# Assessment of gene-disease associations and recommendations for genetic testing for somatic variants in vascular anomalies by VASCERN-VASCA

Nicole Revencu, Astrid Eijkelenboom, Claire Bracquemart, Pia Alhopuro, Judith Armstrong, Eulalia Baselga, Claudia Cesario, Maria Lisa Dentici, Melanie Eyries, Sofia Frisk, et al.

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Nicole Revencu, Astrid Eijkelenboom, Claire Bracquemart, Pia Alhopuro, Judith Armstrong, et al. Assessment of gene-disease associations and recommendations for genetic testing for somatic variants in vascular anomalies by VASCERN-VASCA. Orphanet Journal of Rare Diseases, 2024, 19 (1), pp.213. 10.1186/s13023-024-03196-9. hal-04586607

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# Assessment of gene-disease associations and recommendations for genetic testing for somatic variants in vascular anomalies by VASCERN-VASCA

Nicole Revencu¹†, Astrid Eijkelenboom²†, Claire Bracquemart³†, Pia Alhopuro⁴, Judith Armstrong⁵, Eulalia Baselga⁶, Claudia Cesario⁷, Maria Lisa Dentici⁸, Melanie Eyries⁹, Sofia Frisk¹⁰, Helena Gásdal Karstensen¹¹, Nagore Gene-Olaciregui¹², Sirpa Kivirikko¹³, Cinzia Lavarino¹², Inger-Lise Mero¹⁴, Rodolphe Michiels¹, Elisa Pisanschi¹⁵, Bitten Schönewolf-Greulich¹¹, Ilse Wieland¹⁶, Martin Zenker¹⁶† and Miikka Vikkula¹⁷,18,19*†

**Abstract**

**Background** Vascular anomalies caused by somatic (postzygotic) variants are clinically and genetically heterogeneous diseases with overlapping or distinct entities. The genetic knowledge in this field is rapidly growing, and genetic testing is now part of the diagnostic workup alongside the clinical, radiological and histopathological data. Nonetheless, access to genetic testing is still limited, and there is significant heterogeneity across the approaches used by the diagnostic laboratories, with direct consequences on test sensitivity and accuracy. The clinical utility of genetic testing is expected to increase progressively with improved theragnostics, which will be based on information about the efficacy and safety of the emerging drugs and future molecules. The aim of this study was to make recommendations for optimising and guiding the diagnostic genetic testing for somatic variants in patients with vascular malformations.

**Results** Physicians and lab specialists from 11 multidisciplinary European centres for vascular anomalies reviewed the genes identified to date as being involved in non-hereditary vascular malformations, evaluated gene-disease associations, and made recommendations about the technical aspects for identification of low-level mosaicism and variant interpretation. A core list of 24 genes were selected based on the current practices in the participating laboratories, the ISSVA classification and the literature. In total 45 gene-phenotype associations were evaluated: 16 were considered definitive, 16 strong, 3 moderate, 7 limited and 3 with no evidence.

**Conclusions** This work provides a detailed evidence-based view of the gene-disease associations in the field of vascular malformations caused by somatic variants. Knowing both the gene-phenotype relationships and the strength

<footnote>†Nicole Revencu, Astrid Eijkelenboom and Claire Bracquemart contributed equally to this work.</footnote>

<footnote>†Martin Zenker and Miikka Vikkula contributed equally to this work.</footnote>

<footnote>*Correspondence:</footnote>

<footnote>Miikka Vikkula
miikka.vikkula@uclouvain.be</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
of the associations greatly help laboratories in data interpretation and eventually in the clinical diagnosis. This study reflects the state of knowledge as of mid-2023 and will be regularly updated on the VASCERN-VASCA website (VASCERN-VASCA, https://vascern.eu/groupe/vascular-anomalies/).

**Keywords** ERN, Gene curation, ISSVA, Mosaic, Precision medicine, Postzygotic, Somatic, VASCERN-VASCA, Vascular malformation

**Background**

Vascular anomalies comprise a heterogeneous group of disorders, divided into vascular tumours and vascular malformations [1]. The most widely used classification is that of the International Society for the Study of Vascular Anomalies [2], last updated in 2018. In the majority of cases, vascular malformations occur sporadically, but they may also be observed in several individuals in the same family as part of hereditary diseases or syndromes.

In the last 30 years, significant progress has been made in the elucidation of the genetic causes of vascular malformations. Studies initially focused on the aetiology of the hereditary forms. However, the discovery of somatic TEK variants in venous malformation tissues prompted the community to look for such changes in other types of vascular anomalies [3, 4]. The development of deep next-generation sequencing (NGS) techniques, and their increased use due to reduced costs, has facilitated the identification of somatic variants in various types of non-hereditary vascular anomalies. The major pathways involved in hereditary and non-hereditary forms are: 1) the RAS/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway, 2) the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway, and 3) the G-protein coupled receptor signalling pathway [5].

This work focuses on vascular malformations caused by somatic (postzygotic) variants, which represent the majority of patients with vascular malformations. These individuals often manifest unifocal/isolated or segmental vascular malformations. Body asymmetry (overgrowth, less commonly undergrowth) can be associated. The severity varies largely from harmless malformations to complex entities with significant morbidity, such as in CLOVES (congenital lipomatous overgrowth, vascular malformations, epidermal nevi, skeletal/scoliosis and spinal abnormalities), Klippel-Trenaunay syndrome, and Sturge-Weber syndrome. Multiple (multifocal) lesions are sometimes observed, such as in blue rubber bleb nevus syndrome and multifocal sporadic venous malformations, which complicates the diagnosis, as multiple lesions are usually a feature of a hereditary vascular malformation (e.g.,

capillary malformation-arteriovenous malformation, multiple cutaneous and mucosal venous malformations, cerebral cavernous malformation, etc.).

Pathogenic somatic variants in different genes can be associated with similar phenotypes, such as TEK and PIK3CA in venous malformations [3, 6, 7]. Likewise, somatic variants in the same gene can be associated with a variety of phenotypes. For instance, somatic variants in PIK3CA are associated with isolated lymphatic or venous malformation or with combined slow-flow vascular malformations, collectively known as PIK3CA-related overgrowth spectrum (PROS) [8].

Many genetic variants that have been identified as somatic changes in vascular malformations are also known as oncogenic variants in various cancer types. This has led to the idea that – pathway-specific pharmacological inhibitors known as cancer drugs could be repurposed for the treatment of patients with vascular malformations [9, 10] and some of them are already in off-label use or ongoing clinical trials. In addition, Alpelisib was granted accelerated approval from the Food and Drug Administration in 2022 for the treatment of patients aged 2 years or older with severe manifestations of PROS who require systemic therapy. Confirmatory evidence is needed for continued approval [11]. For these novel targeted treatment approaches, a precise molecular diagnosis is mandatory and requires genetic investigations alongside the clinical, radiological and histological assessment. Nonetheless, identifying the causative somatic genetic variants can be challenging, as they are only present in a subset of cells. Indeed, the tissue of a vascular malformation contains a mixture of mutated and non-mutated cells [12]. The percentage of cells carrying the pathogenic variant can be quite low, requiring representative test material and ultra-deep sequencing, which may limit the breadth of the gene panel content. In addition, there is a significant heterogeneity across the approaches currently used by the diagnostic laboratories in terms of technology, gene/exon/variant content, type and depth of sequencing, with direct consequences on the test sensitivity.

