Final Diagnosis -- Spinal muscular atrophy



Spinal muscular atrophy is one of the most common autosomal recessive diseases, affecting approximately 1 in 10,000 live births and having a carrier frequency of approximately 1 in 50.

SMA is characterized by symmetric proximal muscle weakness and is caused by degeneration of the anterior horn cells of the spinal cord. SMA is classically subdivided into three types based on age of onset and clinical severity.

Type I SMA (Werdnig-Hoffmann disease) is characterized by the onset of severe muscle weakness and hypotonia in the first few months of life and the inability to sit or walk. Fatal respiratory failure usually occurs before the age of 2 years.

Type II SMA (intermediate) is characterized by the onset of proximal muscle weakness before 18 months of age, the ability to sit but not to walk unaided, and survival beyond 4 years of age.

Type III SMA (Kugelberg-Welander disease) is characterized by the onset of proximal muscle weakness after the age of 2 years, the ability to walk independently until the disease progresses, and survival into adulthood.

SMA of all types is associated with homozygous mutations in the survival motor neuron 1 gene (SMN1). SMN1 and its centromeric homolog, SMN2, lie within the telomeric and centromeric halves, respectively, of a large inverted repeat in chromosome region 5q13 (Figure 3). SMN consists of nine exons (exons 1, 2a, 2b, and 3-8), with the stop codon being present near the end of exon 7. SMN1 and SMN2 differ in exons 7 and 8 by only two base pairs, one in exon 7 (Figure 4) and one in exon 8 (Figure 5).

A normal individual can have both SMN1 and SMN2, or can have SMN1 only. Approximately 94% of individuals with clinically typical SMA lack both copies of SMN1 exon 7. A fetus with neither SMN1 nor SMN2 is incompatible with life.

Both SMN1 and SMN2 encode the survival motor neuron protein, the latter is important during the RNA processing. The nucleotide C in the exon 7 of SMN1 serves in the region of a splicing enhancer, the latter ensures that the exon 7 is included in the final mature mRNA product. In SMN2, this C is changed to T, and the splicing enhancer is disrupted. Therefore, 90% of the mature mRNA transcript from SMN2 will be without exon 7. The final protein product that lacks exon 7 is unstable and will be degraded rapidly. Therefore, in a normal individual who have both SMN1 and SMN2 genes, SMN1 contributes 90% of the survival motor neuron protein product, while SMN2 only contributes 10% (Figure 6). An individual with only SMN2 gene apparently can not produce sufficient amount of survival motor neuron protein, and therefore is affected by the SMA.

The most commonly used method to confirm genetically the diagnosis of SMA is a qualitative PCR-RFLP assay (also known as the SMN1 deletion assay) to detect the homozygous absence of SMN1 exon 7. The PCR-RFLP assay takes advantage of the base differences in exons 7 and 8 to distinguish SMN1 from SMN2.

1. Exon 7
By using a mismatch primer on the 3' end of exon 7, we introduce an G to A change (G>A) at position 875 of the cDNA (Figure 7). This change creates a Dra I site (TTTAAA). The third T nucleotide of the sequence is present only in the SMN2 gene and is a C in the SMN1 gene, thus the enzyme cuts only the SMN2 amplified PCR product. The following digestion products are expected (Figure 8):

In normals and heterozygous SMN1 deletion carriers, an undigested product of about 200 bp and a digested product of 176 bp (24 bp shorter) for the SMN1 and the SMN2 genes, respectively.

In normals missing the SMN2 gene (5% of the normal population), we expect only the large 200 bp PCR product representing the SMN1 gene.

In affected patients we expect only the SMN2 gene represented by the 176 bp PCR product. The 200 bp product representing the SMN1 gene is absent

2. Exon 8
The nucleotide that allows one to distinguish between the SMN1 and SMN2 genes is a G>A. The A in the SMN2 gene (located at position 1155 of the cDNA) (Figure 9) creates a Dde I site which will digest the amplified DNA into two products of about 122 bp and 78 bp. The amplified product representing exon 8 of the SMN1 gene (200 bp) does not contain any Dde I sites. Following amplification and digestion of exon 8, the following restriction pattern is expected (Figure 10):

In normals and heterozygous SMN1 deletion carriers, we expect a 200 bp product representing the SMN1 gene and 122 bp and 78 bp digestion products representing the SMN2 gene.

In normals missing the SMN2 gene we expect only the undigested product (200 bp) representing the SMN1 gene.

In affected individuals one expects only the SMN2 gene yielding digestion products of 122 bp and 78 bp. The 200 bp product representing the SMN1 gene is absent.


  1. Procedure manual: Autosomal spinal muscular atrophy (PCR/RFLP), Department of Pathology, Division of Molecular diagnostics, University of Pittsburgh Medical Center.
  2. Van der Steege G, Grootscholten P, van der Vlies P, Draaijers TG, Osinga J, Cobben JM, Scheffer H, Buys CHCM (1995) PCR based DNA test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy. Lancet 345:985-986.
  3. Monani UR, Coovert DD, Burghes AHM (2000) Animal models of spinal muscular atrophy. Human molecular genetics 9: 2451-2457.
  4. Ogino S, Wilson RB (2002) Genetic testing and risk assessment for spinal muscular atrophy (SMA). Human Genetics 111: 477-500.
  5. Lorson CL, Hahnen E, Androphy EJ, Wirth B (1999) A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proceedings of the National Academy of Sciences of the United States of America 96: 6307-6311.

Contributed by Lei Chen, MD, PhD, Alexis Carter, MD, Jeffrey A. Kant, MD, PhD

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