Spinal Muscular Atrophy
the number one genetic killer of infants and toddlers
Leena Shah
Human Anatomy
April 21, 2010
Samir died
at 7 ½
months
Hailey Mae
died at the
age of 2
Owen
died at
19 weeks
and 6
days
Baylee died at 15 months
Spinal Muscular Atrophy (SMA)

A genetic disease in
which loss of nerve
cells in the spinal cord
called motor neurons
affects the part of the
nervous system that
controls voluntary
muscle movement
Mouse Model of Spinal Muscular Atrophy
•Motor neuron
axons (green)
•neuromuscular
junctions (red)
Both mice are 11 days old. The mouse on the left does
not have SMA. The SMA mouse on the right is much
smaller due to effects of the disease, and it is unable to
move normally or fully support its weight
Symptoms and Diagnosis





Weakened muscles
Respiratory problems
Scoliosis
Difficulty walking and sitting
Diagnosis of SMA is made based upon physical
symptoms5





poor muscle tone in the limbs and trunk
feeble movements of the arms and legs
swallowing difficulties
a weak sucking reflex
impaired breathing
Four Types of SMA




SMA Type I
 also called Werdnig-Hoffman disease
 is the most severe form (lifespan 2-3 years)
 symptoms are present before the age of six months and these children
never acquire the power, the strength, and the endurance to sit
independently, to crawl, walk, or breath properly
 SMA I affects 60% of all SMA patients
SMA Type II
 less severe than type 1
 comes to medical attention before the age of 18 months
 Respiratory muscles and feeding problems not as severe as in Type 1
 Can sit, but still cannot walk properly
 Can live into adulthood
SMA Type III
 Known as Kugelberg-Welander disease or Juvenile Spinal Muscular
Atrophy
 Symptoms appear between 18 months and early adulthood
 Difficulty walking, muscles weakness, prone to respiratory infections
 Normal life expectancy
SMA Type IV
 Adult form of SMA – less common
 Affects walking
 Symptoms emerge after age 35
3
The Survival of Motor Neuron (SMN)
Gene

Spinal Muscular Atrophy is caused by
mutations in the SMN gene

SMN gene located on chromosome 5

Autosomal recessive gene

Gene codes for the SMN protein
3
Survival of Motor Neuron Protein


Most SMN proteins are localized in the cytoplasm
They play a crucial role in the assembly of spliceosomal
uridine-rich small nuclear ribonucleoprotein (U snRNP)
complexes



snRNPs are essential to the splicing of introns from pre-mRNA
during the post-transcriptional modification of RNA
snRNPs are made up of small nuclear RNA (snRNA) and a group
of seven proteins known as Sm ribonucleoproteins that make up
the extremely stable Sm core of the snRNP
How do SMN proteins assemble these U snRNP complexes?
4
The Formation of snRNPs


snRNA interacts with the SMN protein to form a SMN
complex
The SMN complex functions as an assembly machine
for snRNPs

It binds with Sm proteins and organizes them so that
they can bind with snRNA to form snRNPs


The Sm core of snRNPs consists of seven proteins: B, D1,
D2, D3, E, F and G
SMN protein acts as a chaperone to ensure that the Sm
core assembles onto the correct snRNA

The Sm proteins form a seven-member ring core structure
that encircles the snRNA
4
Central Tudor Domain of the Human
SMN protein

o
SMN proteins assemble
U snRNPs via binding
to Sm core proteins
The Human SMN
protein contains a
central Tudor domain
that facilitates this
SMN–Sm protein
interaction7
N
Loop 2
β1
C
β2
β5
β3
β4
Figure 1. Tudor Domain of the Human SMN
Protein (residues 83-173). The structure forms a
bent antiparallel β-sheet. Five β strands, β1, β2,
β3, β4, and β5, form a barrel-like fold (labeled in
blue). The strands are connected with short
turns labeled in orange. Loop 1 is in between β1β2, Loop 2 is in between β2-β3, and Loop 3 is in
between β3-β4. The N-terminus and C-terminus
are also labeled in orange.
Loop 3
Loop 1
Interaction with Sm Proteins


