[Quantitative analysis of SMN1 and SMN2 genes based on DHPLC: a reliable method for detection of non-homozygous patients with spinal muscular atrophy].

OBJECTIVE

To develop a rapid and reliable approach for testing the copy number of survival motor neuron (SMN) gene and analyze the compound heterozygous deletions of SMN1 gene.

METHODS

Peripheral blood samples were collected from 38 non-homozygous deletion pediatric patients with SMA, 30 homozygous deletion patients with SMA, and 35 un-related healthy persons. SMN1 and SMN2 genes were amplified separately with allele-specific PCR (AS-PCR). Meanwhile, two irrelevant genes were amplified as internal quality control respectively. The copy numbers of SMN1 and SMN2 were determined by denaturing high-performance liquid chromatography (DHPLC).

RESULTS

(1) A protocol combining multiplex allele-specific PCR and DHPLC was developed to separate SMN1 and SMN2 and to determine the copy numbers of them. The copy numbers of SMN1 and SMN2 varied from 1 to 4 and a clear-cut differentiation among the different copy number ranges could be observed for the two genes. (2) One single copy of SMN1 were detected in 20 of the 38 non-homozygous deletion patients with SMA (52.6%). Heterozygous deletions were determined in these 20 patients. Two copies of SMN2 were detected in 15 of the 20 patients with one copy of SMN1 (75.0%, 15/20). Other 5 of the 20 patients were with 3 copies of SMN2 (25.0%, 5/20). (3) One single copy of SMN1 was detected in 24 of the 30 (80%) parents of SMA patients with homozygous deletion.

CONCLUSION

SMN copy number can be rapidly and reliably determined by the method of multiplex AS-PCR combined with DHPLC.