Mutations in the GGCX and ABCC6 genes in a family with pseudoxanthoma elasticum-like phenotypes

Abstract

A characteristic feature of classic pseudoxanthoma elasticum (PXE), an autosomal recessive disorder caused by mutations in the ABCC6 gene, is aberrant mineralization of connective tissues, particularly the elastic fibers. Here, we report a family with PXE-like cutaneous features in association with multiple coagulation factor deficiency, an autosomal recessive disorder associated with GGCX mutations. The proband and her sister, both with severe skin findings with extensive mineralization, were compound heterozygotes for missense mutations in the GGCX gene, which were shown to result in reduced gamma-glutamyl carboxylase activity and in undercarboxylation of matrix gla protein. The proband's mother and aunt, also manifesting with PXE-like skin changes, were heterozygous carriers of a missense mutation (p.V255M) in GGCX and a null mutation (p.R1141X) in the ABCC6 gene, suggesting digenic nature of their skin findings. Thus, reduced gamma-glutamyl carboxylase activity in individuals either compound heterozygous for a missense mutation in GGCX or with haploinsufficiency in GGCX in combination with heterozygosity for ABCC6 gene expression results in aberrant mineralization of skin leading to PXE-like phenotype. These findings expand the molecular basis of PXE-like phenotypes, and suggest a role for multiple genetic factors in pathologic tissue mineralization in general.

Figure 1. Cutaneous findings in a family with PXE-like phenotype

Note the loose and sagging skin in the 16 year-old proband (III-3, a–c). The proband’s mother (II-2, d) and aunt (II-3, e & f), both 40 years of age, demonstrate redundant folding of the skin in the axillary area (d, e) and popliteal fossa (f), as well as small yellowish papules characteristic of PXE (arrows). The nuclear pedigree of the family with PXE-like skin findings and coagulation factor deficiency. The mutations in the GGCX and ABCC6 genes identified in this family are indicated on the left of panel g. The proband is identified by an arrow.

Figure 2. Histopathology of the cutaneous lesions in the sister (III-1), the proband (III-3) and aunt (II-3)

Staining with hematoxylin-eosin (a–c) demonstrates basophilic abnormal elastic structures in the mid dermis (arrows). Special stains (von Kossa, d–f; Alizarin red, g–i) reveal that these elastotic structures are mineralized (arrows). Scale bar, 0.1 mm.

Figure 3. Mutation detection in the family

(a) Identification of the recurrent nonsense mutation p.R1141X in the ABCC6 gene. Note the heterozygous C→T transition substitution at nucleotide position 3421 (arrow). (b, d) Identification of missense mutations p.V255M and p.S300F in the GGCX gene. The sequences shown are in the complementary strand. (c) The presence of the p.V255M mutation in the control population was examined by restriction enzyme digestion with Apal I. Note that the mutation abolishes the restriction site, resulting in the 243 bp mutant allele, in addition to 163 and 80 bp fragments. In control individuals the 243 bp PCR product is digested to 163 and 80 bp fragments. (e, f) Evolutionary conservation of the amino acids V255 (panel e) and S300 panel (panel f) in the γ-glutamyl carboxylase protein.

Figure 4. Immunohistochemistry of skin in patients with PXE-like cutaneous lesions with mineralization (Patients III-1, III-3 and II-3) as well as in normal skin (control)

Monoclonal antibodies recognizing the under-carboxylated (ucMGP) and carboxylated (cMGP) forms of matrix gla protein, as well as polyclonal antibodies for fetuin-A and osteonectin were utilized. The secondary antibodies, biotin-conjugated anti-IgG, were recognized by avidin-alkaline phosphatase conjugates and visualized by incubation with an alkaline phosphatase substrate yielding red color. Scale bar, 0.1 mm.

Figure 5. Quantitation of different forms of MGP in plasma by ELISA

(a) Total, both phosphorylated and non-phosphorylated forms of under-carboxylated MGP (total ucMGP). (b) Assay of non-phosphorylated, under-carboxylated forms of MGP (ser-ucMGP). (c) The ratio of ser-ucMGP/ser-cMGP. The dashed lines in (a) and (b) represent the mean of control specimens and the coefficient of variation (CV) is indicated by the stippled areas (CV=15.7% in a, n=5; CV=6.0% in b, n=100).