The aim of this work is to make recommendations to harmonise and optimise somatic genetic testing of patients with vascular malformations, performed in the participating centres, all members of
VASCERN-VASCA or providers of genetic testing for VASCERN-VASCA HCP [13]. The harmonisation mainly focused on a recommended gene content (genes, exons, regions and variants to be covered). Technical aspects for identification of low-level mosaicism were also considered, as well as variant interpretation. Germline genetic testing for hereditary vascular malformations was excluded from this work for two reasons: 1) there is a limited overlap of underlying genetic causes, and 2) the sequencing requirements are different.

# Methods

## Expert panel

The group of experts comprised physicians and lab specialists from 11 multidisciplinary centres, members of the working group on vascular anomalies (VASCA) of the European Reference Network on Multisystemic Vascular Diseases (VASCERN) or providers of genetic testing for VASCERN-VASCA HCP [13]. The expert group included geneticists (clinical and research), dermatologists and lab specialists in molecular genetics, pathology or oncology. Two task forces were formed: clinical and laboratory. The clinical task force was responsible for gene selection and evaluation of the gene-phenotype associations, and the laboratory task force, for selection of the variants and regions of interest, as well as the recommendations concerning the technical aspects (tissue selection, DNA isolation, sequencing techniques and variant interpretation). Virtual meetings of subgroups or the entire group were organised on a regular basis. The work started in 2020 and finished in mid-2023.

## Gene and phenotype selection

Genes of interest in the context of vascular malformations caused by somatic variants were identified through the gene lists of participating expert centres, the ISSVA classification, and a literature review. Only the genes asserted in the literature to be involved in vascular malformations caused by somatic events were considered. Genes involved in hereditary vascular malformations were excluded. If a gene had been reported to be involved both in vascular malformations and vascular tumours (ISSVA classification), the latter association was also assessed (e.g., GNAQ). In contrast, genes involved only in vascular tumours were not included (e.g., GNA14, FOS and FOSB). The ISSVA classification was used to define the list of phenotypes. Phenotypes described after the last update of this classification (e.g., GJA4-hepatic hae-mangioma, and GJA4-cutaneous venous malformation with cavernous histology) were added.

## Gene-phenotype association evaluation

The clinical validity of gene-phenotype associations was assessed using a modified framework based on the Gene Clinical Validity Curation Process Standard Operating Procedure version 8 [14], developed by the Clinical Genome Resource Gene Curation Working Group (Table S1). This framework is designed to collect, curate and score the evidence for a gene-disease relationship in two categories of evidence: genetic and experimental. Rules for the assessment and scoring of experimental evidence were adopted from the existing standard operating procedure. In contrast, the assessment of genetic evidence on the basis of case-level data required major adaptations, as the existing framework is not applicable for diseases caused by somatic genetic events. Based on the score, gene-phenotype associations were classified in a five-category system: no evidence (0 points), limited (0,5-5,5 points), moderate (6-11,5 points), strong (12-18 points), and definitive (12-18 points and replication over time).

Data reported in the literature were evaluated. Briefly, a basic score of 0.5 was given for a somatic variant in the respective gene in a case with the respective phenotype, for each curated gene-disease pair. Upgrades of +0.5 to +1.5 were given for different levels of evidence for the pathogenicity of the variant: strong prediction of a pathogenic effect by prediction tools: +0.5; proven functional effect of the variant: +0.5-1; variant known in ClinVar as likely pathogenic: +1; and variant known in ClinVar as pathogenic: +1.5. Additional upgrades of 0.5 to 1 were given if the variant detected in one case affected the neighbouring codons (+/- 2) or the same codon, respectively. Thereby, a somatic variant of known pathogenic effect, which affects the same codon as another scored variant reaches a maximum total score of 3. At least five independent observations were necessary to reach the maximum score of 12 for genetic evidence. The evaluation matrix is available as supplemental material (Table S1).

For the genetic evidence, the literature was reviewed either comprehensively or until the maximum score was reached (12 points). Total scores for genetic and functional evidence were calculated according to the published standard operating procedures. Based on the total score, a final classification of the clinical validity of a gene-disease association was proposed (no evidence – limited – moderate – strong – definitive). Classification as “definitive” evidence required replication by >2 publications over time (>3 yrs.). The evaluation of each gene-disease pair was done by one member of the clinical task force, followed by presentation and discussion in the panel. The final decision on the classification was taken collectively by the clinical task force. The expert panel
had the option to maintain or change (upgrade or down-
grade) the proposed classification that was based on the
scoring system. If the panel decided to change, the rea-
son was documented in the curation summary. This work
led to a list of genes for which the assessed evidence for
a certain phenotype association supported the considera-
tion as a diagnostic gene.

Selection of variants, exons and regions of interest

Based on the gene list constituted by the clinical task
force, the laboratory task force searched for all reported
somatic variants in these genes using the Leiden Open
Variation Database (LOVD: [15]) and ClinVar Miner
[16] databases, as well as PubMed and Google Scholar
with the keywords "gene name + vascular malformation",
"gene name + vascular anomaly" and "gene name + vas-
cular tumour". Variants were considered somatic if the
observed variant allele fraction (VAF) suggested mosaici-
cism or if a variant was present at different allele fractions
among different tissue types from the same individual.
After the literature and public database search, all mem-
bers of the laboratory task force were asked to compare
their in-house databases with the collected data and
complete the list with unreported variants, if any. Vari-
ants were then divided into two groups: 1) variants that
have been clearly associated with vascular anomalies in
the literature and/or databases; and 2) variants that had
emerging yet insufficient evidence, as well as variants
reported as somatic hotspots in cancer or germline hot-
spots in RASopathy genes and TEK. Cancer hotspot vari-
ants were defined through database queries in Catalogue
Of Somatic Mutation In Cancer (COSMIC: [17]) and
CancerHotspots [18]. Variants identified in the COSMIC
database with ≥ 20 documented instances, or in Cancer-
Hotspots with ≥ 10 documented instances, were consid-
ered potential oncogenic driver variants, in accordance
with previous definitions [19–21].

Technical aspects and variant analysis and interpretation

First, the specific approaches and techniques used by the
11 participating laboratories at the beginning of the work
were collected; these included input material, DNA iso-
lation method, sequencing technologies, gene panel con-
tent, sequencing platform, bioinformatics pipelines and
turn-around time. Based on our experience and a litera-
ture review, the laboratory task force drafted analytical
workflows that were discussed in the plenary meetings.
We provide an expert opinion to improve and standard-
ise laboratory approaches for somatic variant detection
and interpretation in the context of vascular malforma-
tions caused by postzygotic events.

Results

Gene curation

The formal curation process was applied to 24 genes (Table 1 and Supplementary Material 2). In total, 45 gene-phenotype associations were evaluated. Twelve genes were associated with more than one phenotype (vascular malformation or vascular tumour). Of the 45 evaluated gene-phenotype associations, 16 were considered definitive, 16 strong, 3 moderate 7 limited and 3 with no evidence.

Curation matrices for each gene-disease association
evaluation are provided as additional file (Supplementary
Material 2). There was at least one "definitive" gene-phe-
notype association for 10 genes (AKT1, GNA11, GNAQ,
IDH1, KRAS, MAP2K1, MAP3K3, NRAS, PIK3CA and
TEK). Out of the 24 genes, 11 were published after the
last update of the ISSVA classification (May 2018), of
which two have at least one "definitive" association
(KRAS and NRAS), five have at least one "strong" assو-
ciation (AKT3, GJA4, HRAS, PIK3R1 and PTPN11), and
four have "limited evidence" (ARAF, CBL, GNB2 and
PIK3CD). When the type of vascular malformation was
mixed or not specified in the reports, it was considered
under the term of "vascular malformation, various types"
(HRAS, NRAS - not linked to KLA).

