Increased bone formation and decreased osteocalcin expression induced by reduced Twist dosage in Saethre-Chotzen syndrome

The Saethre-Chotzen syndrome is characterized by premature fusion of crania l sutures resulting from mutations in Twist, a basic helix-loop-helix (bHLH ) transcription factor. We have identified Twist target genes using human m utant calvaria osteoblastic cells from a child with Saethre-Chotzen syndrom e with a Twist mutation that introduces a stop codon upstream of the bHLH d omain. We observed that Twist mRNA and protein levels were reduced in mutan t cells and that the Twist mutation increased cell growth in mutant osteobl asts compared with control cells. The mutation also caused increased alkali ne phosphatase and type I collagen expression independently of cell growth. During in vitro osteogenesis, Twist mutant cells showed increased ability to form alkaline phosphatase-positive bone-like nodular structures associat ed with increased type I collagen expression. Mutant cells also showed incr eased collagen synthesis and matrix production when cultured in aggregates, as well as an increased capacity to form a collagenous matrix in vivo when transplanted into nude mice. In contrast, Tn ist mutant osteoblasts displa yed a cell-autonomous reduction of osteocalcin mRNA expression in basal con ditions and during osteogenesis. The data show that genetic deletion of Twi st causing reduced Twist dosage increases cell gran th, collagen expression , and osteogenic capability, but inhibits osteocalcin gene expression. This provides one mechanism that may contribute to the premature cranial ossifi cation induced by deletion of the bHLH Tn ist domain in Saethre-Chotzen syn drome.