A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis

Abstract

Familial tumoral calcinosis (FTC) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, which results in painful ulcerative lesions and severe skin and bone infections. Two major types of FTC have been recognized: hyperphosphatemic FTC (HFTC) and normophosphatemic FTC (NFTC). HFTC was recently shown to result from mutations in two different genes: GALNT3, which codes for a glycosyltransferase, and FGF23, which codes for a potent phosphaturic protein. To determine the molecular cause of NFTC, we performed homozygosity mapping in five affected families of Jewish Yemenite origin and mapped NFTC to 7q21-7q21.3. Mutation analysis revealed a homozygous mutation in the SAMD9 gene (K1495E), which was found to segregate with the disease in all families and to interfere with the protein expression. Our data suggest that SAMD9 is involved in the regulation of extraosseous calcification, a process of considerable importance in a wide range of diseases as common as atherosclerosis and autoimmune disorders.

Figure 1.

NFTC maps to 7q21-7q21.3. a, Haplotype analysis of five NFTC-affected families, through use of polymorphic markers on chromosome 7q21. Blackened symbols represent affected individuals. The disease-associated haplotype found in the five families is boxed in red. b, Multipoint linkage analysis, which generated a maximum LOD score of 3.4 between markers D7S2409 and D7S623.

Figure 2.

Clinical features of NFTC—including heralding erythematous papular eruption (a), small calcified tumors ulcerating and discharging chalk-like materials on the surface of the skin (b), and severe gingivitis (c)—and calcified material in the upper dermis, as revealed by histopathological examination (d).

Figure 3.

NFTC caused by a mutation in SAMD9. a, Sequence analysis of SAMD9 reveals an A→G transition at cDNA position 4483 in patient 3 (left panel). The father of the patient carries the mutation in a heterozygous state (middle panel). The wild-type sequence is given for comparison (right panel). b, PCR-RFLP analysis, which confirms segregation of the mutation in family 4. PCR amplification was performed as described in the text. Mutation K1495E creates a novel recognition site for MboII. Thus, affected patients display a 93-bp fragment (an additional 22 bp not shown), and healthy individuals show a 115-bp fragment, whereas both fragments are found in heterozygous carriers of the mutation. c, SAMD9 gene expression assessed using Clontech tissue blot cDNA array and using total RNA extracted from skin biopsy samples obtained from healthy individuals. Expression of SAMD9 was compared with that of ACTB. d, RNA was extracted from fibroblast-cell cultures established from two patients with NFTC and two healthy control individuals. SAMD9 mRNA expression, normalized to that of ACTB, was found to be up-regulated in NFTC fibroblasts (blue column) as compared with normal fibroblasts (orange column). e, ClustalW analysis of the SAMD9 protein region encompassing the mutation site, which demonstrates that K1495 is conserved across species. f, HEK293 cells transfected with wild-type (WT) (left panel) and E1495-carrying (right panel) GFP-SAMD9 constructs and examined using confocal microscopy. Note decreased expression in cells transfected with E1495-carrying GFP-SAMD9. MUT = mutant.