Ph.D Thesis

Ph.D StudentTopaz Orit
SubjectMolecular Genetics of Familial Tumoral Calcinosis
DepartmentDepartment of Medicine
Supervisors PROF. Eli Sprecher
Full Thesis textFull thesis text - English Version


Familial tumoral calcinosis (FTC) is a rare autosomal recessive disorder, characterized by deposition of calcified masses in cutaneous and subcutaneous tissues resulting in painful ulcerative lesions and severe skin and bone infections.  Clinical and metabolic assessment of these families revealed the existence of two forms of FTC:

1). Hyperphosphatemic FTC (HFTC) in families of Druze and Afro-American origins, where elevated levels of phosphate (Pi) were found to precede tumor development.

2) Normophosphatemic FTC (NFTC) in families of Jewish Yemenite origin was found to be characterized by smaller tumors often located in acral areas, and accompanied by inflammatory manifestations.

Consanguinity in the Druze families and Jewish Yemenite families enabled us to use homozygosity mapping to localize the HFTC and NFTC genes to 2q24-q31 and 7q21-7q21.3, respectively. Three deleterious mutations (two splice-site mutations and a non-sense mutation: 1524+1G>A, 1524+5G>A and R162X, respectively) in GALNT3, encoding ppGalNacT3, a glycosyltransferase responsible for initiating mucin-type O-glycosylation, were detected in the HFTC cohort. Using RNA extracted from skin biopsies and blood samples from Druze patients, we observed very low amounts of an aberrant GALNT3 splice variant, lacking exon 7. In situ hybridization and immunohistochemical studies of frozen skin sections of biopsies of Druze patients revealed complete absence of staining for GALNT3 mRNA and protein, respectively. Thus HFTC results from ppGalNacT3 deficiency.

We further demonstrated that hyperphosphatemia-hyperostosis syndrome (HHS), a rare autosomal recessive bone disease, and HFTC are allelic disorders. Using microsatellite analysis, we demonstrated that the mutation had appeared in Israeli Druze and Moslem populations as a result of a founder effect. Hence, these data suggest the existence of gene modifiers of importance.

We then screened candidate genes located within the NFTC critical region on 7q. We identified a missense mutation (K1495E) in Sterile Alpha Motif Domain containing 9 (SAMD9), which was found to segregate with the disease in all Yemenite families. Transfection of HEK293 cells with K1495E-carrying GFP-SAMD9 fusion resulted in remarkably decreased expression of the fusion protein, which was consequently confirmed by western blotting.  Our data indicate that SAMD9 is involved in the regulation of extraosseous calcification, a process of considerable importance in a wide range of common diseases such as atherosclerosis and autoimmune disorders.