SMA - Part 2

Question 1

Describe SMA carrier testing. What problems are associated with carrier testing?

Answer 1

Carrier testing is offered to parents and relatives to see if they have one copy of the SMN1 gene.

Problems:

• Approximately 4% of normal chromosomes carry two copies of the SMN1 gene on the same allele - this could mask a deletion. This can alter in different population.
• Testing cannot distinguish between patients with 1 copy on each allele [1:1] = non carriers, and those with 2 copies on one allele and none on the other [2:0] = carriers.
• De novo mutations occur in 2% of individuals with SMA and the majority are of paternal origin.
• Possible risk of germline mosaicism.

Question 2

What do we do for carrier testing where the parents may have 2 copies of SMN1 on the same allele?

Answer 2

• For those cases where parents may be [2:0] markers that flank the SMN1 region can be used to aid in identification of the at-risk haplotype.
• Alternatively genotyping the grandparents of the apparently non-carrier parent can help determine if they are a carrier as the grandparents will more than likely have [1:0] and [2:0] genotype.
• If the mutation is de novo then the grandparents will have normal dosage levels of [1:1].

Question 3

Have a look at Bayesian risk calculations for SMA.

Answer 3

Do it.

Question 4

What assumptions must we make to simplify risk calculations for complicated SMA cases.

Answer 4

• The sibling of an individual was found to have a single copy of SMN1 [1:0]. The proband has 2 copies of SMN1. What is the probands residual risk of being a carrier?
• We must make the assumptions here that there are no new mutations. We also do not consider alleles with deletion that is masked by SMN2 levels.
• We simplify the risk and ignore rare point mutations and ignore different frequencies about having 2 copies of SMN1 on the same allele.

Question 5

What problems in SMA risk calculations was reported in the 2011 EMQN scheme?

Answer 5

• Problems reported with the 2011 EMQN scheme.
• Vastly different results for the residual risk to the foetus reported.
• 2011 SMA scheme produced risks from <1/100 to 1/8000, and no 2 labs used the same technique. SMA expert calculated at approximately 1 in 2,500.
• Carrier frequency used was 1/45 whereas we would us 1 in 50. Ethnicity plays a role.

Question 6

Describe prenatal testing for SMA. What is required? How is it carried out?

Answer 6

• For testing in Nottingham we require the identification of a homozygous deletion of SMN1 in the proband and that carrier status of the parents has been determined.
• Markers located in and around the SMN1 gene can be used to confirm which haplotype has been inherited by the foetus.
• This requires prenatal DNA and a sample from the proband.
• This result then backs up the restriction digest PCR.
• If one parent had two copies of SMN1 then linkage analysis would help in the interpretation.
• Validated for use on AF and CVS.
• Maternal contamination checks have tested that the restriction digest PCR method can detect approximately 5% maternal contamination. Maternal contamination may give rise to a false negative result.
• There are further complications. For example, a lack of SMN1 has been identified in unaffected siblings of patients with SMA. In such cases there may have been a gene conversion event rather than a straight forward deletion. More linkage would be required for doing prenatal diagnosis. This raises different counselling issues as they are unlikely to be affected if they have the same genotype as unaffected sibling but we can’t really be sure.

Question 7

Describe therapeutic approaches in SMA.

Answer 7

• Try to increase the full length of the SMN2 protein that is being produced (?upreg from 10% levels): aclarubicin, sodium butyrate, valproic acid.
• Looking at administration of agents capable of increasing the expression of SMN levels.
• Transcriptional SMN2 activation and facilitation of correct SMN2 splicing, translational activation and stabilisation of the full length SMN protein are considered as strategies for SMA therapy.

SMN2 isn’t spliced as correctly due to the interruption of ESE by the C to T conversion.

Antisense oligonucleotide mediated enhanced splicing of SMN2 transcript may help.

Blocking of an intron splice silencer (ISS) is one of the most effective methods for increasing incorporation of exon 7 of SMN2.

• Other strategies may include improvement of motor neuron viability by neuroprotecting or neurotrophic agents.
• Gene conversion from SMN2 gene to SMN1 gene in human cells from SMA patients has been reported.
• Stem cell therapy or gene therapy may compensate for the lack of sufficient SMN protein.