SMA - Part 1 Flashcards
Describe Spinal Muscular Atrophy (SMA).
- Progressive muscular weakness due to a loss of lower motor neurons in the spinal cord and the brain cell nuclei.
The brain initiates movement by sending nerve signals down the spinal cord via the anterior horn motor neurons.
These motor neurons delay signals to the muscles which cause the muscles to contract.
The muscles closest to the trunk are more severely and earlier affected.
- Degeneration of these muscles in the spinal cord causes proximal, symmetrical, limb and trunk muscle weakness.
- Autosomal recessive inheritance pattern.
- Incidence is estimated to be approximately 1 in 10,000.
- Carrier frequency is approximately 1 in 50.
Outline the clinical presentations of the different types of SMA.
1) . SMA type I / Werdnig-Hoffmann disease:
- Most severe and most common (approximately 60% of all SMA cases). Onset is less than 6 months. Cannot sit unaided. 50% die before second birthday.
2) . SMA type II / Dubowitz disease:
- Onset is 7-18 months. May sit unaided. Never walk unaided. Have increased risk of respiratory problems. Death after 2 years of age.
3) . SMA type II / Kugelberg-Welander Disease:
- Onset > 18 months. Able to walk, but eventually need to use a wheelchair.
4) . SMA type IV:
- Adult onset after 30 years of age.
5) . Atypical SMA:
- Extended CNS involvement, increased CK values, congenital malformations of other organs and complex syndromes.
Describe the molecular genetics of SMA.
- Survival Motor Neuron (SMN) gene.
- located at 5q13 in two copies; SMN1 and SMN2. Highly unstable region prone to frequent deletions and gene conversion events.
- The copy number of the repeat unit can range from 0-4 per chromosome.
- SMN1 and SMN2 are highly homologous and differ by only 5 base pairs at the 3’ end.
- Two of these base pairs are located in exon 7 and exon 8.
- These differences allow SMN1 to be distinguished from SMN2 and the differences are the basis for the molecular test.
- In the pathogenesis of SMA in addition to SMN1 deletions the presence of this highly homologous SMN2 can also predispose to gene conversion events.
- Gene conversions are the non-reciprocal transfer of sequence information from donor sequence to acceptor sequence and can occur between sister chromatids, homologous chromosomes, or homologous sequences on the same chromatid.
- The base difference in SMN2 is a C>T conversion that results in a synonymous amino acid change.
- This change disrupts an Exon Splice Enhancer (ESE) site preventing efficient splicing of exon 7 in SMN2. Therefore lower levels of protein are produced (approximately 10% lower).
- SMN1 and SMN2 predict identical proteins.
- SMN1 is the more telomeric version of the gene whereas SMN2 is more centromeric.
Describe the differences between SMN1 and SMN2.
- SMN1 and SMN2 are highly homologous and differ by only 5 base pairs at the 3’ end.
- Two of these base pairs are located in exon 7 and exon 8.
- The base difference in SMN2 is a C>T conversion that results in a synonymous amino acid change.
- This change disrupts an Exon Splice Enhancer (ESE) site preventing efficient splicing of exon 7 in SMN2. Therefore lower levels of protein are produced (approximately 10% lower).
- SMN1 and SMN2 predict identical proteins.
- SMN1 is the more telomeric version of the gene whereas SMN2 is more centromeric.
Describe the SMN protein.
- 38kDa.
- Derived from SMN1 (approximately 90% of protein produced).
- Involves per-mRNA splicing.
- SMA patients deleted for SMN1 always have at least one copy of SMN2 which gives about 10% of the protein levels. Complete absence of SMN protein is shown to be embryonic lethal in mice.
- 5-10% of normal patients lack SMN2. Therefore SMN2 acts as a modifying gene. SMN2 can decrease the severity of SMA type 1 patients.
Describe the molecular basis of the different SMA types.
1) . Normal - 2 copies of SMN1 with 1 or 2 copies of SMN2.
2) . Carrier - 2 copies of SMN2 and 1 copy of SMN1 so the patient is a carrier of an SMN1 deletion.
