title: Quantitative analysis of SMN1 and SMN2 genes based on DHPLC: a highly efficient and reliable carrier-screening test.
abstract: Autosomal recessive spinal muscular atrophy (SMA) is a common, fatal neuromuscular disease caused by homozygous absence of the SMN1 gene in approximately 94% of patients. However, a highly homologous SMN2 gene exists in the same chromosome interval, centromeric to SMN1, and hampers detection of SMN1. We present a new, rapid, simple, and highly reliable method for detecting the SMN1 deletion/conversion and for determining the copy numbers of the SMN1 and SMN2 genes by DHPLC. We analyzed SMN1/SMN2 gene exon 7 deletion/conversion by DHPLC. A total of 25 patients with spinal muscular atrophy lacking the SMN1 gene as well as 309 control individuals from the general population and the family members of patients with SMA were analyzed. By DHPLC analysis, we could detect the SMA-affected cases efficiently just by recognizing an SMN2-only peak. Furthermore, after specific primer amplification and adjustment of the oven temperature, all of the SMA carriers with an SMN1/SMN2 ratio not equal to 1 could be identified unambiguously by this simple and efficient detection system. To calculate the total SMN1/SMN2 gene dosages further, we developed a specific multiplex competitive PCR protocol by simultaneously amplifying the CYBB gene (X-linked), the KRIT1 gene (on chromosome arm 7q), and the SMN1/SMN2 gene ratio by DHPLC. By applying this technique, we could successfully designate all of the genotypes with different SMN1/SMN2 gene copy numbers, including equal and unequal amounts of SMN1 and SMN2. We demonstrated that DHPLC is a fast and reliable tool for detection of carriers of SMA.
authors.affiliation: Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Yi-Ning
authors.initials: YN
authors.lastname: Su
authors.forename: Chia-Cheng
authors.initials: CC
authors.lastname: Hung
authors.forename: Hung
authors.initials: H
authors.lastname: Li
authors.forename: Chien-Nan
authors.initials: CN
authors.lastname: Lee
authors.forename: Wen-Fang
authors.initials: WF
authors.lastname: Cheng
authors.forename: Po-Nien
authors.initials: PN
authors.lastname: Tsao
authors.forename: Ming-Cheng
authors.initials: MC
authors.lastname: Chang
authors.forename: Chia-Li
authors.initials: CL
authors.lastname: Yu
authors.forename: Wu-Shiun
authors.initials: WS
authors.lastname: Hsieh
authors.forename: Win-Li
authors.initials: WL
authors.lastname: Lin
authors.forename: Su-Ming
authors.initials: SM
authors.lastname: Hsu
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics*; D005260:Female; D018628:Gene Dosage; D006580:Genetic Carrier Screening / Q000379:methods*; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009134:Muscular Atrophy, Spinal / Q000175:diagnosis*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D010375:Pedigree; D016601:RNA-Binding Proteins / Q000235:genetics*; D055532:SMN Complex Proteins; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein