SMA Flashcards
In how many patients with typical clinical picture and single SMN1 copy number is a second mutation not found? and why could this be?
1/3 Deep intronic muts. sequencing only looking at exons.
What are the two main hypotheses for pathogenesis of low levels of SMN:
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.
explain gene conversion
unidirectional, non-reciprocal exchange of genetic information where one DNA sequence replaces a homologous sequence, resulting in two identical sequences after the event
What can = attenuate the severity of SMA in patients with a low SMN2 copy number
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
Carrier frequency of SMA
1/40 to 1/60 ( ~ 1/50 in the UK)
How can loss of SMN1 exon 7 can occur?
Deletion of gene conversion
what types of mutation are associated with the different types of SMA?
Type 1-homodel, type 2 het del and gene conversion, type3, gene conversion both.
What is the critical difference between SNM1 and 2 resulting in decreased SMN protein levels?
translationally silent C>T transition in SMN2 exon 7 (840C>T)
What is the name of the project to introduce prenatal testing for SMA by NIPD
NIPSIGEN
how many exons does SMN1/2 have ?
9
> 3 SMN2 copy number correlates with a milder phenotype and longer survival. But why is SMN2 copy number rarely reported?
correlation not absolute and not strong enough to have prognostic value
what is the parental origin of most de novo SMN1 mutations?
paternal
What % of SMA are homozygous for a pathogenic inactivating mutations
~1%
6% of parents of a simplex case of SMA will have normal SMN1 dosage for the following two reasons:
(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
what % of SMA are homozygous for a deletion of at least exon 7 of SMN1?
95-98%
Benefits of anti-sense oligonucleotides (ASOs) to enhance SMN expression
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.
Despite high lethality of SMA why does incidence remain high?
high new mutation rate
What is the first level diagnostic test is analysis for SMA?
Exon 7 copy number SMN1
Apart from MLPA, what else could you use for SMA copy number determination?
- 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
differences between full-length SMN protein between SMN1 and 2?
SMN1> 90% full length, SMN2 only 10% full length
Where are the majority of pathogenic inactivating mutations located in SMN1?
exons 3 and 6
Explain the use of modified anti-sense oligonucleotides (ASOs) to enhance SMN expression
target a specific element within SMN2 (ISS-N1) =particularly effective at promoting inclusion of exon 7 in SMN2
How much SMN protein is needed for normal motor function?
23%
how are SMN genes arranged?
in tandem. SMN1 telomeric copy, SMN2 the centromeric copy
How much SMN protein do carriers have?
45%-55%
SMN1 and SMN2 location
5q12.2 5q13.3
What motor neurons degenerate in SMA?
motor neurons in the anterior horn of the spinal cord
What % of SMN1 deletion are de novo?
~2%
Types of SMA?
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.
How can you increase levels of SMN protein?
directly by stabilising SMN RNA and protein or by modulating SMN2 expression
Explain the how stem cell therapy or gene therapy has been used to compensate for the lack of sufficient SMN protein
- 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.
What does the SMN protein do?
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.
What % of SMA are compound heterozygous for a deletion of at least exon 7 of SMN1 and a pathogenic inactivating mutation in SMN1 ?
~2- 5%
how many SMN1 do most people have and in what arrangement?
most 2X SMN1 one on each chromsome. 4% 2 SMN1 in cis.
what are the levels of SMN protein in types 1-3?
type 1 ~10%
type 2 ~14%
type 3 ~18%
SMA phenotypic features:
- 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
How many bp differences between SMN1 and SMN2?
5
Describe SMA pathology
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.
explain MLPA for SMA
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
What can be the basis for families with identical SMN1 and SMN2 and differing severity?
Other genetic modifiers.
Example of a genetic modifier of SMA?
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.
SMA prevalence
~ 1:6,000 to 1:10,000