Diagnostic target regions

Based on the review of reported disease-associated
variants per curated gene, we defined two levels of tar-
get regions per gene to be considered for diagnostic
testing of somatic variants in vascular malformations
(Table 2 and Table S2). The first level represents the mini-
mum content that should be included in a diagnostic
panel (Table 2). This covers pathogenic and likely path-
ogenic (P/LP) variants reported in the literature and/or
databases [15, 16] in patients with non-hereditary vas-
cular anomalies. No additional unreported variants have
been identified in the in-house databases from the par-
ticipating centres. For the delineation of the extended
regions of interest, a broader set of variants was accepted,
such as variants with insufficient evidence of pathogenicity,
and variants in patients with less well-characterised
phenotypes (Table S2). This selection also contains: i)
areas defined as hotspots in cancer but not yet observed
in vascular malformations, and ii) hotspot germline vari-
ants in RASopathy genes and TEK [22].

Technical aspects

The detection of somatic variants has special techni-
cal requirements. The approaches and techniques, from
sample collection to DNA extraction and sequencing
<table><caption>**Table 1** Gene-disease associations and strength of association</caption><thead><tr><th>Gene name</th><th>Disease</th><th>Disease</th><th>Disease</th><th>Disease</th><th>Disease</th></tr></thead><tbody><tr><td>AGGF1</td><td>Klippel-Trenaunay syndrome<br/>NO EVIDENCE</td><td></td><td></td><td></td><td></td></tr><tr><td>AKT1</td><td>Proteus syndrome<br/>DEFINITIVE</td><td></td><td></td><td></td><td></td></tr><tr><td>AKT3</td><td>CMTC variant (w/o (hemi)megalencephaly) or CM (with LM?) with hypotrophy<br/>STRONG</td><td></td><td></td><td></td><td></td></tr><tr><td>ARAF</td><td>CCLA<br/>LIMITED</td><td></td><td></td><td></td><td></td></tr><tr><td>BRAF</td><td>Arteriovenous malformation<br/>STRONG</td><td>(Macrocystic) Lymphatic malformation<br/>MODERATE</td><td>Pyogenic granuloma<br/>STRONG</td><td></td><td></td></tr><tr><td>CBL</td><td>Kaposiform lymphangiomatosis<br/>LIMITED</td><td></td><td></td><td></td><td></td></tr><tr><td>GJA4</td><td>Cutaneous venous malformation<br/>(cavernous histology)<br/>MODERATE</td><td>Hepatic hemangioma<br/>STRONG</td><td></td><td></td><td></td></tr><tr><td>GNA11</td><td>Capillary malformation w/o overgrowth<br/>DEFINITIVE</td><td>Sturge-Weber syndrome<br/>STRONG</td><td>Congenital hemangioma<br/>STRONG</td><td></td><td></td></tr><tr><td>GNAQ</td><td>Capillary malformation w/o overgrowth<br/>DEFINITIVE</td><td>Sturge-Weber syndrome<br/>DEFINITIVE</td><td>Congenital hemangioma<br/>DEFINITIVE</td><td></td><td></td></tr><tr><td>GNAS</td><td>Vascular malformation, any type<br/>NO EVIDENCE</td><td></td><td></td><td></td><td></td></tr><tr><td>GNB2</td><td>Sturge-Weber syndrome<br/>LIMITED</td><td></td><td></td><td></td><td></td></tr><tr><td>HRAS</td><td>Vascular malformations, various type<br/>STRONG</td><td></td><td></td><td></td><td></td></tr><tr><td>IDH1</td><td>Maffucci syndrome<br/>DEFINITIVE</td><td>Spindle cell hemangioma<br/>DEFINITIVE</td><td></td><td></td><td></td></tr><tr><td>IDH2</td><td>Maffucci syndrome<br/>STRONG</td><td>Spindle cell hemangioma<br/>STRONG</td><td></td><td></td><td></td></tr><tr><td>KRAS</td><td>Sporadic arteriovenous malformation<br/>DEFINITIVE</td><td>Gorham-Stout disease<br/>MODERATE</td><td>Epithelioid hemangioma<br/>LIMITED</td><td></td><td></td></tr><tr><td>MAP2K1</td><td>Arteriovenous malformation<br/>DEFINITIVE</td><td>Epithelioid hemangioma<br/>STRONG</td><td></td><td></td><td></td></tr><tr><td>MAP3K3</td><td>Verrucous venous malformation<br/>STRONG</td><td>Cerebral cavernous malformation<br/>DEFINITIVE</td><td></td><td></td><td></td></tr><tr><td>NPM1</td><td>Maffucci syndrome<br/>NO EVIDENCE</td><td></td><td></td><td></td><td></td></tr><tr><td>NRAS</td><td>Vascular malformations, low-flow, various type<br/>LIMITED</td><td>Kaposiform lymphangiomatosis<br/>DEFINITIVE</td><td>Pyogenic granuloma / tufted angioma<br/>LIMITED</td><td></td><td></td></tr><tr><td>PIK3CA</td><td>PIK3CA-related overgrowth spectrum<br/>DEFINITIVE</td><td>Lymphatic malformation<br/>DEFINITIVE</td><td>Common venous malformation<br/>DEFINITIVE</td><td>Megalencephephaly-capillary malformation-polymicrogyria<br/>DEFINITIVE</td><td>Generalised lymphatic anomaly<br/>STRONG</td></tr><tr><td>PIK3CD</td><td>Lymphatic malformation<br/>LIMITED</td><td></td><td></td><td></td><td></td></tr><tr><td>PIK3R1</td><td>Capillary malformation, dilated veins, (mild) overgrowth +/- lymphatic malformation<br/>STRONG</td><td></td><td></td><td></td><td></td></tr><tr><td>PTPN11</td><td>Capillary malformation w/o overgrowth<br/>STRONG</td><td></td><td></td><td></td><td></td></tr><tr><td>TEK</td><td>Common venous malformation<br/>DEFINITIVE</td><td>Blue Rubber Bleb Nevus syndrome<br/>STRONG</td><td>Multifocal sporadic Venous malformation<br/>STRONG</td><td></td><td></td></tr></tbody></table>

technologies, used by the 11 laboratories of the participating centres at the beginning of this study are listed anonymously in Table S3.

