations. Received: 8 September 2017 Revised: 19 December 2017 Accepted: 21 December 2017

Geert Mortier Department of Medical Genetics Antwerp University Hospital Prins Boudewijnlaan 43 B–2650 Antwerp (Belgium) Tel. +32 3 275 9773 (secretary) Tel. +32 3 275 9766 (direct) Fax +32 3 275 9723 E-mail: geert.mortier@ua.ac.be Maximilian Muenke Medical Genetics Branch National Human Genome Research Institute National Institutes of Health 35 Convent Drive, MSC 3717 Building 35, Room 1B-203 Bethesda, MD 20892-3717 (USA) Tel. +1 301 402 8167; Fax +1 301 480 7876 E-mail: mamuenke@mail.nih.gov

Lymphedema is caused by dysfunction of lymphatic vessels, leading to disabling swelling that occurs mostly on the extremities. Lymphedema can be either primary (congenital) or secondary (acquired). Familial primary lymphedema commonly segregates in an autosomal dominant or recessive manner. It can also occur in combination with other clinical features. Nine mutated genes have been identified in different isolated or syndromic forms of lymphedema. However, the prevalence of primary lymphedema that can be explained by these genetic alterations is unknown. In this study, we investigated 7 of these putative genes. We screened 78 index patients from families with inherited lymphedema for mutations in FLT4, GJC2, FOXC2, SOX18, GATA2, CCBE1, and PTPN14. Altogether, we discovered 28 mutations explaining 36% of the cases. Additionally, 149 patients with sporadic primary lymphedema were screened for FLT4, FOXC2, SOX18,CCBE1, and PTPN14. Twelve mutations were found that explain 8% of the cases. Still unidentified is the genetic cause of primary lymphedema in 64% of patients with a family history and 92% of sporadic cases. Identification of those genes is important for understanding of etiopathogenesis, stratification of treatments and generation of disease models. Interestingly, most of the proteins that are encoded by the genes mutated in primary lymphedema seem to act in a single functional pathway involving VEGFR3 signaling. This underscores the important role this pathway plays in lymphatic development and function and suggests that the unknown genes also have a role.