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Two novel SCN9A gene heterozygous mutations may cause partial
deletion of pain perception
Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong
University of Science and Technology, Wuhan, 430030，China.
Ruimei Yuan
Telephone :( 86)15171425469, 13037154560
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Abstract
The physiological sensation of pain and rapid response to stimuli serve as an adaptive
way to avoid harmful situations. While for patients with congenital indifference to
pain (CIP), this protection disappears or almost disappears. Herein, we reported two
patients from two families of China showed insensitivity to pain and diagnosed as CIP
by neurologist. Different from recently reported studies, our patients were not entirely
painless, but did have little pain sensation. Sequence analysis of candidate genes of
two affected individuals identified two novel heterozygous mutations (M899I, M932L)
in
SCN9A
gene.
Furthermore,
a
novel
non-synonymous
single-nucleotide
polymorphism (SNP) within 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. We speculate that the mutations may be the cause of partial deletion
of pain perception in our probands, and the novel polymorphism V1104L may have a
predictive role in the pain sensation of healthy individuals.
Key words: SCN9A; CIP; Nav1.7; pain; anesthesia; mutation
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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 the variability in the 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].
SCN9A, a gene essential for peripheral pain sensation, had been analyzed in
knock-out mice [1,4] before the discovery of naturally occurring mutants in
humans[2]. SCN9A gene knock out 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 null mutants [5]. Interestingly, loss of function caused by SCN9A
human mutants have a defective Nav1.7 channel appear to be completely pain free but
are otherwise normal[5]. Cox et al for the first time demonstrated that mutations in
SCN9A gene resulting in loss of function of Nav1.7 and in congenital indifference to
pain (CIP) [2]. Subsequently, Ahmad et al also evaluated the functional consequences
of in vitro nonsense mutations and their results showed 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
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lowered threshold for activation, causing erythermalgia, a chronic inflammatory
condition [7,8] or paroxysmal extreme pain disorder (PEPD)[9].
So it can be seen that mutations in SCN9A presented as three conditions of
voltage-gated-sodium-channels Nav1.7: channelopathy-associated insensitivity to
pain with insensitive to pain perception, PE with intermittent attacks of red, warm,
painful, swollen extremities and PEED with severe pain episodes. Continuously
several mutations in Nav1.7 have been reported including W897X [2], V136I [10],
I1461T [11], etc, and may result in complete loss or gain of function of Nav1.7. While
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 paper, we reported 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 analysis of three mutations in other members
of the two families and 100 healthy Han Chinese, we found that two mutations
(M899I and M932L) were not presented in the families and healthy individuals, while
a new non-synonymous 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.
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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) However, 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 respectively and 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 were fractured.
They had normal intelligence and could correctly perceive the sensations of
proprioception, touch, temperature, tickle and pressure, but had no favorable
perception of pain. The first proband in family one (Fig. 1) was a nine-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, he 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 was not complete loss of pain perception which protected him
from lip and tongue injury. The second patient in family two (Fig. 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. None of our cases had evident autonomic
nervous system dysfunction.
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Anesthesia for patients with CIP and postoperative pain assessment
These two children underwent orthophoria in our hospital and anesthesia procedures
for these two patients were recorded. Before the surgery, a comprehensive
examination was performed in each patient showing normal. During the operation,
electrocardiography (ECG) and blood pressure and pulse oxygen saturation were
monitored. Total intravenous anesthesia (TIVA) 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 normal and there were no significant changes in blood
pressure and heart rate. The mean time of surgery was about 90 min. Both patients did
not experience nausea and vomiting, and the visual analogue scale (VAS) in the
absence of any analgesics was ≦2 (VAS: 0= no pain, 10= unbearable pain) in the first
24 h postoperatively.
