Abstract
Objective. The physiological sensation of pain and rapid response to stimuli serve as an adaptive way to avoid harmful situations. Our purpose was to investigate why this protection disappears or almost disappears for patients with congenital indifference to pain (CIP).
Design. The study was designed as a case report by scanning the candidate genes within CIP patients.
Setting. The study was set at the Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Patients. We reported patients from two Chinese families that showed insensitivity to pain and were diagnosed with CIP by a neurologist. Different from recently reported studies, our patients were not entirely painless, but demonstrated little pain sensation from injuries.
Measures. The measures made were novel mutations within SCN9A.
Results. Sequence analysis of candidate genes of two affected individuals identified two novel heterozygous mutations (M899I and M932L) in the SCN9A gene. Furthermore, a novel nonsynonymous single-nucleotide polymorphism (SNP) within the SCN9A gene was revealed in affected proband and several unaffected family members. This polymorphism (c. 3312G&T, which produces the amino acid substitution V1104L in human Nav1.7), is present in 6.5% of healthy Chinese.
Conclusions. We speculate that the mutations may be the cause of partial deletion of pain perceptionin in our probands, and the novel polymorphism V1104L may have a predictive role in the pain sensation of healthy individuals.
Introduction
Pain is one of the most common clinical symptoms experienced by humans, but its molecular mechanism remains incompletely understood. Painful stimuli are transmitted in the form of electrical impulses. These impulses, which originate in the periphery, travel from nociceptive dorsal root ganglion (DRG) neurons through ascending spinal pathways to the central nervous system (CNS). Changes in pain perception may be caused by peripheral and/or CNS lesions, and one of the mechanisms is variability in electrical excitability. Voltage-gate sodium channels, some of which are expressed in nociceptors, transmit the local membrane depolarization generated by stimulus transduction along the axon. The channel Nav1.7 (encoded by SCN9A) has been shown to play an important role in nociceptive transmission [1–3].
Nav1.7 a channel essential for peripheral pain sensation encoded by SCN9A, had been analyzed in knockout mice [1,4] before the discovery of naturally occurring mutants in humans [2]. Nav1.7 global knockout mice die probably due to inability to be fed, while just a loss of acute mechanical and inflammatory pain has been found in nociceptor-specific Nav1.7 knockout mice [5]. Interestingly, patients with loss of function mutations in SCN9A can result in a defective Nav1.7 channel; these persons appear to be completely pain free but are otherwise normal [5]. Cox et al. demonstrated for the first time that mutations in SCN9A gene cause loss of function of Nav1.7 and result in congenital inability to experience pain in these patients who were otherwise normal [2]. Subsequently, Ahmad et al. also evaluated the functional consequences of in vitro nonsense mutations; their results showed that the mutant channel displayed no difference in activity over background current, which supported the loss-of-function hypothesis [6]. As an interesting corollary of such observation, gain of function mutations in SCN9A can result in amplified small depolarization and lowered threshold for activation, causing erythermalgia, a chronic inflammatory condition [7,8] or paroxysmal extreme pain disorder (PEPD) [9].
Therefore, it can be seen that mutations in SCN9A presented three conditions of voltage-gated-sodium-channels Nav1.7: 1) channelopathy-associated insensitivity to pain; 2) primary erythermalgia (PE) with intermittent attacks of red, warm, painful, and swollen extremities; and 3) PEPD with severe pain episodes. Continuously several mutations in Nav1.7 have been reported, for example, W897X [2], V136I [10], I1461T [11] and others; these may result in complete loss or gain of function of Nav1.7. We find that different patients with CIP have different mutations which fail to identify a significant shared haplotype, suggesting that each family has a distinct mutation [2]. In this article, we report three novel mutations (M899I, M932L, and V1104L) in two children with CIP. The diagnosis of CIP was independently confirmed by at least two neurologists based on the criteria. After analyzing the three mutations in the other members of the two families and in 100 healthy Han Chinese, we found that two mutations (M899I and M932L) were not present in the families and in healthy individuals, while a new nonsynonymous single-nucleotide polymorphism (SNP), V1104L in SCN9A, was identified and presented in 6.5% of control subjects. We speculate these mutations may be the cause of partial deletion of pain perception in our probands, and the novel SNP V1104L may have a predictive role in the pain sensation of healthy individuals.
