Capillary malformation-arteriovenous malformation, a new clinical and genetic disorder caused by RASA1 mutations

Abstract

Capillary malformation (CM), or "port-wine stain," is a common cutaneous vascular anomaly that initially appears as a red macular stain that darkens over years. CM also occurs in several combined vascular anomalies that exhibit hypertrophy, such as Sturge-Weber syndrome, Klippel-Trenaunay syndrome, and Parkes Weber syndrome. Occasional familial segregation of CM suggests that there is genetic susceptibility, underscored by the identification of a large locus, CMC1, on chromosome 5q. We used genetic fine mapping with polymorphic markers to reduce the size of the CMC1 locus. A positional candidate gene, RASA1, encoding p120-RasGAP, was screened for mutations in 17 families. Heterozygous inactivating RASA1 mutations were detected in six families manifesting atypical CMs that were multiple, small, round to oval in shape, and pinkish red in color. In addition to CM, either arteriovenous malformation, arteriovenous fistula, or Parkes Weber syndrome was documented in all the families with a mutation. We named this newly identified association caused by RASA1 mutations "CM-AVM," for capillary malformation-arteriovenous malformation. The phenotypic variability can be explained by the involvement of p120-RasGAP in signaling for various growth factor receptors that control proliferation, migration, and survival of several cell types, including vascular endothelial cells.

Figure 1

Photographs of vascular malformations linked to RASA1 mutations. Individual numbers refer to pedigrees in figure 2. A, Atypical inherited pink-to-red, round-to-oval CMs (arrows) (patients CM45 III-8, CM45 III-15, and CM45 IV-2, CM8 III-4, and CM8 II-6); patient CM20 III-1, with a circumscribed nasal AVM and a cutaneous capillary blush (arrowhead); patient PW1 III-1, with Parkes Weber syndrome in the lower limb (arrows). B, Photographs of CMs not linked to RASA1 mutations. Typical red-purple facial CM (patient PW1 II-5); typical red CM of the neck (patient PW1 I-3).

Figure 2

Pedigrees, vascular phenotypes, and cosegregation of the identified mutations. Pedigrees of families with detectable RASA1 mutations are shown in order of occurrence of corresponding mutation in the RASA1 gene. Numbered individuals were screened for mutations. The number of lesions is in italic superscript. In families PW1, CM45, CM20, and CM41, cosegregation of deletional mutations with the phenotype was performed using radioactive size-difference gel electrophoresis. In family CM8, the presence of RASA1c.1336C→T was detected by Sau3A1 restriction digestion. The mutation destroys a Sau3A1 restriction-enzyme cutting site that normally splits the 223-bp allele into 134-bp and 89-bp fragments. In family CM11, the allele containing RASA1c.1619G→A yielded a 214-bp fragment in mutation carriers only, in addition to the wild-type control fragment of 485 bp in allele-specific PCR. Question marks indicate that the number of CMs is unknown. RASA1c.475_476delCT and RASA1c.2336_2337delGC were de novo mutations. Altogether, four carriers were identified.

Figure 3

Pedigrees of families without detectable RASA1 mutations. Numbered individuals were screened for mutations. The number of lesions is in italic superscript.

Figure 4

A, Schematic presentation of identified RASA1 mutations. Four deletional mutations—RASA1c.475_476delCT, RASA1c.512delT, RASA1c.1579_1582delGTCT, and RASA1c.2336_2337delGC—cause frame shifts and subsequent premature stop codons. Resulting hypothetical proteins are illustrated. RASA1c.1336C→T is a nonsense mutation, whereas RASA1c.1619G→A results in a Cys-540-Tyr substitution in the PH domain at position 540. SH2 = Src homology 2 domain; SH3 = Src homology 3 domain; C2 = protein kinase conserved region 2; RASGAP = rasGTPase-activating domain. Altered amino acid sequences due to frame shifts are shown by diagonal lines. B, Multiple alignment of PH domains. The figure illustrates the alignment of 15 PH-domain amino acid sequences around the p.C540Y mutation. Highly conserved phenylalanine is represented by a boldface F. Only 12 of 37 PH domains containing the mutated cysteine p.C540Y at this position are shown (box). Also shown are the three orthologous PH-domain sequences, all from RASGRF1, that contain a tyrosine (Y) at this position.

Figure 5

Signaling pathways implicating p120-RasGAP. Growth-factor binding to RTKs on the cell surface activates the Ras/MAPK-signaling pathway, which mediates signals to transcription factors in the nucleus. Changes in gene expression lead to cellular responses, such as differentiation and proliferation. p120-RasGAP (RasGAP) downregulates Ras signaling by catalyzing intrinsic GTPase activity of Ras. p120-RasGAP also is implicated in signaling to the cytoskeleton by binding Rap1a and/or p190-RhoGAP (RhoGAP).