SMA carrier testing--validation of hemizygous SMN exon 7 deletion test for the identification of proximal spinal muscular atrophy carriers and patients with a single allele deletion.

To facilitate the detection of carriers of a hemizygous survival motor neuron (SMN) exon 7 deletion we have modified the quantitative SMN exon 7 assay described by McAndrew et al (1997). The major changes include quantitative analysis of the amount of SMN exon 7-specific fluorescently-labelled PCR product on an automated sequencer, and the monitoring of the completeness of a DraI digestion necessary to distinguish the PCR products of exons 7 of SMN and its copy gene. In our method the amount of SMN exon 7 PCR product is compared with the amount of a co-amplified PCR product of the retinoblastoma (RB1) exon containing a DraI restriction site. By co-amplification using the same primers of plasmids included in the reaction as internal standards containing SMN exon 7 with a 36-nucleotide deletion and RB1 exon 13 with a 19-nucleotide deletion, respectively, the relative amplification efficacy can be monitored. The assay has been validated in 63 ascertained carriers and 28 ascertained non-carriers. The sensitivity of the test is approximately 97%, the specificity approaches 100%. In four out of six SMA patients without a homozygous deletion we detected a hemizygous deletion. The implications of the use of this assay for carrier testing and for confirmation of the clinical diagnosis of SMA are discussed.