Normophosphatemic familial tumoral calcinosis is caused by deleterious mutations in SAMD9, encoding a TNF-alpha responsive protein

Abstract

Normophosphatemic familial tumoral calcinosis (NFTC) is an autosomal recessive disorder characterized by calcium deposition in skin and mucosae and associated with unremitting pain and life-threatening skin infections. A homozygous missense mutation (p.K1495E), resulting in SAMD9 protein degradation, was recently shown to cause NFTC in five families of Jewish-Yemenite origin. In this study, we evaluated another Jewish-Yemenite NFTC kindred. All patients were compound heterozygous for two mutations in SAMD9: K1495E and a previously unreported nonsense mutation, R344X, predicted to result in a markedly truncated molecule. Screening of unaffected population-matched controls revealed heterozygosity for K1495E and R344X only in individuals of Jewish-Yemenite ancestry, but not in more than 700 control samples of other origins, including 93 non-Jewish Yemenite. These data may be suggestive of positive selection, considering the rarity of NFTC and the small size of the Jewish-Yemenite population; alternatively, they may reflect genetic drift or the effect of a population-specific modifier trait. Calcifications in NFTC generally develop over areas subjected to repeated trauma and are associated with marked inflammatory manifestations, indicating that SAMD9 may play a role in the inflammatory response to tissue injury. We therefore assessed the effect of cellular stress and tumor necrosis factor-alpha (TNF-alpha), a potent pro-inflammatory cytokine, on SAMD9 gene expression. Whereas exogenous hydrogen peroxide and heat shock did not affect SAMD9 transcription, osmotic shock was found to markedly upregulate SAMD9 expression. In addition, incubation of endothelial cells with TNF-alpha caused a dose-related, p38-dependant increase in SAMD9 expression. These data link NFTC and SAMD9 to the TNF-alpha signaling pathway, suggesting a role for this system in the regulation of extra-osseous calcification.

Figure 1. Identification of a new mutation in SAMD9

(a) Pedigree and haplotype analysis of an NFTC family and four healthy unrelated control individuals (C1–C4) carrying mutations p.K1495E and p.R344X. The p.K1495E- and p.R344X-associated haplotypes are boxed in red and blue, respectively. Filled symbols represent affected individuals. (b) Subepidermal calcified nodule over the left elbow of the male patient. (c) Severe gingivitis displayed by the female patient. (d) Sequence analysis revealed two distinct mutations in the patients: an A>G transition at cDNA position 4483, which is predicted to result in missense mutation p.K1495E (right upper panel); and a C>T transition at cDNA position 1030, resulting in premature termination codon p.R344X (left upper panel). The corresponding wild-type sequences are given in lower panels. (e) PCR–restriction fragment length polymorphism analysis confirms segregation of the mutations in the family. PCR amplification was performed as described in the text. Mutation p.K1495E creates a novel recognition site for MboII. Thus, heterozygous carriers display a 93 bp fragment and healthy homozygous individuals show only a 115 bp fragment; mutation p.R344X abolishes a recognition site for endonuclease BSh1236I. Thus healthy individuals display a 170 bp fragment, whereas carriers of the mutation also display a 200 bp fragment.

Figure 2. Effect of TNF-α on SAMD9 gene expression

(a) EaHy cells were cultured in the presence of increasing concentrations of TNF-α, and SAMD9 gene expression was measured as described in Materials and Methods; (b) EaHy cells were exposed for increasing periods of time to 10 ng ml⁻¹ of recombinant TNF-α, and SAMD9 gene expression was quantified by quantitative real-time PCR; (c) EaHy cells were cultured for 6 hours in the presence of 0 (C), 10 ng ml⁻¹ of recombinant TNF-α (T), or 10 ng ml⁻¹ of recombinant TNF-α and 0.5mM of methylprednisolone (T+M), and SAMD9 gene expression was assessed by quantitative real-time PCR.

Figure 3. TNF-α-mediated SAMD9 induction

(a) EaHy cells were cultured in the presence (+) or absence (−) of 10ngml⁻¹ TNF-α, p38 inhibitor (SB203580), or c-Jun N-terminal kinase inhibitor (SP600126), and SAMD9 gene expression was measured after 6 hours; (b) EaHy cells were cultured in the presence (+) or absence (−) of 10ngml⁻¹ TNF-α and NF-κB inhibitor JSH-23, and SAMD9 gene expression was assessed as described above.

Figure 4. Effect of cellular stress on SAMD9 gene expression

EaHy cells were cultured in the presence of increasing concentrations of (a) sorbitol or (b) hydrogen peroxide, and SAMD9 gene expression was monitored using quantitative real-time PCR.