Patients and Methods
Patients
We studied two Chinese families with sporadic patients who were diagnosed as “congenital analgesia” during the first year of life (Table 1). There were no known histories of neurological disorders in their family members. In addition, neurological examination was performed in two affected individuals at the age of five and four and were revealed to be otherwise normal. These patients, however, had little pain perception with significantly high pain threshold at any time and in any part of their body. They can even walk or jump when their lower limbs are fractured. They had normal intelligence and could correctly perceive the sensations of proprioception, touch, temperature, tickle, and pressure, but had no perception of pain. The first proband in family one (Figure 1) was a 9-year-old boy, who was frequently treated for bruises and bone fracture at the local hospital. At the age of 4 he had a femoral bone fracture in his right leg, resulting in claudication, which was later corrected through orthopedic surgery. In addition, other injuries were found in his daily life; he also suffered several complications due to lack of pain sensation such as repeated infections around the left caput femoris, fractures in the cataclasis region after removal of external fixation, and arthrosis in his left ankle and forefoot. Fortunately, this boy did not have complete loss of pain perception, which protected him from lip and tongue injury. The second patient in family two (Figure 1) was an 8-year-old girl. She had injuries in her lip and tongue (with a loss of the distal third) caused by biting herself during the first 3 years without any pain. In addition, bone fracture and other injuries such as right hip dislocation, bilateral tibial fractures, and right talus bone necrosis also occurred. Neither of these cases had evidence of autonomic nervous system dysfunction.
The status and mutations of patients with insensitivity to pain
| Patients | Age (years) | Manifestation of Pain Sensitivity | Previous Injures | cDNA Genotype | Protein Mutation | Exon |
| Subject 1 | ||||||
| Boy | 9 | Significantly high pain threshold; walks or jumps with lower limbs fractured | Femoral bone fracture in right leg; repeated infections around the left caput femoris; repeated fractures in the cataclasis region after removal of external fixation and arthrosis in left ankle and forefoot; continuous soft tissue injury | G2697A | M988I | 15 |
| Subject 2 | ||||||
| Girl | 8 | Bitten her lip or tongue bleeding but did not cry; walk with fractured lower limbs | A loss of the distal third tongue; bone fracture and other injuries such as right hip dislocation, bilateral tibial fractures and right talus bone necrosis; continuous soft tissue injury | A2796C | M932L | 15 |
| G3312T | V1104L | 16 |
| Patients | Age (years) | Manifestation of Pain Sensitivity | Previous Injures | cDNA Genotype | Protein Mutation | Exon |
| Subject 1 | ||||||
| Boy | 9 | Significantly high pain threshold; walks or jumps with lower limbs fractured | Femoral bone fracture in right leg; repeated infections around the left caput femoris; repeated fractures in the cataclasis region after removal of external fixation and arthrosis in left ankle and forefoot; continuous soft tissue injury | G2697A | M988I | 15 |
| Subject 2 | ||||||
| Girl | 8 | Bitten her lip or tongue bleeding but did not cry; walk with fractured lower limbs | A loss of the distal third tongue; bone fracture and other injuries such as right hip dislocation, bilateral tibial fractures and right talus bone necrosis; continuous soft tissue injury | A2796C | M932L | 15 |
| G3312T | V1104L | 16 |
The status and mutations of patients with insensitivity to pain
| Patients | Age (years) | Manifestation of Pain Sensitivity | Previous Injures | cDNA Genotype | Protein Mutation | Exon |
| Subject 1 | ||||||
| Boy | 9 | Significantly high pain threshold; walks or jumps with lower limbs fractured | Femoral bone fracture in right leg; repeated infections around the left caput femoris; repeated fractures in the cataclasis region after removal of external fixation and arthrosis in left ankle and forefoot; continuous soft tissue injury | G2697A | M988I | 15 |
| Subject 2 | ||||||
