Spinal Muscular Atrophy

Question 1

What is the function of the SMN1 gene?

Answer 1

It encodes survival motor neuron protein (SMN) which is a critical part of the complex that interacts with small nuclear ribonucleoproteins (snRNPs)

Question 2

What are snRNPs and what do they do?

Answer 2

Small Nuclear Ribonucleoproteins
U1, U2, U4, U5, U6
They are a key part of the spliceosome, which carries out splicing

Question 3

What type of mutation in the SMN1 gene is usually the cause of Spinal Muscular Atrophy?

Answer 3

Deletion in exon 7 or exon 8

Question 4

How is SMA inherited?

Answer 4

It is autosomal recessive, as it is a loss of function mutation

Question 5

What is the prevalence of SMA?

Answer 5

Affects 1 in every 6,000-10,000

It is the second most common autosomal recessive disorder, behind Cystic Fibrosis

Question 6

What is the pathophysiology behind SMA?

Answer 6

No/less abundance of Survival Motor Neuron (SMN) protein leads to atrophy of the alpha-motor neurons in the anterior horn of the spinal cord
Muscles innervated by these neurons receive no stimulation, so undergo atrophy

Question 7

SMA has phenotypic heterogeneity: what does this mean?

Answer 7

The phenotype of the disease varies depending on the specific genetic make-up
In the case of SMA, the severity depends on the copy number of the SMN2 gene

Question 8

What is the function of the SMN2 gene?

Answer 8

The SMN2 gene is almost identical to SMN1, except it undergoes alternative splicing to miss out exon 7, so only codes a functional SMN protein 10-20% of the time

Question 9

Features of Type 1 SMA

Answer 9

Most severe form of SMA
Signs and symptoms appear shortly after birth
Patients do not survive past 2 years of age
Patients have 1 or 2 copies of the SMN2 gene

Question 10

Features of Type 2 SMA

Answer 10

Develops around age 1
Can shorten life expectancy, but most survive into adulthood
Patients usually have 3 copies of the SMN2 gene

Question 11

Features of Type 3 SMA

Answer 11

May not appear until late childhood or adulthood
Life expectancy not affected
Patients usually have 3 copies of the SMN2 gene

Question 12

Features of Type 4 SMA

Answer 12

The least severe form of SMA
Symptoms do not occur until adult life
Symptoms are much milder
Life expectancy not affected
Patients have 4 copies of the SMN2 gene

Question 13

What are the general symptoms of SMA?

Answer 13

Problems swallowing
Increased incidence of respiratory illnesses due to difficulty coughing
Weak arms and legs
Shaking

Question 14

How is SMA diagnosed (after birth)?

Answer 14

Multiplex Ligation-Dependent Probe Amplification Assay (MLPA)
Blood sample taken, look for common deletions in exon 7 and exon 8

Question 15

How to interpret the results of MLPA?

Answer 15

Peaks form for all the exons of a gene on a graph
Full peak = both copies of gene functioning normally
Reduced height peak = one copy of the gene has the exons deleted
No peaks = both copies of the gene have the exons deleted

Question 16

3 possible methods by which fetal DNA can be obtained for prenatal testing for SMA

Answer 16

Amniocentesis
Chorionic Villus sampling
Cell-free fetal DNA

Question 17

What happens during amniocentesis?

Answer 17

Cells taken from amniotic fluid by a needle inserted through the abdominal wall
Chance of miscarriage or damaging placenta

Question 18

What happens during chorionic villus sampling?

Answer 18

Cells taken from the placenta by a needle inserted through the abdominal wall or cervix
Chance of miscarriage
Only offered if there is family history of a severe genetic disorder

Question 19

What happens during cffDNA testing?

Answer 19

Cell free fetal DNA circulates in the mothers blood

Much less invasive but has a low PPV

Question 20

How is fetal DNA analysed for SMA?

Answer 20

Sequenced using NGS
OR
Amplified using qPCR with primers for SMN1 exon 7. Compare the fluorescence over cycles against a control gene

Question 21

What is constitutive splicing?

Answer 21

Splicing of sites that are always used (as opposed to alternative splicing)

Question 22

What percentage of genes undergo alternative splicing?

Answer 22

95%

Question 23

What are the 3 important binding sites in an intron and what sequence do they contain?

Answer 23

5’ Splice site: GU
Branch point: A
3’ Splice site: AG

Question 24

Which snRNP binds to which splice site?

Answer 24

U1 binds 5’ splice site
U2 bind branch point
The tri-snRNP (U4, U5, U6) binds the region in between

Question 25

What is hnRNA?

Answer 25

Heterogenous nuclear RNA
The term for the mRNA transcript before it has undergo splicing
Also know as pre-mRNA

Question 26

RNA splicing is a form of genomic plasticity. What does this mean?

Answer 26

The ability of the genome to change and adapt

Question 27

How is alternative splicing regulated?

Answer 27

Splicing regulatory proteins and Exon/Intron Enhancers/Silencers

Question 28

What are the splicing regulatory proteins?

Answer 28

Serine Arginine rich (SR) proteins which bind to enhancers

Heterogenous nuclear ribonucleoparticles (hnRNPs) which bind to silencers

Question 29

What determines whether or not a splice site is used?

Answer 29

The balance of enhancer and silencer proteins

Question 30

Where are enhancer/silencer elements located?

Answer 30

They can be located far away from the splice site, but are brought into proximity by looped 3D structures of RNA molecules