Sensation: It is the rapid response to represent stimuli from the environment. Essential for survival because these environmental factors may cause physical damage There are numerous receptors, ion channel and other proteins involved in perception and transmission of painful stimuli.

1. A stop codon mutation in
SCN9A causes
lack of pain sensation
By:
Hadiah Bassam Al Mahdi
Bachelor in Science
May
2010

2.  It is the rapid response to represent stimuli from
the environment.
 Essential for survival because these
environmental factors may cause physical
damage
 There are numerous receptors, ion channel
and other proteins involved in perception
and transmission of painful stimuli.
Introduction
Sensation:

3.  Is a gene located on chromosome 2 and
coded for voltage gated sodium channel
Nav1.7 which plays a significant role in
nociception signalling
 The mutation C A transvertion at
nucleotide 984 transforming the codon
tyrosine at 328 to a stop codon truncated
protein that can’t perform its function.
SCN9A

4. erythermalgia
Different mutations in
voltage-gated channel
Nav1.7 have been shown to
be associated with tow
congenital disorder
erythromelalgia,
paroxysmal extreme pain.
Mutation other than stop codon

5. Structure:

6. Action potential is electrical
membrane potential cells.
In neurons, it plays central role
in cell to cell communications.
Nerves impulse

7. Nav1.7 play a critical role in generation
and conduction of action potential

8.  Screning of stop codon mution in
SCN9A and its relation with lack pain
sensation .
 Knock out of Nav1.7 in mice to see
what is the effect and compare it with
human.
Objectives:

10. Mutational analysis and
bioinformatics
Ensembl used for deriving a list of
exons sequence.
Primer3 to design amplified exons
primers.
PCR touch down PCR were used.
Mutation detection was performed by
comparing the sequence of two affected
individuals with healthy control.

11. • Human NAv1.7 gene mutation assembled from
overlapping pcr fragments.
• the mutant,Nav1.7 stop 328 was generated by
replacing the codon for residue 328 with a stop
codon.
• the resulting construct was cloned into
pcDNA(pZsGreen1-N1) and was used in
co-transfection to visualize transfected cells.
Construct and site directed
mutagenesis

12.  The transformed cells(HEK293 and
ND7/23 ) were cultured in an
appropriate media after transfection by
lipofection.
Cell culture:

13. uses a
micropipette
attached to the
cell membrane to
allow recording
from a single ion
channel
cell-
attached
patch
clamp
Electrophysiology study
Membrane capacitance were contineously
monitored using a short -10mv and -100mv
pulse
Membrane capacitance were contineously
monitored using a short -10mv and -100mv pulse

14. Probes and in situ hybridization
Used to
defect
Nav1.7
mRNA on
selected
tissue .

15.  Hybridization signal intensity was
assessed using both the film
autoradiogram and the emulsion-
dipped slides.
Image analysis and
photography

16.  Canadian family parents noticed when
your child infection or injury did not
report pain and they never cried.
 The neurological examination of
sensory & mental function was normal
and can discriminate between hot &
cold or sharp & dull .
Results:

17. Genotyping of Canadian family:
Pedigree structure and genotype of Canadian
family members with congenital inability to
experience pain

18. Sequencing

19. Functional evaluation of the
mutation
Schematic representation of the Nav1.7 protein and
localization of the identified stop codon mutation
after the fifth transmembrane region in domain 1.

20. 1. Nonsense –messenger-mediated RNA
decay which have no functional
consequences.
2. Truncated protein lacking all
pore-forming regions of the full-length
channel.
Expected results of mutation

21. Re-engineered the mutation (Nav1.7 stop328) and full
length human Nav1.7 cDNA in pZsGreen1-N1
Test of the functionality of truncated
protein
Transient transfected in HEK293 and ND7/23
cell lines
Performed whole-cell patch-clamp experiments

22. Functional effects of mutation
Typical voltage
activated sodium
current
No current
produced

23. Transfection of NaV1.7stop328 did not alter
the voltage gated current
control
Transfected
cells

24.  Global deficiency of Nav1.7 in human appear
healthy but in mice is lethal.
 To test this difference they compared the
expression of this gene in rat, mouse,
monkey and human tissue using in situ
hybridization technique.
Why Nav1.7 serves different functions
in the species ?

25.  First: they examined the expression of
Nav1.7 mRNA in the brain.
 Second: they also measure the
expression of mRNA in the endocrine
gland.
Two experiments were
performed

26. Strong hybridization signals of Nav1.7 mRNA
in all four species
In dorsal root gangelia (DRG)In dorsal root gangelia (DRG)

27. Prominent mRNA hybridization signal in both
rodent
ratmouse
signal in the
paraventricular
hypothalamic
nucleus(Pa)
and supraoptic
nucleus(SO)

28. mRNA hybridization signal in (Pa and SO)
primate is not prominent
monkey Human
The ISH
signal
observed
in primate
was much
weaker
than
strong
labeling
present in
both
rodent
species

29.  Nav1.7 mRNA hybridization signal is
detected in the rat but not in human.
Nav1.7 mRNA expression in adrenal
gland

30.  The interior lobe of the human pituitary was
devoid of Nav1.7 mRNA hybridization signal
but in the rat are labeled.
Nav1.7 mRNA expression in pituitary
gland
rat Human

31.  Congenital inability to experience pain phenotype is
linked to loss of function mutation in SCN9A gene.
 The patients claim to start to feel pain during
adolescence may be reflective of the emergence of a
true pain sensation, but could also be a learned
behavior.
 The difference in expression of Nav 1.7 mRNA in
rodent than primate explains why global deficiency
of Nav1.7 is lethal in mice but not in humans.
conclusion

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Refrences

33. Questions????