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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 study. Then, 5 ml of heparin anti-coagulated
blood were obtain from the right elbow veins from members of 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 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, California, USA). PCR products were purified and direct sequencing was
performed with an ABI PRISM 377 automated sequencer (PE Applied Biosystems,
USA). To confirm whether the identified mutations were present in the general
population or not, peripheral blood was collected from 100 health Chinese and
SCN9A mutations were detected. All mutant sites were re-genotyped
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Results
All PCR products had the expected lengths. Sequence analysis of SCN9A revealed
three novel heterozygous nonsense mutations in two children. (Fig. 2) For the boy in
the 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 the family 2, two
novels heterozygous mutations were noted in exon 15 (2796 A
amino acid change M932L and in exon 16 (3312G
C) resulting in the
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.
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Discussion
SCN9A has previously been displayed to be involved in nociception in both humans
and rodents. SCN9A encodes the α-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 the nociceptive transmission [1-3]. Gain-of-function mutations in SCN9A, such
as the autosomal-dominant pain disorder primary erythermalgia (OMIM 133020) [5],
produce a lower current threshold for activation of Nav1.7 sodium channel and result
in hyperexcitability of nociceptive neurons [16-18]. As an interesting corollary of
such observation, 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
examinations show almost normal. Now it has been confirmed that CIP is relevant
with 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 SCN9A gene of two individuals with CIP. The mutations M899I
and M932L were found in two affected individuals respectively but absent in the other
families members and 100 healthy adults. So we postulated 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 affects about 6.5% of control adults. As it is not unique for CIP
patients, more investigations are required to clarify whether it is a cause of incomplete
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deletion of pain and whether it can explain the variations in pain thresholds between
individuals.
Furthermore, some researchers had put their eyes on the 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 to
12.7% of control chromosomes may increase nociceptor excitability. They speculated
that individuals with different genotype of rs6746030 may experience differing
amounts of pain [21,22] which is strong evidence to our assumption, the novel
non-synonymous V1104L may be associated with a decrease nociceptor excitability
and lower pain perception in general population.
It is reported that CIP is an autosomal recessive disorder [23], 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
mutation of gene dosage. Our patients were not completely painless but did have mild
pain perception. Some researchers had found that anosmia or hyposmia is also a
common feature in patients with CIP. While in our study it had not been found.
Further laboratory studies such as whole cell patch clamp are required to clarify
whether each mutation causing a functional alteration in the mature protein or not.
Also we need some clinical results to demonstrate the perhaps relevance between
V1104L and pain threshold. Once these observations suggest positive results that
polymorphisms of Nav1.7 sodium channel may alter the pain perception in general
population, it may predict the potential pain perception in general individual which is
very important for the field of pain medicine. It also underscores the potential
importance of Nav1.7 as a molecular target for pain treatment.
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Acknowledgements
We thank these two children and other family members for their co-operation. We
thank all the medical staff in this study for their intelligence and endeavor. And lastly
we expressed our gratitude to our sponsor B. Braun Anesthesia Research Fund of
China.
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References
1. Nassar MA, Stirling LC, Forlani G, et al. Nociceptor-specific gene deletion reveals
a major role for Nav1.7 (PN1) in acute and inflammatory pain. Proc Natl Acad Sci
USA 2004;101: 12706-11.
2. Cox JJ, Reimann F, Nicholas AK, et al. An SCN9A channelopathy causes
ccongenital inability to experience pain. Nature 2006; 444: 894-8.
3. Akopian AN, Souslova V, England S, et al. The tetrodotoxin-resistant sodium `
channel SNS has a specialized function in pain pathways. Nat Neurosci 1999; 2:
541-8.
4. Nassar MA, Levato A, Stirling LC, Wood JN. Neuropathic pain develops normally
in mice lacking both Na(v)1.7 and Na(v)1.8. Mol Pain 2005;1:24.
5. Young FB. A life without pain? Hedonists take note. Clin Genet 2008;73:31-3.
6. Ahmad S, Dahllund L, Eriksson AB, et al. A stop codon mutation in SCN9A
causes lack of pain sensation. Hum Mol Genet 2007;16: 2114-21.
7. Yang Y, Wang Y, Li S, et al. Mutations in SCN9A, encoding a sodium channel a
subunit, in patients with primary erythermalgia. J. Med. Genet 2004; 41: 171-4.