## Tissue selection and DNA requirements

In participating centres, sequencing analysis for somatic variants was preferably performed on DNA from affected
<table><caption>**Table 2** Variants, exons and regions of interest for vascular malformations caused by somatic events</caption><thead><tr><th rowspan="2">Gene Name</th><th rowspan="2">Transcript Reference</th><th colspan="2">Minimal required regions</th><th colspan="2">Optional regions</th></tr><tr><th>Exons</th><th>amino acid residues of interest</th><th>Exons</th><th>amino acid residues of interest</th></tr></thead><tbody><tr><td>AKT1</td><td>NM_001382430.1</td><td>3</td><td>E17</td><td>3<br>4</td><td>L52<br>Q79</td></tr><tr><td>AKT3</td><td>NM_5465.7</td><td>3</td><td>E17</td><td>None</td><td></td></tr><tr><td>ARAF</td><td>NM_001654.5</td><td>7</td><td>S214</td><td>9</td><td>R255</td></tr><tr><td>BRAF</td><td>NM_004333.4</td><td>15</td><td>V600</td><td>6<br>11<br>14<br>15<br>16</td><td>T244 to F247, Q257<br>G466, G469<br>N581<br>N581 to K601<br>Q636</td></tr><tr><td>CBL</td><td>NM_005188.4</td><td>15</td><td>Y774</td><td>2<br>7<br>8<br>9<br>16</td><td>Y102<br>D359 to K362<br>Multiple hotspots, splice<br>C404 to R420, splice<br>A877</td></tr><tr><td>GJA4</td><td>NM_002060.3</td><td>2</td><td>G41</td><td>None</td><td></td></tr><tr><td>GNA11</td><td>NM_002067.5</td><td>4<br>5</td><td>R183<br>Q209</td><td>None</td><td></td></tr><tr><td>GNA14</td><td>NM_004297.4</td><td>4</td><td>Q205</td><td>None</td><td></td></tr><tr><td>GNAQ</td><td>NM_002072.5</td><td>4<br>5</td><td>R183<br>Q209</td><td>2</td><td>M59, T96</td></tr><tr><td>GNB2</td><td>NM_005273.4</td><td>5</td><td>K78</td><td>None</td><td></td></tr><tr><td>HRAS</td><td>NM_005343.4</td><td>2<br>3</td><td>G12, G13<br>T58 to M72</td><td>3<br>5</td><td>Q61<br>F156</td></tr><tr><td>IDH1</td><td>NM_005896.4</td><td>4</td><td>R132</td><td>None</td><td></td></tr><tr><td>IDH2</td><td>NM_002168.4</td><td>4</td><td>R172</td><td>4</td><td>R140</td></tr><tr><td>KRAS</td><td>NM_004985.5</td><td>2<br>3<br>4</td><td>K5, G12, G13, Q22<br>Q61, Y64<br>A146</td><td>2<br>3<br>4<br>5</td><td>V14, L19<br>T58 to G60<br>K117<br>D153, F156</td></tr><tr><td>MAP2K1</td><td>NM_002755.4</td><td>2<br>3</td><td>F53 to E62<br>P105 to 107, C121 to G128</td><td>2<br>3<br>5<br>6<br>7<br>8</td><td>L42 to D67<br>E102 to M146<br>L177<br>E203<br>P264, P294<br>G301, S331</td></tr><tr><td>MAP3K3</td><td>NM_002401.5</td><td>13</td><td>I441</td><td>15</td><td>Y544</td></tr><tr><td>NRAS</td><td>NM_002524.5</td><td>3<br>4</td><td>Q61<br>A146</td><td>2<br>3</td><td>G12 to P34<br>T50 to G60</td></tr><tr><td>PIK3CA</td><td>NM_006218.4</td><td>Full gene</td><td></td><td>Full gene</td><td></td></tr><tr><td>PIK3CD</td><td>NM_005026.5</td><td>16</td><td>L666</td><td>13<br>24</td><td>S520<br>E1021</td></tr><tr><td>PIK3R1</td><td>NM_181523.3</td><td>11<br>13<br>14</td><td>Y452 to Y467<br>N564 to Y580<br>W583, splice</td><td>9<br>10<br>13</td><td>R348<br>G376<br>N564, K567, splice</td></tr><tr><td>PTPN11</td><td>NM_002834.5</td><td>3<br>12<br>13</td><td>E76<br>V428, A461<br>T507</td><td>1_13</td><td>Multiple hotspots</td></tr><tr><td>TEK</td><td>NM_00459.5</td><td>17<br>22<br>23</td><td>Y897 to A925<br>R1099X<br>T1105 to G1115</td><td>15<br>17<br>18<br>23</td><td>R849<br>L920<br>F960<br>K1100 to Y1113</td></tr></tbody></table>

This table describes regions of interest (genes, exons, amino acids) that should be studied for somatic exploration of vascular anomalies. The regions of interest are divided into two groups: "Minimal required regions" and "Optional regions". The first one represents what is well described and implicated in the pathogenicity of somatic vascular anomalies, and the second one represents hotspot variants reported in cancer, or germline hotspots in *RASopathy genes* and *TEK*, with insufficient evidence of pathogenicity in non-hereditary vascular anomalies. Reference transcripts are from ensembl.org. Data are from literature and databases (LOVD, Clinvar Miner, Cosmic (v96) and CancerHotspots (v2))
tissue, as the variants are usually absent or present at only
very low allele fraction in other sources, such as blood or
saliva. Affected tissue samples were obtained by lesional
biopsies or from surgical resections. They were further
processed in various ways until DNA extraction, mainly
as native tissue samples in saline solution, frozen or in
RNAlater solution, but some laboratories also used for-
malin-fixed paraffin embedded (FFPE) tissue (Table S3).
The specifics of tissue processing were matched to the
laboratory protocols for subsequent analysis (for exam-
ple, not all the library preparation methods are compat-
ible with genomic DNA (gDNA) from FFPE tissue). For
most centres, histological examination was considered
in the diagnostic work-up in a pathology lab, especially
if the diagnosis was unclear or to ensure that the biopsy
is representative (deep malformation). Some laboratories
performed tissue dissection (removing unaffected tissue,
e. g. epidermidis) to increase the sensitivity of somatic
testing (Table S3).

### Minimum tissue requirements

The minimum amount of tissue was dependent on the
input criteria (quantity and quality) of the laboratory
protocols for subsequent analysis. A punch or excisional
biopsy size of 4 mm was usually sufficient for subsequent
analysis in the laboratory's protocols.

### DNA isolation methods

A variety of commercial tissue DNA extraction kits and
protocols were used by the participating laboratories to
isolate gDNA from the affected tissue. The specifics of
the DNA isolation protocols were adapted according to
both the tissue preparation and the laboratory protocols
for subsequent analysis. The minimum DNA input for
analysis ranged from 5 to 200 ng (Table S3). Quality con-
trol of gDNA were included in each laboratory protocol
depending on the methods used for sequencing part.

### Sequencing analysis by NGS

Most centres were using custom targeted panels, while
three centres (9, 10 and 11) were using whole-exome
sequencing (WES) and applied a virtual in silico panel of
genes of interest for the analysis of WES data (Table S3).
Two laboratories were using unique molecular identifiers
(UMIs) to increase sequencing accuracy. For those cen-
tres using WES, the analysed gene panels also included
genes for hereditary vascular malformations, to enable
germline and somatic testing in the same analysis flow.
Higher read depth was used (120X-400X minimal read
depth) compared to standard germline WES testing.
However, the target regions may have suboptimal cov-
erage compared to custom panels and low-frequency
variants can thereby go undetected. Minimal sequencing

depth for custom panels ranged from 100 to 2000X
(Table S3).

### Read alignment and variant detection

In house pipelines as well as commercial pipelines
were used by the participating centres to align and call
the variants (Table S3). Bioinformatics pipelines were
adapted to the special requirements of somatic variant
calling (with a low VAF).

### Technical sensitivity and specificity

Pathogenic variants that are detected in vascular mal-
formations caused by somatic events are generally
present only in a subset of cells and the mutant allele
frequency in DNA from mixed lesional tissue samples
may be quite low. In the participating diagnostic rou-
tine laboratories, the indicated cut off for the minimum
VAF varied from 1 to 3% (Table S3). If depth for detect-
ing low VAF is not reached due to technical limitations,
it will reduce the analytical sensitivity of the assay.
Notably, approximately 15% (n=41/262) of cases with
a pathogenic variant in *PIK3CA* detected by the mem-
bers of this working group had a VAF of <3% (internal
data from the participating centres).

