SMA

Question 1

In how many patients with typical clinical picture and single SMN1 copy number is a second mutation not found? and why could this be?

Answer 1

1/3 Deep intronic muts. sequencing only looking at exons.

Question 2

What are the two main hypotheses for pathogenesis of low levels of SMN:

Answer 2

a) involved in the synthesis of U snRNPs and assembly into the spliceosome. SMN deficiency = impaired mRNA production and neurons become deficient in proteins necessary for normal growth and functioning.
b) SMN has a motor neuron specific function such as mRNA transport along the axon and maintaining the integrity of neuromuscular junctions.

Question 3

explain gene conversion

Answer 3

unidirectional, non-reciprocal exchange of genetic information where one DNA sequence replaces a homologous sequence, resulting in two identical sequences after the event

Question 4

What can = attenuate the severity of SMA in patients with a low SMN2 copy number

Answer 4

A rare SMN2 exon 7 variant, c.859G>C creates a new ESE increasing the amount of exon 7 inclusion and full-length transcript from SMN

Question 5

Carrier frequency of SMA

Answer 5

1/40 to 1/60 ( ~ 1/50 in the UK)

Question 6

How can loss of SMN1 exon 7 can occur?

Answer 6

Deletion of gene conversion

Question 7

what types of mutation are associated with the different types of SMA?

Answer 7

Type 1-homodel, type 2 het del and gene conversion, type3, gene conversion both.

Question 8

What is the critical difference between SNM1 and 2 resulting in decreased SMN protein levels?

Answer 8

translationally silent C>T transition in SMN2 exon 7 (840C>T)

Question 9

What is the name of the project to introduce prenatal testing for SMA by NIPD

Answer 9

NIPSIGEN

Question 10

how many exons does SMN1/2 have ?

Answer 10

9

Question 11

> 3 SMN2 copy number correlates with a milder phenotype and longer survival. But why is SMN2 copy number rarely reported?

Answer 11

correlation not absolute and not strong enough to have prognostic value

Question 12

what is the parental origin of most de novo SMN1 mutations?

Answer 12

paternal

Question 13

What % of SMA are homozygous for a pathogenic inactivating mutations

Answer 13

~1%

Question 14

6% of parents of a simplex case of SMA will have normal SMN1 dosage for the following two reasons:

Answer 14

(1) 4% of the general population has two copies of SMN1 on a single chromosome (false negative result)-
Linkage can resolve carrier status of parents of affected child with 2 copies of SMN1 exon 7
(2) De novo deletion of exon 7 on one SMN1 allele occurs in 2% of SMA patients; only one parent is a carrier

• Risk calculation (Bayesian) = residual risk to potential carriers after a negative dosage test

Question 15

what % of SMA are homozygous for a deletion of at least exon 7 of SMN1?

Answer 15

95-98%

Question 16

Benefits of anti-sense oligonucleotides (ASOs) to enhance SMN expression

Answer 16

ASOs highly stable for months and have little or no toxicity. A single administration of ASO splicing modifiers, either systemically or CNS of severe SMA mice, rescues disease phenotype. 18mer ASO currently in phase 3 clinical trials for SMA infants and children.

Question 17

Despite high lethality of SMA why does incidence remain high?

Answer 17

high new mutation rate

Question 18

What is the first level diagnostic test is analysis for SMA?

Answer 18

Exon 7 copy number SMN1

Question 19

Apart from MLPA, what else could you use for SMA copy number determination?

Answer 19

• Real time PCR - multiplex assay using TaqMan probes specific to exon 7 and control probe to RNAse P.
• Quantitative PCR¬ – Multiplex PCR using primers, specific to the nucleotide differences in exons 7 and 8, followed by restriction digest to distinguish between the exons of the 2 genes. PCR products are then analysed by capillary electrophoresis and the copy number of each exon is compared to amplified controls

Question 20

differences between full-length SMN protein between SMN1 and 2?

Answer 20

SMN1> 90% full length, SMN2 only 10% full length

Question 21

Where are the majority of pathogenic inactivating mutations located in SMN1?