| Girl | 8 | Bitten her lip or tongue bleeding but did not cry; walk with fractured lower limbs | A loss of the distal third tongue; bone fracture and other injuries such as right hip dislocation, bilateral tibial fractures and right talus bone necrosis; continuous soft tissue injury | A2796C | M932L | 15 |
| G3312T | V1104L | 16 |
| Patients | Age (years) | Manifestation of Pain Sensitivity | Previous Injures | cDNA Genotype | Protein Mutation | Exon |
| Subject 1 | ||||||
| Boy | 9 | Significantly high pain threshold; walks or jumps with lower limbs fractured | Femoral bone fracture in right leg; repeated infections around the left caput femoris; repeated fractures in the cataclasis region after removal of external fixation and arthrosis in left ankle and forefoot; continuous soft tissue injury | G2697A | M988I | 15 |
| Subject 2 | ||||||
| Girl | 8 | Bitten her lip or tongue bleeding but did not cry; walk with fractured lower limbs | A loss of the distal third tongue; bone fracture and other injuries such as right hip dislocation, bilateral tibial fractures and right talus bone necrosis; continuous soft tissue injury | A2796C | M932L | 15 |
| G3312T | V1104L | 16 |
Families used to map the locus for CIP. Female family members are indicated by circles and males by squares. Affected individuals are indicated with filled symbols.
Anesthesia for Patients with CIP and Postoperative Pain Assessment
These two children underwent orthopedic surgeries (plate fixation of lower limb fractures) in our hospital and anesthesia procedures for these two patients were recorded. Before the surgery, a comprehensive examination was performed in each patient, which showed normal results. During the operation, electrocardiography, blood pressure, and pulse oxygen saturation were monitored. Total intravenous anesthesia was applied with 1.5 mg of midazolam, 2 mg/kg propofol, and 2 µg/kg fentanyl for anesthesia induction. Anesthesia was maintained with 6∼10 mg/kg/h propofol. During the surgery, body temperature was maintained at normal and there were no significant changes in blood pressure and heart rate. The mean time of surgery was about 90 minutes.
Both patients did not experience nausea and vomiting, and the visual analog scale (VAS) in the absence of any analgesics was ≦ (VAS: 0 = no pain, 10 = unbearable pain) in the first 24 hours postoperatively.
Genotype Assays
This study was approved by the Institutional Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and informed consent was obtained before the study. Then, 5 mL of heparin anti-coagulated blood were obtained from the right elbow veins from members of the two families including their grandparents, parents, and other members. Genomic DNA was extracted from the blood samples using a guanidinium isothiocyanate method.
Genomic DNA from the patients and their family members (including their parents, brothers or sisters, and their grandparents) were screened for mutations in SCN9A (GenBank accession number, NG_012798.1). All the 26 exons of SCN9A were amplified by polymerase chain reaction (PCR) (all primers were designed by Drenth JP [12], except for exons 12, 23, 24, and 25 [13]) followed by direct sequencing. The PCR reaction was performed in a Gene Amp PCR system 9700 (Applied Biosystems, Foster City, CA). PCR products were purified and direct sequencing was performed with an ABI PRISM 377 automated sequencer (PE Applied Biosystems). To confirm whether the identified mutations were present in the general population or not, peripheral blood was collected from 100 healthy Chinese and SCN9A mutations were detected. All mutant sites were re-genotyped
Results
All PCR products had the expected lengths. Sequence analysis of SCN9A revealed three novel heterozygous nonsense mutations in two children (Figure 2). For the boy in family 1, a new heterozygous base substitution was identified in exon 15 (2697 G → A), resulting in the amino acid change M899I. For the girl in family 2, two novels heterozygous mutations were noted in exon 15 (2796 A → C) resulting in the amino acid change M932L and in exon 16 (3312 G → T) causing the amino acid change V1104L. The first two mutations showed disease segregation within the other members of the two families and 100 Chinese normal control subjects. The mutation V1104L was also absent in all the other members of the two families, while it was presented in 6.5% of healthy Chinese.