Each laboratory established empirically its sequenc-
ing requirements, such as the minimum sequenc-
ing depth, a minimum number of variant reads and
the minimum VAF to be reported (Table S3). Bino-
mial distribution-based coverage calculation can aid
in determining the minimum sequencing depth [23].
UMI-based sequence analysis was successfully used by
two of the participating laboratories to increase test
accuracy (Table S3). One laboratory used strand-spe-
cific amplification, which may help to discriminate gen-
uine C:G > T > A variants from deamination artefacts
in FFPE samples. If tissue handling and NGS library
flows are combined with the processing of solid tumour
specimens, there is a possibility of cross-contamination
from solid tumour specimens as the origin of patho-
genic variants with a low VAF. One laboratory added
common single nucleotide polymorphisms (SNPs) in
the library preparation to trace this cross-contamina-
tion during analysis of sequences.

### Alternative methods of somatic variant detection/
validation

Some laboratories used digital PCR (dPCR)/droplet
digital PCR (ddPCR) to confirm low frequency variants
or detect recurrent hotspots variants in genes such as
GNAQ, GNA11, PIK3CA, and TEK.
## Variant interpretation

### Variant filtering

As usually performed in NGS-based analysis, common benign variants and artefacts were removed from the detected list of variants prior to interpretation.

### Variant classification and reporting

All centres indicated that their classification of variant pathogenicity was essentially based on ACMG recommendations [24]. The AMP/ACMG/ASCO recommendations were used by the centres to report somatic test results for vascular malformations [25]. All the information required for unambiguous clinical interpretation of the test result was reported according to the guidelines. The Human Genome Variation Society (HGVS) standard nomenclature [26] providing the transcript reference sequence was used to annotate all detected variants. Coverage of the NGS assay, VAF, exons/hotspots that do not meet the quality standards, description of methods, assay performance characteristics, limitations of the assays used and a list of genes covered are the minimal data content indicated in reports [25]. All reported variants were classified as described above, and pathogenic and likely pathogenic alterations were generally disclosed. Unexpected findings or variants of unknown significance were discussed with clinicians prior to reporting. Recommendations for any supplemental testing were made when appropriate.

## Discussion

The aim of this work was to propose a performant and accurate genetic testing approach for patients with vascular malformations caused by somatic genetic events. At the beginning of this work, there was a large heterogeneity between the centres in the VASCERN-VASCA regarding the genes and the exonic regions that were screened, as well as in the methodological approaches. Therefore, our first aim was to harmonise the content of the diagnostic gene panel, by proposing a core gene list based on evidence of associations with vascular malformations. Our second aim was to make recommendations for the technical approaches.

## Scope of diagnostic somatic testing in vascular malformations

By systematic evaluation of gene-disease associations, we came up with a list of 24 genes, for which there was usable published evidence for their role in vascular malformations driven by somatic variants. For a subset of 17 genes the evidence was rated “strong” or “definitive” for at least one gene-phenotype association. We consider these core genes to be a mandatory content of any diagnostic panel used for a broad spectrum of vascular malformations. We

also recommend the inclusion of a further 4 genes with “limited evidence”, as it can be expected that these genes may be upgraded as new published observations emerge. In contrast, we do not recommend the inclusion of the remaining 3 genes, for which “no evidence” of an association with vascular malformations was found (Table 1).

Since somatic (gain-of-function) variants that are associated with vascular malformations are typically restricted to specific hotspot regions within the respective genes, we also aimed to provide recommendations on variants and regions of interest for each gene. The sites of variants reported in vascular malformations should be covered by a diagnostic test as a minimum requirement (Table 2). As the variants associated with vascular malformations overlap with recurrent oncogenic variants found in cancer in the respective genes, a broader definition of the regions of interest also includes these (Table S2). In general, laboratories using targeted gene panels that only partially cover genes according to their defined hotspots should be aware that novel regions of interest may rapidly emerge.

Notably, the list of genes evaluated in this work is significantly larger than the number of genes mentioned in the last ISSVA classification, illustrating the major progress that has been made since the last update (May 2018). There are 11 new genes (AKT3, ARAF, CBL, GJA4, GNB2, HRAS, KRAS, NRAS, PIK3CD, PIK3R1 and PTPN11) that are not in the ISSVA classification (2018). Of these, seven have at least one “definitive” (KRAS and NRAS) or “strong” (AKT3, GJA4, HRAS, PIK3R1 and PTPN11) gene-phenotype association. The remaining four genes (ARAF, CBL, GNB2 and PIK3CD) have been associated with vascular malformations with “limited evidence”. For ARAF, two patients have been reported by the same group in 2019 [27] with lymphangiomatosis or central conducting lymphatic anomaly, respectively. Based on the available data, it is not clear whether both variants were somatic or not. The observation of ARAF variants in lymphatic malformations has not been replicated since. Case reports have been published for GNB2, CBL and PIK3CD, [28–30] describing patients with Sturge-Weber syndrome (GNB2) or lymphatic anomalies (CBL and PIK3CD). Further evidence is needed to confirm the involvement of these genes in vascular malformations. In the most recent version of the ISSVA classification, the NPM1 gene was indicated as being associated with Maffucci syndrome. However, a careful review of the literature does not support this association, as the only patient with a variant in this gene reported to date was a 7-year-old girl with Maffucci syndrome and acute myeloid leukaemia [31]. This patient had two somatic variants, both in the leukemic cells and in the vascular lesion: a frameshift variant in the NPM1 gene and a missense
variant in the *IDH1* gene. As *IDH1* is strongly associ-
ated with Maffucci syndrome, the variant identified in
this gene can explain this part of the phenotype. Somatic
*NPM1* variants are instead associated with acute myeloid
leukaemia (OMIM #601,626). Thus, we conclude that the
*NPM1* variant is not involved in the pathogenesis of Maf-
fucci syndrome.

There is also increasing evidence that several genes (particularly the core genes of the RAS-MAPK and PI3K-AKT cascades) are involved in a wider range of vascular anomalies than previously recognised, probably reflecting variability in the timing of the mutation, the cell type affected and other factors. For example, the E542K variant in *PIK3CA* has been identified in isolated LM, isolated VM and PROS. New phenotypes were added for a total of five genes: *BRAF* with sporadic AVM (strong evidence) and LM (moderate evidence), *GNA11* with Sturge-Weber syndrome (strong evidence), *MAP2K1* with epithelioid haemangioma (strong evidence), *MAP3K3* with cerebral cavernous malformation (definitive evidence) and *PIK3CA* with generalised lymphatic anomaly (strong evidence).

We evaluated gene-disease associations until vari-
ant level, when relevant. As mentioned above, the same
pathogenic variants can be associated with different or
overlapping phenotypes and with different extension
and severity of the disease. However, different variants in
the same gene can also be associated with specific phe-
notypes; for example, the R183Q variant in GNAQ has
been associated with CM and Sturge-Weber syndrome,
but not with non-involuting congenital haemangiomas
(NICH) or rapid-involuting congenital haemangiomas
(RICH), which are instead associated with the Q209H/
L/P variants.

The expanding genetic heterogeneity and phenotypic
diversity associated with individual genes challenges the
use of narrowly focused diagnostic assays that include
only one or a small panel of genes targeted to a defined
clinical phenotype. However, the predominance of recur-
rent variants in some distinctive phenotypes still leaves a
possible rationale for the use of targeted methods such as
digital PCR, which can be an efficient and cost-effective
first-tier approach to search for highly recurrent hotspot
variants or to analyse genes with a narrow mutational
spectrum, such as GNAQ, GNA11, PIK3CA and TEK
hotspot variants. However, negative results from such an
approach, should be followed by testing of a more com-
prehensive gene panel.

