Detection of Survival Motor Neuron (SMN) Gene Deletions

Homozygous deletion of exons 7 and/or 8 of the SMN gene is found in greater than 95% of affected spinal muscular atrophy patients (SMA Types I, II, and III) (original citation by Lefebvre et al., (1995) Cell 80:155-165; clinical reviews in Morrison (1996) Neuromuscul Disord 6:397-408 and Bertini (1996) J Mol Med 74:555-562). It has recently been shown that the various clinical forms of SMA correlate well with the amount of SMN protein that can be detected by Western blot analyses (Lefebvre et al., (1997) Nature Genetics 16:265-269). In addition, the data of this paper suggests that SMN protein produced from SMNc is functionally active and the levels of its expression may ameliorate symptoms in some cases of SMA.

The presence of an homologous, centromeric copy of SMN, (SMNc), complicates the analysis of this disorder. CompGene uses a PCR-based method that permits direct evaluation of the SMN gene without interference by SMNc. In the figure shown below, SMN exon 7 is detected in normal individuals by allele-specific amplification (ie. only SMN and not SMNc is amplified and a negative result is obtained for the SMN exon 7 in affected individuals with a homozygous deletion). The figure shows pairs of SMN exon 7 PCR products from a normal and an affected individual at increasing PCR cycle number. No SMN exon 7 PCR product is detectable in the affected individual even after 38 PCR cycles. Typical diagnostic SMA assays are performed at 26 - 30 PCR cycles depending on the diagnostic situation. An internal positive control PCR product is provided by the simultaneous amplification of exon 11 of the cystic fibrosis transmembrane conductance regulator gene (CFTR). Lane M shows DNA size standards while lane 1 shows the no DNA control after 38 PCR cycles. This DNA based test will provide a confirmation of diagnosis in the majority of suspected cases of spinal muscular atrophy Types I, II, or III.

The method of analysis of SMN exon 8 is different than SMN exon 7 as the former is distinguished from SMNc exon 8 by the presence of a Dde I site only in the SMNc exon 8 PCR product. Hence, an affected individual with a homozygous deletion will not have the SMN exon 8 PCR product and should have the SMNc Dde I fragments as an internal control for the PCR amplification.

The figure below shows examples of evaluation of SMN exon 7 and exon 8. Lane 1: DNA size standards; Lane 2: Exon 7 - No DNA control; Lane 3: Exon 7 - negative control DNA; Lane 4: Exon 7 - SMA I positive control DNA; Lane 5: Exon 7 - Query SMA I patient; Lane 6: Exon 8 - No DNA control; Lane 7: Exon 8 - undigested negative control DNA; Lane 8: Exon 8 - negative control DNA digested with Dde I; Lane 9: Exon 8 - SMA I positive control DNA digested with Dde I; Lane 10: Exon 8 - query SMA I patient DNA digested with Dde I. The positions of absent functional SMN exons 7 and 8 are indicated by the red arrowheads adjacent to lanes 4 and 9.

A common clinical situation with SMA I families is the parents' desire to have prenatal testing for a subsequent pregnancy after a previous affected child has died in infancy. In many cases, no biological material from the proband is available. Always keep in mind that archival pathology specimens may provide suitable DNA for molecular genetic analysis. As nearly all SMA I (Werdnig-Hoffman) cases appear to have homozygous deletion of at least SMN exon 7, it would be anticipated that the parents are heterozygotes for a SMN exon 7 deletion. CompGene can provide a quantitative PCR analysis to determine SMN exon 7 copy number in the parents. In the case shown below, a quantitative PCR analysis of SMN exon 7 showed that the parents (lanes 2 and 3) of the fetus (lane 4) are carriers of a SMN exon 7 deletion (reduced intensity of the SMN exon 7 PCR product as compared to normal in lane 1 - confirmed by quantitative densitometry) and that the fetus has a homozygous deletion of SMN exon 7 consistent with its being predicted to be affected with SMA.

NB: It is important to appreciate that a few very rare situations have been reported where individuals homozygous for SMN deletions are phenotypically normal. These individuals have been ascertained due either to their obligate carrier status or as the asymptomatic sibs of index cases (Cobben et al. (1995) Am J Hum Genet 57:805-808; Hahnen et al. (1995) Hum Mol Genet 4:1927-1933; McAndrew et al. (1997) Am J Hum Genet 60:1411-1422). In addition, a few very rare cases of point mutations and other microdeletions of SMN have been observed in SMA families (summarized in McAndrew et al. (1997) Am J Hum Genet 60:1411-1422). The possibility of such situations must always be in mind and testing of obligate carrier parents in prenatal situations to demonstrate anticipated SMN deletion heterozygosity is required.