SMN binds to the Arg and Gly-rich C-terminal tails
of the Sm D1 and D3 proteins
When the Tudor domain has mutation on Glutamic
Acid residue 134 [E134K], (a point mutation that
causes SMA), SMN binds to neither


Glutamic Acid (E) is changed to Lysine (K)
Therefore it can be concluded that the Tudor
domain binds to the C-terminal tails of Sm D1 and
D3
7
The E134K Mutation
N
C
Loop 2
β5
E134K
β1
β2
β3
β4
Figure 2.
The Tudor Domain of the Human SMN
Protein is shown. Individual β strands are
labeled in cyan. Turns between β strands
(Loops 1, 2, and 3) are labeled in orange
along with the N-terminus and C-terminus.
In blue is the Glutamic Acid residue 134.
During an E134K mutation, the Glutamic
Acid residue (E) would be changed to a
Lysine residue (K).
Loop 3
Loop 1
How does the E134K mutation impair
Sm binding?



E134K mutant does not disrupt the threedimensional structure of the Tudor
domain
several negatively charged amino acids
are located in loop 1 (Glu 104, Asp 105),
β4 (Glu 134) and the helical turn
connecting β4 and β5 (Asp 140)
These residues are conserved in all SMN
homologs causing the structure to exhibit
an overall negatively charged surface
7
Negatively Charged Amino Acids
β5
Asp
140
β4
3b
Figure 3.
Glu
134
Asp
105
3a
Glu
104
Loop 1
(a) The Tudor Domain of the Human SMN
Protein with negatively charged amino
acids highlighted. Loop 1 has Glutamic
Acid residue 104 in orange and Aspartic
Acid residue 105 in cyan. Glutamic Acid
residue 134 is in blue on β4. The
Aspartic Acid residue 140 is located
between β4-β5 in yellow.
(b) Structure of the Tudor Domain like 3a.
Figure shows all the side chains of the
negatively charged amino acids
highlighted in 3a.
Mutation Disrupts Charge Distribution


the E134K mutation changes the
local charge distribution at the Sm
binding site
This affects electrostatic interactions
with the positively charged Cterminal Sm tails7
Asp
140
Glu 134
Glu 104
Asp 105
Figure 4. Surface structure of the Tudor Domain of the Human SMN Protein. On the left, negatively
charged residues Glu 134 (in blue), Glu 104 (in orange), Asp 105 (in cyan), and Asp 140 (in yellow)
are shown. On the right, the charge distribution of the same Tudor domain is shown, with the
negatively charged surfaces in red. Therefore if Glu 134 were to be mutated, it would affect the charge
distribution and electrostatic interactions with other positively charged proteins.
Distal Spinal Muscular Atrophy
Type V

Other forms of spinal muscular atrophy are caused
by mutation of other genes

One form is the disease Distal SMA, type V



caused by missense mutations on the enzyme glycyltRNA synthetase (GlyRS)
This progressive disorder affects nerve cells in the
spinal cord and weakens muscles in the hands and
feet
Also known as Distal SMA with upper limb
predominance
2
Symptoms of Distal SMA Type V

Usually begin during adolescence

Initial symptom – cramps in the hand induced by
exposure to cold temperatures

Weakness and wasting away (atrophy) of hand
muscles, especially between index finger and thumb

Foot abnormalities



High arch (pes cavus)
affected individuals eventually develop problems with
walking (gait disturbance)
People have normal life expectancies
2
Distal SMA Type V
Glycyl-tRNA Synthetase (GlyRS)


GlyRS is an enzyme in humans
coded by GARS gene
autosomal dominant pattern


one copy of the altered gene in each
cell is sufficient to cause the disorder
GlyRS is a type of aminoacyl-tRNA
synthetase (ARS)
1
Aminoacyl-tRNA Synthetase (ARS)


ARS are enzymes which couple
amino acids with their cognate
tRNAs to form an aminoacyl-tRNA
This allows the translation from
genetic code to amino acid code
during translation
GlyRS with mutation S581L