Answer 21

exons 3 and 6

Question 22

Explain the use of modified anti-sense oligonucleotides (ASOs) to enhance SMN expression

Answer 22

target a specific element within SMN2 (ISS-N1) =particularly effective at promoting inclusion of exon 7 in SMN2

Question 23

How much SMN protein is needed for normal motor function?

Answer 23

23%

Question 24

how are SMN genes arranged?

Answer 24

in tandem. SMN1 telomeric copy, SMN2 the centromeric copy

Question 25

How much SMN protein do carriers have?

Answer 25

45%-55%

Question 26

SMN1 and SMN2 location

Answer 26

5q12.2 5q13.3

Question 27

What motor neurons degenerate in SMA?

Answer 27

motor neurons in the anterior horn of the spinal cord

Question 28

What % of SMN1 deletion are de novo?

Answer 28

~2%

Question 29

Types of SMA?

Answer 29

Arthrogyposis multiple congentia-prenatal ( Absence of movement except for extraocular and facial movement. Death from respiratory < one month)
Type 1 Werndig-Hoffman, diagnosed <6 months profound hypotonia and symmetrical flaccid paralysis (“floppy baby”).
Type 2: Dubrowitz. diagnosed 6-12 months.sit unaided, but never be able to walk without support.
Type 3. Kugelberg-Welander. Diagnosed < 3 years 3a
3b > 3 years. stand and walk alone, but may show difficulty. proximal muscle weakness.
Adult SMA-second or third decade of life.

Question 30

How can you increase levels of SMN protein?

Answer 30

directly by stabilising SMN RNA and protein or by modulating SMN2 expression

Question 31

Explain the how stem cell therapy or gene therapy has been used to compensate for the lack of sufficient SMN protein

Answer 31

• AAV9 can infect numerous cell types and transverse the blood brain barrier.
• Neonatal SMA model mice injected systemically with AAV9 engineered to carry WT SMN gene, expressed high levels of SMN protein in multiple tissues and derived remarkable therapeutic benefit phase 1 clinical trials to assess the safety.
• Current research identifying microRNA biomarkers which can indicate how well a treatment is working, monitored by a blood samples which is a minimally invasive procedure.

Question 32

What does the SMN protein do?

Answer 32

expressed ubiquitously andabundant in motor neurons of the spinal cord. Acts as a chaperone to assist in the assembly of U snRNPs and is an essential component of the spliceosome.

Question 33

What % of SMA are compound heterozygous for a deletion of at least exon 7 of SMN1 and a pathogenic inactivating mutation in SMN1 ?

Answer 33

~2- 5%

Question 34

how many SMN1 do most people have and in what arrangement?

Answer 34

most 2X SMN1 one on each chromsome. 4% 2 SMN1 in cis.

Question 35

what are the levels of SMN protein in types 1-3?

Answer 35

type 1 ~10%
type 2 ~14%
type 3 ~18%

Question 36

SMA phenotypic features:

Answer 36

• Progressive proximal, symmetrical limb and trunk muscle weakness (typically lower limbs before upper)
• Intercostal muscle weakness = breathing difficulties.
• Fine tremor in the fingers
• Muscle twitches in tongue (fasciculation) = poor suck and swallow with increasing swallowing and feeding difficulty over time
• Facial weakness

Question 37

How many bp differences between SMN1 and SMN2?

Answer 37

5

Question 38

Describe SMA pathology

Answer 38

weakness and atrophy of voluntary muscles due to spinal cord and motor neuron degeneration. Widespread synaptic defects in neuromuscular junctions (NMJs) = motor neuron death and muscle atrophy.

Question 39

explain MLPA for SMA

Answer 39

probes specific to the single nucleotide differences of exons 7 and 8 of the SMN1 and SMN2 genes. MLPA kit from MRC-Holland detects dels/dups and gene conversions of exon 7 and 8 of SMN1 and SMN2

Question 40

What can be the basis for families with identical SMN1 and SMN2 and differing severity?

Answer 40

Other genetic modifiers.

Question 41

Example of a genetic modifier of SMA?

Answer 41

PTEN depletion = increased survival of SMN deficient neurons=ameliorate severity
Plastin 3 (PLS3): upregulated in unaffected SMN1 del females with identical SMN2 copies vs  affected females.

Question 42

SMA prevalence

Answer 42

~ 1:6,000 to 1:10,000