Received on October 2, 2005
Accepted on December 29, 2005

General Clinical Chemistry

Determination of SMN1/SMN2 Gene Dosage by a Quantitative Genotyping Platform Combining Capillary Electrophoresis and MALDI-TOF Mass Spectrometry

¹ Institute of Chemistry, and Genomics Research Center, Academia Sinica, Taipei, Taiwan, ROC, and Department of Bioinformatics and Structure Biology, National Tsing Hua University, Hsinchu, Taiwan, ROC
² Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan, ROC
³ Institute of Chemistry, and Genomics Research Center, Academia Sinica, Taipei, Taiwan, ROC
⁴ Institute eGene, Inc., Irvine, CA
⁵ Institute of Chemistry, and Genomics Research Center, Academia Sinica, Taipei, Taiwan, ROC, and Institute of Molecular Biology, National Chung-Hsing University, Taichun, Taiwan

^* To whom correspondence should be addressed. E-mail: yjchen{at}chem.sinica.edu.tw.

Background: Spinal muscular atrophy (SMA) is a common inherited and fatal neuromuscular disease caused by deletions and/or mutations that lead to altered concentrations of proteins encoded by the survival motor neuron genes SMN1 and SMN2. Because of the high incidence (at least 1 in 10 000 live births and a carrier frequency of 1 in 35 to 1 in 50) and severity of the disease, precise quantification of SMN1 and SMN2 gene copy numbers is essential for diagnosis and genetic counseling.

Methods: We developed a genotyping platform combining capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to quantify absolute gene dosage. The absolute gene dosage can be determined by a multiplexed competitive PCR protocol followed by capillary electrophoresis analysis. The relative SMN1/SMN2 ratio can be analyzed by PinPoint assay followed by MALDI-TOF MS analysis.

Results: The complementary assays were evaluated in confirmed cases including 9 affected patients, 33 carriers, and 478 healthy individuals from the general population. We were able to determine all of genotypes with different SMN1/SMN2 gene copy number ratios, which unambiguously diagnosed carrier status and the severity of SMA with 100% specificity.

Conclusions: This quantitative genotyping platform is suitable for detection of SMA. The described approach may serve as a general quantitative genotyping method for molecular diagnosis of other inheritable diseases.