Genes involved in vascular malformations caused by
somatic events may also be analysed in combination with
genes involved in hereditary vascular malformations
(germline events), although this is not specifically recom-
mended. Instead, the expert panel agreed that the analysis

of genes involved in hereditary vascular malformations is
generally not essential in sporadic patients with a type of
vascular malformation that is usually caused by somatic
events, as the underlying genes are mostly different,
and the clinical presentation can usually guide towards
somatic or germline testing, respectively. Moreover, for
laboratories using custom panels, the inclusion of the
genes for hereditary forms would greatly affect the size
of the panel, which may conflict with the need for very
deep sequencing to detect somatic variants with very low
VAFs. The combination of germline and somatic testing
in one assay should take into account cost-effectiveness,
which also depends on the local laboratory equipment
and organisation. Nevertheless, the limitation to a very
strictly-defined gene content will become obsolete with
the advent (and cost efficiency) of new, very large-scale
sequencing technologies. For laboratories using a WES-
based approach it is easy to open their analysis to the
genes involved in hereditary vascular malformations
based on the patient's phenotype (e.g. KRIT1 for patients
with hyperkeratotic cutaneous capillary-venous malfor-
mations, and RASA1, EPHB4, ENG1, ACVRL1, SMAD4,
PTEN for AVM). However, it has to be noted that WES is
usually not performed at a sequencing depth that is suffi-
cient to reach a reliable mosaic detection threshold of 1%
VAF. For laboratories using a custom panel, on the other
hand, a low threshold for extending the analysis toward
the genes involved in hereditary forms can be suggested,
if the phenotype is compatible with such a disease. It has
to be noted, that multiplicity of vascular lesions or a posi-
tive family history are not always present in hereditary
vascular malformations. If somatic testing is performed
exclusively, potentially pathogenic germline variants
should be reported and the patient referred for genetic
counselling and germline testing.

Somatic variants in overlapping groups of genes may
also be involved in other disorders, for example genes of
the PI3K-AKT-mTOR pathway in segmental overgrowth
or brain malformations or genes of the RAS-MAPK
pathway in neurocutaneous mosaic disorders. Thus,
laboratories may find it more useful and efficient to use
a combined panel rather than separate panels to test for
these groups of conditions.

Technical recommendations for genetic testing of somatic
variants

Recommendations for technical aspects of somatic vari-
ant testing are summarised in Table 3. The recommen-
dations represent a consensus of expert opinion based
on the practice and experience of the reference centres
involved in this study, and are intended to assist clinical
laboratories in the detection of somatic variants and to
ensure a high quality of sequencing results.
<table><caption>**Table 3** Technical recommendations for genetic testing of somatic variants</caption><thead><tr><td>Technical aspect</td><td>Recommendation of the VASCERN-VASCA expert group</td></tr></thead><tbody><tr><td>Tissue sampling</td><td>4 mm of affected tissue specimen from debulking/reduction surgery, or skin biopsy of affected tissue containing abnormal vasculature is the minimum tissue size recommended for adequate DNA yield<br>Fresh/frozen and FFPE samples can be considered<br>Blood samples are generally not recommended, as the diagnostic yield is low. Cell-free DNA from blood plasma or lymphatic fluid are a promising alternative source, but need further evaluation</td></tr><tr><td>Histologic examination</td><td>Recommended for differential diagnostics and/or estimation of the representativeness of the sample. Dissection (removal of epidermis) can be performed to increase the percentage of affected tissue</td></tr><tr><td>Tissue culturing</td><td>Tissue culturing is not necessary, but culturing endothelial cells from affected tissue may aid subsequent variant detection</td></tr><tr><td>DNA extraction</td><td>Standard DNA extraction protocols</td></tr><tr><td>DNA quality</td><td>Low-quality samples can be analysed; however, it reduces sensitivity and may introduce sequencing artefacts. Cost-effectiveness of prior DNA quality control must be determined in each laboratory</td></tr><tr><td>Gene-disease validity</td><td>Core gene list (Table 1) and updates thereof https://vascern.eu/</td></tr><tr><td>Expected VAFs</td><td>Mosaic variants can be present at multiple frequencies, expected VAFs are mostly lower than 10%</td></tr><tr><td>Target region</td><td>Minimum required regions that must be covered for the detection of somatic variants associated with vascular malformation are reported in Table 2. Recommended optional regions are also listed in Table S2</td></tr><tr><td>Sequencing analysis by NGS</td><td>Various platforms can be used<br>UMI-based sequencing is recommended especially for amplicon-based, but also for hybridisation-based capture technologies. For the latter, if UMI s are not used, read deduplication is recommended, which can be based on read start and end coordinates; Strand-specific amplification or capture can help to discriminate genuine C:G > T > A variants from deamination artefacts in FFPE samples</td></tr><tr><td>Technical sensitivity and specificity</td><td>Sequencing depth and minimum number of variant reads should be empirically defined. Detection threshold of 1% is recommended. Including common SNPs in the library preparation is advised to detect cross-contamination from tumour samples when handled in parallel</td></tr><tr><td>Read alignment, variant detection, and variant filtering</td><td>Diagnostically validated pipelines for somatic calling are recommended. Prefiltering is advised to remove benign variants and artefacts</td></tr><tr><td>Validation of NGS findings</td><td>Recommended for all variants with VAF less than 5% using dPCR or a second NGS analysis</td></tr><tr><td>Alternative methods of somatic variant detection</td><td>dPCR can be used for the detection of recurrent variants associated with specific phenotypes</td></tr><tr><td>Variant classification</td><td>ACMG classification with modifications suggested in PMID: 35,997,716 and PMID: 34,040,190 are recommended. Literature/database searches should extend beyond cancer variant data annotation and interpretation</td></tr><tr><td>Reporting</td><td>AMP/ACMG/ASCO recommendations are largely applicable (PMID: 27,993,330). Variants should be annotated using the HGVS standard nomenclature. Reports should provide VAF, coverage, regions that do not meet quality standards, description of methods, limitations and list of genes analysed. Pathogenic/likely pathogenic variants should be disclosed. Recommendations for supplemental testing should be made, such as germline panel testing, based on the indication and clinical context</td></tr><tr><td>Identification of potential germline variants</td><td>Potential pathogenic/likely pathogenic germline variants should be included in the report and referral for germline testing and genetic counselling should be advised</td></tr><tr><td>Data sharing</td><td>Submission of variants with phenotypic, clinical significance, and classification criteria to DNA variant databases, such as ClinVar and LOVD, is recommended to increase knowledge of the genetics of vascular anomalies</td></tr></tbody></table>

Regarding the source of DNA for diagnostic testing, a tissue sample containing abnormal vasculature is the material of choice and gold standard. Since surgical resection tissue is not always available, a skin biopsy with a punch from the site of the lesion can be obtained. Cell culturing from tissue samples is not routinely necessary, but can help increase the VAF if endothelial cells

from the affected tissue are isolated and used for culture [32, 33]. Blood leucocyte DNA is not recommended as the diagnostic yield is relatively low [34], with a few exceptions, such as for some patients with MCAP [35] or MSVM (multiple sporadic venous malformations) [36]. Depending on the phenotype, other non-invasive specimens, such as urine (urinary epithelial cells) [37]
or saliva (buccal cells) [38] may also contain the causative variant, but their use cannot be recommended for the general routine. Cell-free DNA (cfDNA) analysis is emerging as an attractive alternative to tissue-based testing. This would be particularly beneficial when an invasive procedure is not possible and for young patients. Nevertheless, the VAF of disease-causing variants in cfDNA is usually much lower than in lesional tissue DNA (often less than 1%) and requires highly sensitive techniques, such as dPCR [39, 40]. The diagnostic yield of cfDNA in vascular malformations is currently rather low. It may be increased by using cfDNA from an effluent vein [41] or lymphatic fluid [40]. However, the use of cfDNA for diagnostic purposes needs to be further developed and validated.

