Comparison of three methods for the genetic diagnosis of spinal muscular atrophy

Objective To evaluate the value of PCR-restriction fragment length polymorphism (PCR-RFLP), real-time PCR and multiplex ligation-dependent probe amplification (MLPA) in the genetic diagnosis of spinal muscular atrophy (SMA) and make laboratory support accessible to clinicians for the molecular diagnosis of SMA. Methods Methodological evaluation. Forty-one suspected SMA cases and 359 control individuals received in Shanghai Children′s Medical Centre from March 2013 to June 2014 were detected for the deletion of exon 7 and 8 in the survival motor neuron gene 1 (SMN 1) by PCR-RFLP, real-time PCR and MLPA, respectively. Then the results of the three methods were compared and the benefits and limitations of the three methods were evaluated. Results The result of real-time PCR was in complete agreement with that of MLPA, showing that 29 suspected cases harbored homozygous deletions of SMN1 and 1 case possessed heterozygous deletion. Among the homozygous deletions, 27 patients demonstrated absence of exon 7 and 8, and 2 cases demonstrated only the absence of exon 7. Meanwhile, both PCR-RFLP and MLPA analysis showed the same results that only 5 out of 395 control cases carried heterozygous deletion. As for cases without heterozygous deletions, PCR-RFLP demonstrated the same result with real-time PCR and MLPA but it missed all the heterozygous ones. Conclusions PCR-RFLP, the conventional SMA gene diagnosis method, was only capable of detecting homozygous deletion of exon 7 and/or 8 of SMN1, but was not as sensitive as to find out the carriers with heterozygous deletions. MLPA could detect the deletion and quantify the copy numbers of exon 7 and 8 of SMN1, efficiently, while the price was relatively high, which brings challenges for its application in the carrier screening of SMA. Compared with these two methods, real-time PCR with high throughput and low input was a rapid, accurate and economic method for the genetic diagnosis of SMA and carrier screening in large populations. (Chin J Lab Med, 2015, 38: 16-20) Key words: Spinal muscular atrophies of childhood; Polymorphism, restriction fragment length; Real-time polymerase chain reaction