Loss of transmembrane lipid asymmetry in apoptotic cells or in activated platelets is thought to be catalyzed by a specific membrane protein named phospholipid scramblase. 1 Recently, the transmembrane protein TMEM16F was shown to be required for Ca2-induced lipid scrambling and exposure of phosphatidylserine at the cell surface. 2 The TMEM16F gene is located on chromosome 12 (12q12) and comprises 20 exons encoding a 910–amino acid protein with 8 transmembrane segments. 2, 3 A patient with Scott syndrome, an inherited bleeding disorder caused by defective scramblase activity, was reported to be homozygous for a TMEM16F mutation (IVS12–1G3T) causing exon 13 skipping, frame shift, and premature termination of translation. 2 After obtaining informed consent, we investigated another patient with Scott syndrome, a 64-year-old Welsh female with a moderate bleeding tendency. The scramblase defect in her platelets, erythrocytes, and B lymphocytes has been previously characterized. 4 The patient’s genomic DNA was isolated from peripheral blood leukocytes and all TMEM16F exons (including splicing junctions) were amplified and sequenced. Two different mutations were identified (Figure 1A): a transition at the first nucleotide of intron 6 (IVS6 1G3A), disrupting the donor splice site consensus sequence of intron 6, and a single-nucleotide insertion in exon 11 (c. 1219insT, cDNA numbering from the ATG), predicting a frame shift and premature termination of translation at codon 411 (Figure 1C).

To clarify the impact of the IVS6 1G3A mutation on TMEM16F pre-mRNA splicing, total RNA was isolated from the patient’s blood cells using TRIzol Reagent (Invitrogen) and reverse-transcribed with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). A TMEM16F cDNA amplicon spanning exons 5-8 was amplified and analyzed by agarose gel electrophoresis (Figure 1B). Whereas the normal control showed only the expected 459-bp fragment, the patient showed both the normal fragment (likely the product of the c. 1219insT allele) and a shorter (345-bp) fragment. The low intensity of the patient’s 459-bp band suggests that the mRNA transcribed from the c. 1219insT allele is partially degraded in vivo. Sequencing of the shorter fragment showed that it lacked exon 6 (Figure 1B). Exon 6 skipping, which is attributable to the IVS6 1G3A mutation, predicts the in-frame deletion of 38 amino acids (residues 212-249)