Serine residue 581 missense mutation is 50 amino
acids from the active site


Yet gives reduced aminoacylation activity



Serine (S) is replaced with Leucine (L) at residue 581
Interesting because patients carrying this mutation
develop disease younger age than for other GlyRS
mutations
The S581L mutation is located on an alpha-helix in the
anticodon-binding domain and induces a local
conformation change
Overall structure between mutant and wildtype similar
but residues 567-575 of the anticodon-binding domain
have shift position
1
Residues
567-575
Glycyl-tRNA Synthetase
GlyRS with mutation S581L
Figure 5.
Molecular Structure of wildtype and S581L GlyRS mutant. On the left, the wildtype structure is present
with residues 567-575 highlighted in white. On the right, the S581L mutant structure is present with,
again, residues 567-575 in white. A small shift in position resulting in the localized conformation of the
protein is seen. The area where the conformation occurs is known as the anticodon-binding domain of
the GlyRS protein.
Residues
567-575
Glycyl-tRNA Synthetase
GlyRS with mutation S581L
Figure 6.
Surface Structure of wildtype and S581L GlyRS mutant. On the left, the wildtype surface structure is
present with residues 567-575 highlighted in white. On the right, the S581L mutant surface structure
is present with, again, residues 567-575 in white. A more prominent shift in position resulting in the
localized conformation of the protein is seen. The conformation occurs in the anticodon-binding domain
of the GlyRS protein.
Reduced GlyRS Activity

The S581L mutation results in the loss of contact with the following
atoms:


Additional contacts are made with following atoms:


V564, V577, and H591
The mutation therefore causes a small shift in the adjoining beta-turn
of residues 567– 575 which is a position that interacts with the tRNA
anticodon


L580, A583 and T585
The shifted residues are displaced away from the tRNA in S581L-GlyRS
compared to wildtype
This shift can affect glycine binding to the tRNA
reduced enzyme activity of GlyRS = reduced aminoacylation
1
Affected Residues
V564
H591
L580
T585
V577
A583
S581L
S
L
Figure 7.
A close up of the wildtype GlyRS Protein is shown on the left. The Serine residue 581, (where the S581L mutation would occur), is
highlighted in green along with its side chain. In cyan are residues L580, A583, and T585 with their side chains. Contact with these
residues is lost during an S581L mutation. In yellow are residues V564, V577, and H591 with their side chains. New contacts with these
residues are made during an S581L mutation. Side chains are coded by their elemental makeup (red for oxygen, gray for carbon, blue
for nitrogen). At the top right the structure of the GlyRS S581L mutant is shown. The picture is identical to the wildtype with the
exception of the Serine (S) 581 residue mutated to Lysine (L). The difference in the side chains can be see on the bottom right as
Lysine has no oxygen and is a nonpolar residue as opposed to Serine, which is polar.
Treatment



There is no treatment for the
progressive muscle weakness caused
by the disease
Respiratory complications are common
Physical therapy is important to
prevent contractions of muscles and
tendons and abnormal curvature of the
spine (scoliosis)
3
A Therapeutic Target:
Scavenger Decapping Enzyme (DcpS)



SMA is caused by mutation in SMN1 gene
The severity of SMA involves a second
gene, SMN2
DcpS is a potential binder to the SMN2
promoter


DcpS functions in the last step of mRNA decay
to hydrolyze the cap structure following 3’ to
5’ exonucleolytic decay
DcpS can be used to modulate gene
expression of SMN2 gene
8
DcpS Basics


Before translation, mRNA Processing
One of the alterations is the addition of a 5’ cap


DcpS binds to the SMN2 transcript


Capping provides stability to mRNA as well as a
point of attachment to the ribosome during
translation
Decapping enzyme DcpS degrades RNA by
hydrolyzing the cap structure and thereby splicing
the last exon (7), the last coding exon in the SMN2
transcript
This results in the translation of an incorrectly
spliced SMN2 mRNA and the formation of less
SMN2 proteins than normal
8
DcpS bounded to Inhibitor Drug
D156844

DcpS has two active sites: a closed one and an open
one

D156844 is C5-substituted quinazoline

It inhibits the closed active site of DcpS while a point
mutation His277Asn inhibits the open active site