Sequence chromatograms with mutations in the two families A: Affected member in the family 1 carries the heterozygous mutation of G2697A. B and C: Affected member in the family 2 carries the heterozygous mutations of A2796C and G3312T. Arrows indicated the mutation sites.
Discussion
SCN9A has previously been shown to be involved in nociception in both humans and rodents. SCN9A encodes the á-subunit of Nav1.7 that is predominately expressed in peripheral sensory neurons, especially in nociceptive small-diameter DRG neurons [14,15]. Recent studies have demonstrated that the channel Nav1.7 plays an important role in nociceptive transmission [1–3]. Gain-of-function mutations in SCN9A, such as the autosomal-dominant pain disorder PE (OMIM 133020) [5], produce a lower current threshold for activation of Nav1.7 sodium channels, and results in hyperexcitability of nociceptive neurons [16–18]. As an interesting corollary of such observation, in loss-of-function mutations in Nav1.7, as a cause of CIP, the firing of action potentials may be substantially compromised and accompanied by hypo-excitability of nociceptive neurons [2,19]. Based on the studies above, we speculated that mutations in SCN9A may alter the functions of Nav1.7 to different extents, thus resulting in hyperalgesia or analgesia.
CIP is characterized by lack of physical sensation of pain while other neurological functions are almost normal. Now it has been confirmed that CIP is related to the SCN9A gene and the function of Nav1.7. It is surprising that “the presence of pain perception depends on a sodium channel”[20]. In our cases, we identified three novel mutations in the SCN9A genes of two individuals with CIP. The mutations M899I and M932L were found in these two affected individuals but were absent in the other families members and 100 healthy adults. Therefore, we postulate that these two mutation sites may be associated with the partial deletion of pain perception and lead to changes in Nav1.7 function. The third mutation was a novel SNP site V1104L founded in one of patients and had a frequency of about 6.5% of control adults. As this site is not unique for CIP patients, more investigations are required to clarify whether it is a cause of incomplete deletion of pain and whether it can explain the variations in pain thresholds between individuals. Furthermore, some researchers have investigated pain perception altered by nucleotide polymorphism in SCN9A and found that rs6746030 (3448 C → T, resulting in the amino acid change R1150W in exon 18) with a certain frequency of 1.1–12.7% of control chromosomes, may increase nociceptor excitability. They speculated that individuals with a different genotype of rs6746030 may experience differing amounts of pain [8,21], which would be a strong evidence for our assumption. The novel nonsynonymous V1104L may be associated with diminished nociceptor excitability and lower pain perception in the general population.
It is reported that CIP is an autosomal recessive disorder [22], while our patients with heterozygous mutations in SCN9A showed partial deletion of pain perception. Based on our results, we speculate that the occurrence of clinical features of CIP depends on incomplete dominance and the extent of loss of pain perception may be related to the percentage of mutation gene in the whole gene dosage. Our patients were not completely painless but did have mild pain perception. Little anesthetic was needed during the operation to maintain normal blood pressure and heart rate. Postoperative pain assessment was ≤2 in the first 24 hours without any pain treatment. Anosmia or hyposmia is also a common feature in patients with CIP, while it was not found in our cases.
Further laboratory studies such as whole cell patch clamp are required to clarify whether each mutation causes a functional alteration in the mature protein or not. Also we need some clinical results to demonstrate the possible relationship between V1104L and pain threshold. Once these observations suggest that polymorphisms of Nav1.7 sodium channel may alter pain perception in general population, it may predict pain perception in individuals within the general population, which would be an important advance for the field of pain medicine. Also this finding would underscores the potential importance of Nav1.7 as a molecular target for pain treatment.
Acknowledgments
We thank the two children and other family members for their cooperation. We thank all the medical staff in this study for their intelligence and endeavor. And lastly, we express our gratitude to our sponsor, B. Braun Anesthesia Research Fund of China.
Competing Interest
None to declare.
