SCN9A Loss of Function Mutations E1671 analgesic drugs and treatments with up to 50% of treated subjects receiving inadequate pain relief [Stewart et al., 2003]. Thus there exists a significant need to develop better therapies. In this paper we explore one method to achieve this goal: the analysis of gene mutations in humans with altered pain perception. Over the past several years, elucidation of the genetic defects underlying three monogenic pain disorders has provided important insights about an unexpected component of human pain [Yang et al., 2004; Fertleman et al., 2006; Cox et al., 2006]. Channelopathy-associated Insensitivity to Pain (CIP) is a rare condition in which patients have no pain perception and anosmia, but are otherwise essentially normal (MIM# 243000). Recently, genetic studies in families demonstrating recessively inherited CIP have identified nonsense mutations that result in truncation of the voltage-gated sodium channel type IX α subunit (SCN9A), a 113.5-kb gene comprising 26 exons (MIM# 603415) [Cox et al., 2006; Goldberg et al., 2007; Ahmad et al., 2007; Nilsen et al., 2009]. The encoded sodium channel is composed of 1977 amino acids and is organized into 4 domains, each with 6 transmembrane segments [Klugbauer et al., 1995]. The SCNA family of sodium channels (SCN1A-SCN11A) evolved from an archetypal potassium channel by quadruplication, where four potassium subunits have to coalesce to form the functional potassium channel. SCN9A is predominantly expressed in the dorsal root ganglion (DRG) neurons and sympathetic ganglion neurons [Rush et al., 2006]. Functional studies, performed for only some mutations to date, have shown that CIP-associated mutations lead to a loss of function of Nav1.7 [Cox et al., 2006]. Heterozygous mutations that cause changes of amino acids in Nav1.7 result in very different pain phenotypes; Primary Erythermalgia (PE; MIM# 133020) and Paroxysmal Extreme Pain Disorder (PEPD; MIM# 167400) [Yang et al., 2004; Fertleman et al., 2006]. The primary symptoms associated with PE are severe chronic burning pain sensations in the hands and feet. The missense mutations causing PE are gain of function mutations that predominantly enhance the activation of Nav1.7 [Cummins et al., 2004]. PEPD is characterized by severe burning rectal, ocular, and submandibular pain sensations. Eight distinct Nav1.7 missense mutations were reported in diﬀerent PEPD families that are different from those associated with erythermalgia [Fertleman et al., 2006]. The PEPD mutations that have been functionally characterized cause depolarizing shifts in voltage-dependence of steady-state inactivation and cause incomplete inactivation of Nav1.7, leading to prolonged persistent currents. Recently, heterozygous missense mutations in SCN9A have been reported to cause febrile seizures, but without altered pain perception (MIM# 603415) [Singh et al, 2009]. These mutations are all in highly conserved amino acids and are postulated to alter the function of brain-expressed Nav1.7. Analysis of a knock-in mouse for one of the reported mutations (N641Y) showed homozygous mice to be significantly more susceptible to seizures [Singh et al, 2009], supporting the hypothesis that this mutation is disease-causing. Thus prior to this study, missense mutations were only reported as leading to either excess or paroxysmal pain, or febrile seizures. Here we report for the first time missense mutations in SCN9A that cause CIP and demonstrate that the amino acid changes result in loss of function of Nav1.7. MATERIALS AND METHODS Patients and pedigrees Bedouin Patients The patients belong to a consanguineous family, one father with 2 first degree cousin wives (Figure 1a). We studied three sisters of the same nuclear family. Two of the girls were the offspring of one wife and another patient was the daughter of the second wife to the same father. Their ages were 15, 5 and 3 years respectively. The pregnancy and delivery were normal. All presented with a history of indifference to pain in early childhood, e.g. pin pricks or falls, with recurrent trauma including skin burns, fracture of bones and amputation of toes which caused them little distress. All patients had old skin scars due to burns, frequent cuts and bruises. Deformities of the limbs due to osteomyelitis of the upper and lower limbs and old bone fractures were evident, including a fracture of the shoulder which was identified by local swelling in one patient and a tibial bone fracture which was noticed due to limping in another. Trauma of the oral region was frequent in all patients. The tips of the tongues were amputated due to recurrent biting. The patients extracted their teeth and suffered from recurrent bouts of mandibular osteomyelitis. On examination the children were able to feel fine touch and pin prick but were indifferent to painful stimuli caused by squeezing the Achilles tendon and finger tips. In addition, they showed a