SCN9A Loss of Function Mutations E1673 metatarsal. The lack of pain response coupled with erythema initially resulted in misdiagnosis of cellulitis and there was a delay of weeks in radiographic assessment (Supp. Figure S1). Now aged 5 years she has been taught and reasoned with to no longer bite her fingers. She gets upset at the presence of blood but does not respond normally to painful events. She seems to have preservation of temperature perception. Autonomic function has been normal including normal sweat production, pupillary reflexes, temperature control, bladder and bowel function. Histamine flare test was normal. Cognitive development has been normal. On examination at one year there were scars from bites on her fingers, ulceration of her tongue and scarring from injury on her toes. Deep tendon reflexes and light touch sensation were normal. Fungiform papillae of the tongue were present. Corneal reflexes were absent. Motor nerve conduction studies were normal, peroneal sensory action potential (SAP) was absent and median and ulnar SAPs reduced. Sural nerve biopsy was normal histologically. Electron microscopy did reveal a few thinly myelinated axons surrounded by redundant Schwann cell basement membrane and although these could be interpreted to be in keeping with a demyelinating process, classical onion bulb formation was not present and there were no definitive abnormalities. A diagnosis of congenital insensitivity to pain was made and the normal development with absence of autonomic involvement led to consideration of SCN9A-associated pain insensitivity. Linkage analysis Mutation screening For the Bedouin family polymorphic markers for the region were developed using Tandem Repeats Finder [Gelfand et al., 2007] and PCR primers designed by the use of the "Primer3" web site (http://frodo.wi.mit.edu/primer3/). PCR was performed on 50 ng DNA with the addition of 0.01 μCi α- [32P] dCTP to enable visualization of the PCR products which were separated on 6% polyacrylamide gels and visualized by a Phosphor–Imager [Parvari et al., 1998]. Genomic DNA was isolated from blood by standard methods. All coding exons and flanking splice sites of SCN9A were bi-directionally sequenced using either previously reported primers [Cox et al., 2006] or SCN9A ex 15f: GAGATATTGAAAATTGATGAAAATGA and SCN9A ex 15r: CAAAATTTCGTTCTCTTTCCTG. The c.2687G>A, c.4108_4122delCGATGGAAAAACCTG and c.4474delA mutations (NM_002977.3) were sought in 260 ethnically matched control chromosomes by amplicon restriction analysis with BseDI (Fermentas, Lithwania) (c.2687G>A) or bi-directional sequencing of genomic DNA. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). Verification of splicing Direct PCR amplification of reverse transcribed RNA from patient's lymphoblastoid cells RNA was extracted from lymphoblastoid cells of patient ip15 using the EZ-RNA II kit of Biological Industries (Israel) according to manufacturer instructions. cDNA was synthesized from 5 microgram RNA by the Reverse – iT 1st strand synthesis kit of ABgene (Surrey, U.K.) with a decamer random primer. The PCR product confirming correct splicing was amplified using primers SCN9A-2310f: CCACCCAATGACTGAGGAAT and SCN9A- 2977r: GGTTGTTTGCATCAGGGTCT, PCR conditions were: 45 cycles of 95o 1', 57o 1', 72o 1' and elongation of 5' at 72 o. The PCR product was directly sequenced. Minigene splicing assay A DNA fragment of 1037 bp containing the wild-type coding exon 15 along with 321 bp of upstream and 360 bp of downstream intronic flanking regions was amplified from genomic DNA, sequenced and cloned into the NdeI site of the PTB minigene construct. The c.2687G>A mutation was created by PCR mutagenesis. Wild-type and mutant minigenes were transfected into HeLa cells using lipofectamine (Invitrogen). RNA was analysed by RT-PCR using primers specific for the minigene ALFA and BRA, followed by sequencing of the PCR products.