Low VAFs of the disease-causing somatic variants in mixed tissue specimens or other diagnostic samples from patients with vascular malformations present a major diagnostic challenge. The VAFs of somatic driver variants are generally lower than those typically found in solid tumours. Therefore, routine settings adopted from molecular oncology may be insufficient for vascular malformations. In large patient cohorts, most pathogenic variants are present in lesional tissue samples with a VAF less than 10%, even in patients with severe phenotypes [42, 43]. The expected VAF range depends on the sample type (e.g. [34] and clinical context (e.g. [44] and may even fall below 1%. Enrichment of mutant cells in a given sample might be achieved by tissue dissection, microdissection, endothelial cell culturing or cell separation [45, 46]. These approaches may be considered when faced with a negative result. However, the current experience is insufficient to recommend their routine use. In samples of poor DNA quality or quantity, the sensitivity for low-level mosaicism is reduced and the probability of ambiguous results is increased. The expert group recommends to include a quality control of the gDNA starting material to relate the DNA quality assessment with the sequencing results. If tissue handling and NGS library flows are combined with the processing of solid tumour specimens, there is possibility of cross-contamination from solid tumour specimens as the origin of pathogenic variants with a low VAF. Different methods using polymorphic tags in the library preparation and/or post-sequencing bio-informatic analysis can be applied to detect such cross-contamination [47, 48].

Concerning the sensitivity threshold for somatic variants in lesional tissue samples, the expert group recommendation for the minimum VAF is set at 1%. Sequencing depth and minimum number of variant reads needed to reach this threshold should be empirically defined for each work-flow. Standardized set of DNA samples carrying mosaic variants with different

allele fractions are made available by companies helping in panel development. It is also possible to exchange DNA samples between laboratories for genetic tests that are not yet covered by an external quality assessment scheme. Binomial distribution-based coverage calculations can be used to determine the probability to detect a variant at a certain allele ratio [23]. Notably, increasing sequencing depth of libraries, especially those derived from poor quality material, per se has limited effect on sensitivity because of the background level of technical artefacts that are inherent to the technology. Artefacts can arise from different sources, including tissue fixation with formalin, PCR and sequencing; they are therefore dependent on sample type, library preparation and sequencing technique [49]. When FFPE tissue samples are used, common base substitution artefacts, particularly C>T/G>A caused by cytosine deamination, significantly limit the detection threshold [50]. An elegant option to overcome some of these limitations of current NGS technologies and thereby lower the detection threshold is the use of UMI-based sequencing. These are complex molecular barcodes that are added to DNA prior to any PCR amplification steps. This enables the definition of the consensus sequence of PCR replicates derived from a single template DNA molecule, thereby eliminating amplification and sequencing artefacts [51]. The use of UMIs is recommended especially for amplicon-based, but also for hybridisation-based capture technologies. For the latter, if UMIs are not used, read deduplication is recommended, which can be based on read start and end coordinates.

Generally, ambiguous findings and results close to the detection threshold of the given test should be interpreted with caution in the context of the clinical indication, and independent confirmation should be sought. This can preferably be achieved by an alternative technique, such as dPCR, or if not available, by repeating the NGS analysis [52]. Digital PCR provides a high depth of genotyping allowing the detection of specific variants at VAFs as low as 0.1%, with a minimal gDNA input requirements (5 ng of gDNA per test) and can also be used if the gDNA quality or quantity is suboptimal (e.g. DNA from FFPE tissue). The use of dPCR as a first-tier test for specific indications is discussed above. The need for specific assays for each change at the nucleotide level and limited multiplexing capabilities constrain the use of dPCR in vascular malformation diagnostics. The use of blocker displacement amplification as an alternative method to confirm variants with very low VAF in vascular malformations has recently been reported [46].

Bioinformatic pipelines for NGS data analysis need to be adapted for the specific needs of low-level mosaic detection. For variant callers the threshold may need
to be adjusted for the desired minimum calling VAF. In addition, for some recurrent hotspot positions, it may be considered to program a systematic variant calling and/or to manually inspect the aligned sequences in a genome viewer such as integrative genomics viewer (IGV: [53]). On the other hand, a more agnostic approach with an exploratory setting based on deep WES may be useful for detecting disease causing variants in novel genes [54].

The interpretation and classification of causal somatic variants in vascular malformations can sometimes be challenging. Although disease-causing variants in several genes largely overlap with oncogenic drivers detected in solid tumours, other variants in specific genes can be found (almost) exclusively in vascular malformations (e.g., variants in the receptor tyrosine kinase TEK gene have not been reported in cancer). Furthermore, atypical variants in some oncogenes are (almost) exclusively found in vascular malformations (e.g. atypical HRAS and KRAS indel variants [55]).

In our experience, the American College of Medical Genetics/Association for Molecular Pathology (ACMG/AMP) general criteria of classification of germline variants [24] were not completely applicable for somatic variant classification. Other classification guidance schemes from oncology, such as the joint consensus recommendations of AMP, ACMG, American Society of Clinical Oncology (ASCO) [25, 56] and European Society for Medical Oncology (ESMO) [57] can be used, but were found to focus on clinical relevance and to lack guidance on variant classification. An exception was the Belgian ComPerMed, which proposed guidelines for biological classification and clinical interpretation of somatic variants [58]. Gene-specific recommendations were recently reported for PIK3R1 [21], and for AKT3, MTOR, PIK3CA and PIK3R2 by the ClinGen Brain Malformation Variant Curation Expert Panel [59]. Such gene specific recommendations should be tested and validated for vascular malformations.

## Conclusion

Based on the VASCERN-VASCA expertise, our aim was to optimise and guide the diagnostic genetic testing performed for non-hereditary vascular malformations caused by somatic variants. We defined the core gene list that should be tested and evaluated, the level of evidence of the gene-phenotype associations and the variants/regions of interest that should be included in a (virtual) panel. Knowing the gene-phenotype relationship and the strength of this association can greatly help laboratories to interpret the data and contribute to clinical diagnosis. This study reflects the state of knowledge as of mid-2023 and will be regularly updated on the VASCERN-VASCA website [13]. Laboratories around the world should be able to use these data.

