The majority of patients with Saethre-Chotzen syndrome have mutations in the TWIST gene, which codes for a basic helix-loop-helix transcription factor. Of the genetic alterations identified in TWIST, nonsense mutations, frameshifts secondary to small deletions or insertions, and large deletions implicate haploinsufficiency as the pathogenic mechanism. We identified three novel intragenic mutations and six deletions in our patients by using a new strategy to screen for TWIST mutations. We used polymerase chain reaction (PCR) amplification with subsequent sequencing to identify point mutations and small insertions or deletions in the coding region, and real-time PCR-based gene dosage analysis to identify large deletions encompassing the gene, with confirmation by microsatellite and fluorescence in situ hybridization (FISH) analyses. The size of the deletions can also be analyzed by using the gene dosage assay with "PCR walking" across the critical region. In 55 patients with features of Saethre-Chotzen syndrome, 11% were detected to have deletions by real-time gene dosage analysis. Two patients had a translocation or inversion at least 260kb 3′ of the gene, suggesting they had position-effect mutations. Of the 37 patients with classic features of Saethre-Chotzen syndrome, the overall detection rate for TWIST mutations was 68%. The risk for developmental delay in patients with deletions involving the TWIST gene is approximately 90% or eight times more common than in patients with intragenic mutations.
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