Compound heterozygosity for 2 novel TMEM16F mutations in a patient with Scott syndrome

To the editor:

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 (IVS12–1G→T) 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 + 1G→A), 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 + 1G→A 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 + 1G→A mutation, predicts the in-frame deletion of 38 amino acids (residues 212-249) from the N-terminal cytoplasmic domain of the protein (Figure 1C). The normal intensity of this band suggests that the stability of the mRNA transcribed from this allele is normal.

To definitely exclude the possibility that the identified mutations are polymorphisms present in the general population, 100 unrelated white individuals were genotyped for all 3 mutations by restriction analysis with BstY I (IVS12–1G→T), Rsa I (IVS6 + 1G→A, restriction site introduced with a mutagenic reverse primer) and Tsp509 I (c.1219insT), respectively (New England Biolabs). No instance of any of these mutations was found.

In conclusion, we have identified 2 novel mutations in the TMEM16F gene in a patient with Scott syndrome. This finding confirms the recent report²  that the product of this gene is required for Ca²⁺-dependent phospholipid scrambling and that loss-of-function mutations in this gene can give rise to Scott syndrome.