Mutations in a novel factor, glomulin, are responsible for glomuvenous malformations ("glomangiomas")

Abstract

Glomuvenous malformations (GVMs) are cutaneous venous lesions characterized by the presence of smooth-muscle--like glomus cells in the media surrounding distended vascular lumens. We have shown that heritable GVMs link to a 4--6-cM region in chromosome 1p21-22. We also identified linkage disequilibrium that allowed a narrowing of this VMGLOM locus to 1.48 Mb. Herein, we report the identification of the mutated gene, glomulin, localized on the basis of the YAC and PAC maps. An incomplete cDNA sequence for glomulin had previously been designated "FAP48," for "FKBP-associated protein of 48 kD." The complete cDNA for glomulin contains an open reading frame of 1,785 nt encoding a predicted protein of 68 kD. The gene consists of 19 exons in which we identified 14 different germline mutations in patients with GVM. In addition, we found a somatic "second hit" mutation in affected tissue of a patient with an inherited genomic deletion. Since all but one of the mutations result in premature stop codons, and since the localized nature of the lesions could be explained by Knudson's two-hit model, GVMs are likely caused by complete loss of function of glomulin. The abnormal phenotype of vascular smooth-muscle cells (VSMCs) in GVMs suggests that glomulin plays an important role in differentiation of these cells--and, thereby, in vascular morphogenesis--especially in cutaneous veins.

Figure 1

Typical GVMs. A and B, Cutaneous GVMs of right lower extremity in two patients, showing phenotypic variability. C, Cross-section of normal vein (magnification 40×), stained with antibody against SMC α-actin (red). D–F, Cross-sections of GVM (magnifications 10×, 40×, and 40×, respectively), stained with either (D and E) hematoxylin and eosin or (F) antibody to SMC α-actin (reddish brown). Arrows indicate glomus cells; L = vascular lumens. A lesion with somatic mutation is presented in panels A and D.

Figure 2

Pedigrees of 20 families with GVMs, presented in order of occurrence of the mutations in glomulin. Individuals tested are indicated by numerals. Blackened symbols denote affected patients, symbols containing a dot denote unaffected carriers, the half-blackened symbol (in families Bln and Wi) denotes a phenocopy, and question marks (?) indicate that the status is unknown. The second-hit mutation, 980delCAGAA, in family Ad, also is indicated.

Figure 3

Glomulin mutations. Control sequence (upper chromatograms) is compared with mutant sequence (lower chromatograms). Asterisks (*) indicate chromatograms showing reverse-strand sequence. Sequences presented for large genomic deletions and for the second-hit mutation, 980delCAGAA, were obtained from cloned fragments. Arrows indicate site(s) of mutation.

Figure 4

Glomulin gene—structure and mutations. The sizes of exons and of the three largest introns are given; other introns are to scale. Exon 1 is noncoding (n c), exon 2 contains the translation start site, and exon 19 contains the TGA stop codon. Above the sequence line, the white arrowheads indicate differences versus FAP48 cDNA (i.e., a new, 85-bp exon and an extra G), the single asterisk (*) indicates a single amino acid deletion, the double asterisks (**) indicate a splice-site mutation, and the box indicates the second-hit mutation, 980delCAGAA; the other three mutations cause immediate stop codons. Below the sequence line, frameshift mutations leading to premature stop codons are indicated, as are sequences of breakpoints of 8.4-kb deletion with GG insertion. The “FAP48” line indicates exons encoding FAP48.

Figure 5

Glomulin northern blot analysis—human MTN blot (Clontech) hybridized with a 482-bp 5′ probe of glomulin. Each lane contains 1 μg polyA+ RNA from adult tissue. Similar results were obtained with a full-length glomulin probe. Lower gel, β-actin control.