3) . Embryonic Lethal - No copies of SMN1 present and 1 copy of SMN2.
4) . SMA Type II and III - No copies of SMN1 but 3 copies of SMN2. This has been the result of a gene conversion event. Results in an apparently homozygous deletion of SMN1, however, what has actually happened is that 2 of the copies of SMN1 have become SMN2 via a gene conversion event. This genotype is more commonly seen in SMA types II and III.
5) . SMA Type II and III - 1 normal copy of SMN2, 2 copies of SMN2 with a small part of SMN1 attached to the ends of the genes. These are hybrid genes created by gene conversion events. Also seen in those types of SMA that are slightly less severe than SMA type I - i.e. SMA types II and III.
Outline the genotype to phenotype correlations that are seen in SMA.
- SMN2: The greater the number of SMN2 copies the less severe the disease.
- The number of copies of SMN2 acts as a dose dependent disease modifier, as the number of SMN2 copies can vary between 0 and 5 (however genotype/phenotype correlations are imprecise).
- Homozygous deletions of SMN1 with 5 copies of SMN2 have been reported in asymptomatic individuals with family histories of SMA.
- SMN2 copy numbers overlap in SMA types I, II and III.
- SMA type I = deletion in both alleles.
- SMA type II = deletion in one SMA1 gene + gene conversion in other.
- SMA type III = gene conversion in both.
- Cannot be used to predict clinical course.
How do we test for the deletion in SMN1? What methods may be used to test for SMA?
- We look at deletions in exons 7/8.
- Around 94% of SMA patients have homozygous deletions of SMN1 (or more correctly lack SMN1 either due to deletion or gene conversion).
- SMN1 sequence analysis is also available. Less than 1% of patients will be homozygous for a pathogenic sequence variant.
- Approximately 4% of SMA patients are compound heterozygotes for a deletion on one allele and a point mutation on the other allele.
- Testing by restriction digest for diagnostic cases - not quantitative but rapid and cheap. Only looks for the absence of SMN1 exon 7/8.
TESTING METHODS:
- Single Strand Confirmation Polymorphism Analysis (SSCP). Has the advantage of being able to distinguish between sequence variants and tells us whether the mutation has arisen via a gene conversion event or not. One disadvantage is that it will detect polymorphisms and unclassified variants in SMN1.
- MLPA - quantitative method. Can identify homozygous loss of SMN1. Can identify those with one copy of SMN1. These could just be carriers or possibly have a point mutation on the other allele.
- The MLPA kit P060-B2 also detects copy number for SMN2.
Describe diagnostic SMN1 exon 7 and exon 8 PCR.
- Uses restriction enzymes. If the exon is present then the enzyme will cut the fragment and you will see bands present on the gel. If the exon is not present then the corresponding fragments will not be visible on the gel.
EXON 7 PCR:
- Uses a restriction enzyme digest using Hinf1.
- Before the digest the SMN1 exon 7 fragment will be 102kDa and afterwards it will be 78kDa + 24kDa.
- The SMN2 exon 7 fragment will remain at 102kDa.
- DMD is used as a control for Hinf1 activity and will digest a 181kDa fragment to 127kDa + 54kDa.
EXON 8 PCR:
- Uses a restriction enzyme digest using DdeI.
- SMN1 will be 189kDa both before and after digestion.
- SMN2 will be 189kDa before digestion and 123kDa + 66kDa after digestion.
- DMD exon 34 is used as a DdeI action control and will be digested from a 426kDa fragment to 213kDa + 19kDa + 96kDa.
INTERPRETATION:
- The majority of normal individuals have at least 1 copy of SMN1 and 1 copy of SMN2 per chromosome.
- 10% of normal patients lack SMN2 copy of the gene.
- 94-97% of SMA patients have homozygous deletion of the SMN1 gene.
- If a diagnosis of SMA is suspected the MLPA can be performed to determine the copy number of SMN1. If only one copy is detected then point mutation analysis can be performed.
