SMN Flashcards
What are the clinical features of Spinal Muscular Atrophy?
- Proximal muscle weakness
- Fasciculation (twitching)
- Dysphagia, dysarthria (a disorder of speech/ language)
- Lost of deep tendon reflex
- Scoliosis
Affects the anterior horn cells of the spinal cord:
SMN1 disease types
Clinical type:
Type I (Werdnig-Hoffman Dis.):
•Cannot sit unsupported or lift heads
•Survive < 2 yrs
•Comprises 60-70% of SMA pt.
Type II:
•Can sit; cannot stand and walk unaided
•Survive > 4 yrs
Type III (Kugelberg-Welanderdisease):
•Stand-alone and walk but sometimes lose the ability to walk later in lifetime
•Type IIIa: Onset before 3 yrs age
•Type IIIb: Onset at age ≥3 yrs
Type IV:
•First symptom at age 20-30 yrs
Molecular genetics of SMN1 mutations
Common Cause (Freq. 95-98%)
HMZ Del of SMN1Ex 7
Gene Conversion of SMN1to SMN2
Genotype designation: [0+0] or a2
Rare Mutations (Freq. 2-5%)
Nonsense, frameshift, or missense mutation
Compound HTZ for SMN1 Del
Genotype designation: [1d+0] or a2
Disease of SMN
The homozygous absence of the SMN1 exon 7 has been observed in the majority of patients and is being used as a reliable and sensitive spinal muscular atrophy diagnostic test.
Although SMN2 produces less full-length transcript than SMN1, the number of SMN2 copies has been shown to modulate the clinical phenotype.
SMA results from a reduction
in the amount of the SMN protein, and there is a strong
correlation between the disease severity and SMN protein levels
The coding sequence of
SMN2 differs from that of SMN1 by a single nucleotide
(840C>T), which does not alter the amino acid but has been
shown to be important in splicing.
SMN1 vs. SMN2
SMN1 exon 7 is absent in the majority of patients independent
of the severity of SMA.
Several studies have shown that
the SMN2 copy number modifies the severity of the disease
SMN2 modifier gene
The majority of patients
with the severe type I form have one or two copies of SMN2;
most patients with type II have three SMN2 copies; and most
patients with type III have three or four SMN2 copies.
SMA mechanism
A single SMN2 gene produces
less functional protein compared with a single SMN1
gene. Therefore, SMA arises because the SMN2 gene
cannot fully compensate for the lack of functional SMN when SMN1 is mutated.
However, small amounts of full-length transcripts
generated by SMN2 are able to produce milder type II
or III phenotypes when the copy number of the SMN2 gene is increased.
SMA is, therefore, caused by low levels of SMN protein, rather than a complete absence of the protein
Point mutations in SMA
a patient with SMA possesses only a single copy of SMN1, it is likely that the remaining copy contains a more subtle mutation, including nonsense mutations, missense mutations, splice site mutation insertions, and small deletions.
most frequently reported mutations are the
p. Tyr272Cys (c.815A>G),
c. 399_402delAGAG, c.770-780dup11,
and p.Thr274Ile (c.821C>T)
Population information for SMA
1/40 –1/60 carrier frequency of SMA
The estimated frequency of alleles with two or more
copies of SMN1 is 3–8 times more prevalent in African Americans, when compared with other ethnic groups
More frequent in African American populations
Detection rate varies by ethnicity ranging
from 71% in African Americans to 95% in whites. The major contributor to this ethnicity-based variation in detection rate is the occurrence of two (or more) SMN1 genes in tandem on a single chromosome 5 (i.e., the [2 + 0] Category 2 carrier genotype).
The estimated frequency of alleles with two or more
copies of SMN1 is 3–8 times more prevalent in African Americans, when compared with other ethnic groups.34 This translates to a much higher frequency of individuals with the SMA carrier [2 + 0] genotype among African Americans compared with other races.
Specificity of the carrier test
As with the diagnostic test, analytical specificity of the carrier test (proportion of negative test results if not a carrier) is >99% if the presence of polymorphisms underneath the primer and/or probe binding sites are ruled out.
Laboratory requirements for SMN testing
The laboratory must establish validated, nonoverlapping cutoff values that can accurately and reliably distinguish SMN1copy numbers of 0, 1, 2, and >3.
Therefore, the copy number of genomic
internal standard reference gene should be constant at two copies within the genome, and the PCR amplification efficiency of the SMN1 gene relative to the chosen internal reference standard gene should be consistent between analyses. Performing replicate copy number measurements with two independent two copy number internal standard reference genes can help assure the accuracy of copy number analysis.
Prenatal Testing
Indications for prenatal diagnosis of SMA include a 25% risk
for the fetus to be affected (when both carrier parents are
identified as a result of family history or following carrier
identification by population screening) or the presence of abnormal findings such as decreased fetal movements and contractures in utero or increased NT on fetal ultrasound.
Laboratory procedures
multiplex ligation-dependent probe amplification
(MLPA) and quantitative PCR (qPCR) being the most
common.
RLFP procedure for SMN
Although this is a highly repetitive
region, the exon 7 base pair difference (840C>T) alters a DraI restriction enzyme site (due to a mismatched primer) and allows one to easily distinguish SMN1 from SMN2 on digestion of the PCR products.
Clinical disadvantages of SMA RFLP include the
inability to detect carrier status and determine SMN2 copy
number.
MLPA
MLPA technology has several key advantages,
including (1) allowance for simultaneous detection of SMN1
and SMN2 copy numbers, therefore, helping differentiate SMA type 1 from SMA types 2 and 3, (2) a high degree of precision for the quantitative detection of three or fewer SMN1 copies, (3) 20 independent control loci are able to be assayed in one reaction, (4) all reactions are performed in a single tube, and (5) probe sets are easy to obtain commercially, (6) a high degree of reproducibility and a large number of samples can be tested simultaneously, and (7) only 20 ng of genomic DNA is required, but 5–6-fold more DNA can be successfully technically validated.
MLPA technology has several important limitations
including (1) DNA sequence variants located under probe binding sites of SMN1 alleles may interfere with probe hybridization and might result in a false-positive carrier (one copy) or false-positive diagnostic (zero copies) result, (2) reactions are sensitive to contaminants but generate uninterpretable results, (3) MLPA cannot yet be used to investigate single cells, which is important for preimplantation genetic diagnosis testing, (4)
MLPA is not a suitable method to detect unknown point mutations,
(5) MLPA probes are sensitive to small deletions, insertions, and mismatches, and (6) MLPA requires a CE analyzer, which is a higher cost option compared with slab gel electrophoresis for RFLP.