8. Estacion M, Harty TP, Choi JS, Tyrrell L, Dib-Hajj SD, Waxman SG. A sodium
channel gene SCN9A polymorphism that increases nociceptor excitability. Ann
Neurol 2009; 66: 862-6.
9. Dib-Hajj SD, Estacion M, Jarecki BW, et al. Paroxysmal extreme pain disorder
M1627K mutation in human Nav1.7 renders DRG neurons hyperexcitable.Mol
Pain 2008;4:37.
10. Cheng X, Dib-Hajj SD, Tyrrell L, Waxman SG. Mutation I136V alters
electrophysiological properties of the NaV1.7 channel in a family with onset of
erythromelalgia in the second decade. Molecular Pain 2008; 4:1.
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11. Fertleman CR, Baker MD, Parker KA, et al. SCN9A mutations in paroxysmal
extreme pain disorder: Allelic variants underlie distinct channel defects and
phenotypes. Neuron 2006; 52:767–74.
12. Drenth JP, te Morsche RH, Guillet G, Taieb A, Kirby RL, Jansen JB. SCN9A
mutations define primary erythermalgia as a neuropathic disorder of voltage gated
sodium channels. J Invest Dermatol 2005; 124:1333-8.
13. Wang Y, Yang Y, Li S, Fan JF, Xu Z, Liu Bo, Fan ZM, Jin J, Wu GD, Pu DF, Shen
Y, Zhu XJ. Gene mapping and mutation indentification in patients with primary
erythromelalgia. Chin J Dermatol, 2004; 37: 383-6.
14. Klugbauer N, Lacinova L, Flockerzi V, Hofmann F. Structure and functional
expression of a new member of the tetrodotoxin-sensitive voltage-activated
sodium channel family from human neuroendocrine cells. EMBO J 1995;
14:1084-90.
15. Toledo-Aral JJ, Moss BL, He ZJ, et al. Identification of PN1, a predominant
voltage-dependent sodium channel expressed principally in peripheral neurons.
Proc Natl Acad Sci U S A 1997; 94:1527-32.
16. Cummins TR, Dib-Hajj SD, Waxman SG. Electrophysiological properties of
mutant Nav1.7 sodium channels in a painful inherited neuropathy. J Neurosci
2004; 24:8232-6.
17. Dib-Hajj SD, Rush AM, Cummins TR, et al. Gain-of-function mutation in Nav1.7
in familial erythromelalgia induces bursting of sensory neurons. Brain
2005;128:1847-54.
18. Han C, Rush AM, Dib-Hajj SD, et al. Sporadic onset of erythermalgia: a
gain-of-function mutation in Nav1.7. Ann Neurol 2006; 59: 553-8.
19. Goldberg YP, MacFarlane J, MacDonald ML, et al. Loss-of-function mutations in
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the Nav1.7 gene underlie congenital indifference to pain in multiple human
populations. Clin Genet 2007; 71: 311-319.
20. Waxman SG. Neurobiology: a channel sets the gain on pain. Nature 2006;
444:831-2.
21. Reimann F, Cox JJ, Belfer I, et al. Pain perception is altered by a nucleotide
polymorphism in SCN9A. Proc Natl Acad Sci U S A 2010; 107:5148-53.
22. Estacion M, Harty TP, Choi JS, et al. A sodium channel gene SCN9A
polymorphism that increases nociceptor excitability. Ann Neurol 2009; 66:862-6.
23. Dyck PJ. Peripheral Neuropathy. WB Saunders, Philadelphia 1984: 1557–99.
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Figure legends
Figure 1| Families used to map the locus for CIP .Females are indicated by circles and
males by squares. Affected individuals are indicated with filled symbols.
Figure 2| 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.
Table 1 The status and mutations of patients with insensitivity to pain
Patients
Subject 1
Age
Boy
9
Manifestation of
pain sensitivity
significantly high pain threshold;
walk or jump with lower limbs
fractured
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bitten her lip or tongue bleeding
but did not cry; walk with
fractured lower limbs
Subject 2
Girl
Previous injures
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
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
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protein
mutation
Exon
G2697A
M988I
15
A2796C
G3312T
M932L
V1104L
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cDNA genotype