## Abbreviations

<table><tr><td>ACMG</td><td>American College of Medical Genetics</td></tr><tr><td>AMP</td><td>Association for Molecular Pathology</td></tr><tr><td>ASCO</td><td>American Society of Clinical Oncology</td></tr><tr><td>cfDNA</td><td>Cell-free DNA</td></tr><tr><td>ESMO</td><td>European Society for Medical Oncology</td></tr><tr><td>gDNA</td><td>Genomic DNA</td></tr><tr><td>COSMIC</td><td>Catalogue Of Somatic Mutation In Cancer</td></tr><tr><td>CLOVES</td><td>Congenital lipomatous overgrowth, vascular malformations, epidermal nevi, skeletal/scoliosis and spinal abnormalities</td></tr><tr><td>dPCR</td><td>Digital PCR</td></tr><tr><td>ddPCR</td><td>Droplet digital PCR</td></tr><tr><td>HGVS</td><td>Human Genome Variation Society</td></tr><tr><td>ISSVA</td><td>International Society for the Study of Vascular Anomalies</td></tr><tr><td>IGV</td><td>Integrative genomics viewer</td></tr><tr><td>LOVD</td><td>Leiden Open Variation Database</td></tr><tr><td>MCAP</td><td>Megalencephaly-capillary malformation</td></tr><tr><td>MSVM</td><td>Multiple sporadic venous malformations</td></tr><tr><td>NGS</td><td>Next-generation sequencing</td></tr><tr><td>NICH</td><td>Non-involuting congenital haemangiomas</td></tr><tr><td>PHTS</td><td>PTEN Hamartoma tumour syndrome</td></tr><tr><td>PROS</td><td>PIK3CA-related overgrowth spectrum</td></tr><tr><td>SNP</td><td>Single-nucleotide polymorphism</td></tr><tr><td>RICH</td><td>Rapidly involuting congenital haemangiomas</td></tr><tr><td>VAF</td><td>Variant allele fraction</td></tr><tr><td>UMI</td><td>Unique molecular identifier</td></tr><tr><td>WES</td><td>Whole-exome sequencing</td></tr></table>

## Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13023-024-03196-9.

**Supplementary Material 1.**

**Supplementary Material 2.**

**Supplementary Material 3.**

**Supplementary Material 4.**

## Acknowledgements

The authors of this publication are members of the Vascular Anomaly Working Group (VASCA WG) of the European Reference Network for Rare Multisystemic Vascular Diseases (VASCERN) or providers of genetic testing for VASCERN-VASCA HCP. This project has been supported by the European Reference Network on Rare Multisystemic Vascular Diseases (VASCERN)—Project ID: 101085076, which is funded by the European Union within the framework of the EU for Health Programme. MZ is a member of ERN-ITHACA, funded by the EU4Health programme of the European Union, under GA nr. 101085231. ILM thanks Marte Kirkevold and Mariann Kristensen for their assistance in the acquisition of data. MLD thanks the Dermatology Unit and Genodermatosis Unit of Bambino Gesù Children's Hospital, Rome, Italy. The authors thank Mr. Clovis Lessuise for excellent secretarial help.

## Authors' contributions

Conceptualization: NR, AE, CB, JA, PA, EB, CEL, IW, MZ, MV. Curation matrix adaptation for somatic variants: MZ. Data acquisition: CB, PA, EB, CEL, NGO, SF, ME, HGK, ILM, RM, NR, IW, MV, MZ. Data analysis & interpretation: CB, PA, EB, SF, ME, CEL, ILM, NR, IW, MV, MZ. Data curation: CB, PA, EB, HGK, CEL, ILM, RM, NR, MV, MZ. Writing-original draft (major contributors): NR, AE, CB, MZ, PA, CL. Writing-review & editing: all.

## Funding

This project has been supported by the European Reference Network on Rare Multisystemic Vascular Diseases (VASCERN)—Project ID: 101085076, which is funded by the European Union within the framework of the EU for Health Programme. MZ received funding from the German Federal Ministry of Education and Research (BMBF; grant numbers: FKZ 01GM1902A and FKZ 01GM1921A) and is a member of ERN-ITHACA, funded by the EU4Health programme of the European Union, under GA nr. 101085231. PA received special funding for
university-level health research in Finland, University of Helsinki. MV received funding from the Fonds de la Recherche Scientifique—FNRS Grants T.0240.23 & PC005.22, the Fund Generet managed by the King Baudouin Foundation (Grant 2018-J1810250-211305), and the Walloon Region through the FRFS-WELBIO strategic research programme (WELBIO-CR-2019C-06).

### Availability of data and materials

All data analysed during this study are included in the article and its supplementary information files.

### Declarations

### Ethics approval and consent to participate

Not applicable.

### Consent for publication

Not applicable.

### Competing interests

EB: PI for Novartis and Founder of Venthera. All other authors declare no conflicts of interest.

### Author details

¹Center for Human Genetics, Cliniques universitaires Saint-Luc, University of Louvain, VASCERN VASCA European Reference Centre, Brussels, Belgium.
²Department of Pathology, Radboud University Medical Center, VASCERN VASCA European Reference Centre, PO Box 9101, 6500, HB, Nijmegen, the Netherlands.
³Normandie Univ, UNICAEN, Service de Génétique, CHU Caen Normandie, BIOTARGEN EA 7450, VASCERN VASCA European Reference Centre, Caen 14000, France.
⁴HUS Diagnostic Center, Laboratory of Genetics, University of Helsinki and Helsinki University Hospital, VASCERN VASCA European Reference Centre, Helsinki, Finland.
⁵Institut de Recerca Sant Joan de Déu, Esplugues de Llobregat, CIBER-ER (Biomedical Network Research Center for Rare Diseases), Instituto de Salud Carlos III (ISCIII), Madrid, and Genomic Unit, Molecular and Genetic Medicine Section, Hospital Sant Joan de Déu, VASCERN VASCA European Reference Centre, Barcelona, Spain.
⁶Department of Dermatology, Hospital Sant Joan de Deu, VASCERN VASCA European Reference Centre, Barcelona, Spain.
⁷Laboratory of Medical Genetics, Translational Cytogenomics Research Unit, Bambino Gesù Children Hospital and Research Institute, IRCCS, VASCERN VASCA European Reference Centre, Rome, Italy.
⁸Medical Genetics Unit, Bambino Gesù Children's Hospital, IRCCS, VASCERN VASCA European Reference Centre, 00165 Rome, Italy.
⁹Sorbonne Université, Département de Génétique, Assistance Publique-Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, VASCERN VASCA European Reference Centre, Paris, France.
¹⁰Department of Molecular Medicine and Surgery, Karolinska Institutet and Department of Clinical Genetics, Karolinska University Hospital, VASCERN VASCA European Reference Centre, Stockholm, Sweden.
¹¹Department of Genetics, Center of Diagnostics, Copenhagen University Hospital - Rigshospitalet, VASCERN VASCA European Reference Centre, Copenhagen, Denmark.
¹²Laboratory of Molecular Oncology, Pediatric Cancer Center Barcelona, Hospital Sant Joan de Déu, VASCERN VASCA European Reference Centre, Barcelona, Spain.
¹³Department of Clinical Genetics, HUS Diagnostic Center, University of Helsinki and Helsinki University Hospital, VASCERN VASCA European Reference Centre, Helsinki, Finland.
¹⁴Department of Medical Genetics, Oslo University Hospital, VASCERN VASCA European Reference Centre, Oslo, Norway.
¹⁵Laboratory of Medical Genetics, Translational Cytogenomics Research Unit, Bambino Gesù Children Hospital and Research Institute, IRCCS, VASCERN VASCA European Reference Centre, Rome, Italy.
¹⁶Institute of Human Genetics, University Hospital Otto-Von-Guericke-University, Magdeburg, Germany.
¹⁷Center for Vascular Anomalies, Cliniques Universitaires Saint-Luc, Brussels, Belgium.
¹⁸Human Molecular Genetics, de Duve Institute, University of Louvain, VASCERN VASCA European Reference Centre, Brussels, Belgium.
¹⁹WELBIO Department, WEL Research Institute, Avenue Pasteur, 6, 1300 Wavre, Belgium.

Received: 12 December 2023 Accepted: 19 April 2024

Published: 22 May 2024

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