SCN9A Loss of Function Mutations E1675 acid change R896Q (reviewed but not shown). Arginine at position 896 is highly conserved, both evolutionary (Supp. Figure S2a) and in all sodium channels of the family (Supp. Figure S2b). This DNA change is not present in SNP or genomic databases. To exclude the possibility that the change is a population polymorphism, we determined its prevalence in healthy Bedouins of the same geographic region. The mutation eliminates a restriction site for the enzyme BseDI (Supp. Figure S3a). We analyzed 130 healthy Bedouins, using the elimination of a BseDI restriction site by the mutation, and found it was not present in any control individual (Supp. Figure S3b). However, since all 13 presently known mutations causing CIP truncate the protein, we verified that the substitution does not cause aberrant splicing. Firstly, bioinformatics analysis of the c.2687G>A coding exon 15 mutation using a battery of splice site/enhancer prediction algorithms (http://www.fruitfly.org/seq_tools/splice.html; http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi?process=home) showed that splicing control was unlikely to be disrupted by the mutation. Secondly, RNA was extracted from lymphoblastoid cells of patient ip15, and an RT- PCR product was produced with primers fitting sequences in exons 14 and 16. The sequence of this product demonstrated that splicing was not altered (Supp. Figure S4a). Thirdly, we assessed the effect of the mutation on the control of splicing of exon 15 by a minigene assay. This showed that there was no difference between the wild- type and mutant minigenes with both producing mRNA with normal inclusion of coding exon 15 (Supp. Figure S4b). DNA sequence analysis in the British patient Sequence analysis of SCN9A in the British proband revealed that she was a compound heterozygote for a de novo five amino acid in-frame deletion (Nav1.7 -ΔR1370-L1374) and a truncating mutation (Nav1.7-I1493SfsX8) (Figure 1b). Neither mutation is present in SNP or genomic databases or is predicted to disrupt splicing signals. Furthermore, these mutations were shown to be absent from 130 healthy Caucasian control individuals. Reduced cell surface expression of mutant channels All channelopathy-associated insensitivity to pain mutations reported to date cause premature truncation of the Nav1.7 sodium channel [Cox et al., 2006; Goldberg et al., 2007; Ahmad et al., 2007; Nilsen et al., 2009]. Similarly, one of the mutant alleles in the compound heterozygote (Nav1.7-I1493SfsX8) leads to truncation of the protein prior to domain 4 (Figure 1c), which would be predicted to cause a complete loss of function. However, the British five amino acid in-frame deletion (Nav1.7-ΔR1370-L1374) and the Bedouin missense mutation (Nav1.7-R896Q) are not expected to cause truncation but instead change the amino acid sequence in the S5-S6 extracellular loop of domains III and II of Nav1.7 respectively (Figure 1c). These loops form part of the sodium channel pore and both mutations are located just prior to the ion selectivity filter domain [Marban et al., 1998]. To study the effect of these mutations on the cellular localization of Nav1.7, FLAG-tagged SCN9A constructs were generated. The FLAG tag was inserted in the first extracellular loop in domain I of Nav1.7, similar to previous FLAG-tagging experiments performed for Nav1.5 [Makita et al., 2008; Liu et al., 2005]. Comparison of non-FLAG tagged and FLAG-tagged WT channels by patch clamping in HEK293 cells showed that epitope tagging did not affect the current densities or the gating properties of the sodium channel (Supp. Figure S5). Unfortunately, positively transfected cells could not be detected by immunostaining in a range of fixatives using a reliable anti-FLAG monoclonal antibody (data not shown). In HEK293 cells, we were also unable to see a convincing plasma membrane stain using a monoclonal anti-Nav1.7 antibody in cells transfected with the WT construct (data not shown). We therefore switched to analysis of the FLAG-tagged SCN9A constructs in undifferentiated PC12 cells. Using the monoclonal anti-Nav1.7 antibody we could see an obvious intracellular staining in cells transfected with the wild-type construct (Figure 2a and Supp. Figure S6). Furthermore, in a proportion of cells we could see a faint but distinct ‘rim’ of staining which co-localized with the plasma membrane marker pan cadherin (Figure 2a). In contrast, cells transfected with the mutant channels Nav1.7-ΔR1370-L1374 and Nav1.7-R896Q typically showed no plasma membrane staining for Nav1.7, although a similar number of cells as in the wild-type transfected cells showed strong intracellular staining. This suggests that the mutations caused abnormal trafficking of the channels to the plasma membrane compared to WT (Figure 2a and Supp. Figure S6). However, this phenotype was not the same in every cell as a minority of mutant-transfected cells appeared to show some Nav1.7 staining at the plasma membrane. To quantify this result we counted at least 300 transfected cells over 3 coverslips for each construct (Figure 2b). This showed that there were significantly more WT-transfected cells with Nav1.7 plasma membrane staining than the number of mutant-transfected cells with Nav1.7 plasma membrane staining. This data suggests