By inhibiting DcpS, the SMN2 mRNA is not decapped
and exon 7 is not spliced, resulting in increased SMN2
mRNA levels

This causes increased SMN2 levels and a decrease in the
severity of SMA disease
8
DcpS bound to Inhibiter D156844
D156844
His 277
Figure 8.
Molecular
Structure of
Scavenger
Decapping
Enzyme (DcpS)
bound with
inhibitor
D156844. Also,
highlighted in
blue, is residue
277 – the location
of where the point
mutation of
Histidine to
Asparanine would
occur.
DcpS Inhibited by D156844 at the
Closed Active Site
D156844
Figure 9.
A close up of the Molecular
Structure of DcpS. Bound to the
molecule is the inhibiter D156844.
The position of the inhibitor is on an
active site that is “closed” because
of its tight conformation.
DcpS Open Active Site
His277
Figure 10.
A close up of the
molecular
structure of DcpS
with the potential
location of
mutation
His277Asn
highlighted. The
mutation would
occur in the “open”
active site of the
DcpS. (open
because of loose
conformation).
Social Implications of SMA

Mutation causing SMA can be detected via prenatal testing5



Genetic counselor – informs of choices, risks, benefits
Prepare for an affected baby with early intervention breathing and
physical therapies for the newborn
SMA affects every 1 in 6000 babies5

In Vitro Fertilization (IVF)


Chorionic Villus Sampling (CVS)




Newborn screening can allow parents to start with intervention
strategies right away before waiting for symptoms to make a diagnosis
Is there enough benefits from early testing to counteract the cost of
testing every newborn?


a sample of the placenta is removed via catheter at 10-12 weeks of
gestation
Test during ongoing pregnancy and then faced with the decision of whether
or not to terminate the pregnancy
Early vs. Late Diagnosis


only those embryos without SMA are transferred back to the mother's uterus
No effective treatments to delay or prevent symptoms found yet 5
In 2009 costs were estimated to be $400 for a carrier screen and
$260,000 for the lifetime cost of a child with severe SMA6
Researchers concluded that 11,000 women would have to be
screened to prevent one case of SMA – a cost of $4.7 million per
every averted SMA case6
References
1.
2.
3.
4.
5.
6.
7.
8.
Cader, Muhammed Z., et al. "Crystal Structure of Human Wildtype and
S581L-Mutant Glycyl-tRNA Synthetase, an Enzyme Underlying Distal
Spinal Muscular Atrophy." FEBS letters 581.16 (2007): 2959-64. Print.
"Distal Hereditary Motor Neuropathy, Type V - Genetics Home
Reference." Genetics Home Reference - Your Guide to Understanding
Genetic Conditions. U.S. National Library of Medicine, Aug. 2009. Web.
21 Apr. 2010.
<http://ghr.nlm.nih.gov/condition=distalhereditarymotorneuropathytype
v>.
"Frequently Asked Questions." SMA Foundation | Spinal Muscular
Atrophy. Spinal Muscular Atrophy Foundation. Web. 21 Apr. 2010.
<http://www.smafoundation.org>.
McDowall, Jennifer. "Sm Ribonucleoproteins." European Bioinformatics
Institute | Homepage | EBI. Protein Data Bank. Web. 21 Apr. 2010.
<http://www.ebi.ac.uk/interpro/potm/2005_5/Page1.htm>.
Norrgard, Karen. “Medical Ethics: Genetic Testing and Spinal Muscular
Atrophy.” Scitable. Web. 2008.
Preidt, Robert. "Screening for Spinal Muscular Atrophy Not CostEffective: Study: MedlinePlus." National Library of Medicine - National
Institutes of Health. 5 Feb. 2010. Web. 21 Apr. 2010.
<http://www.nlm.nih.gov/medlineplus/news/fullstory_94965.html>.
Selenko, Philipp, et al. "SMN Tudor Domain Structure and its Interaction
with the Sm Proteins." Nat Struct Mol Biol 8.1 (2001): 27-31. Print.
Singh, Jasbir, et al. "DcpS as a Therapeutic Target for Spinal Muscular
Atrophy." ACS Chemical Biology 3.11 (2008): 711-22. Print.
Thank You