Quantitative analysis ofSMN1andSMN2genes based on DHPLC: A highly efficient and reliable carrier-screening test
- 1 May 2005
- journal article
- research article
- Published by Hindawi Limited in Human Mutation
- Vol. 25 (5), 460-467
- https://doi.org/10.1002/humu.20160
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. Hum Mutat 25:460–467, 2005.Keywords
This publication has 20 references indexed in Scilit:
- Determination of SMN1 and SMN2 copy number using TaqMan™ technologyHuman Mutation, 2003
- A simple method for diagnosis of autosomal recessive spinal muscular atrophy by denaturing high-performance liquid chromatography.Journal of Child Neurology, 2003
- Prevalence of SMN1 deletion and duplication in carrier and normal populations: implication for genetic counsellingJournal of Medical Genetics, 2003
- Genetic testing and risk assessment for spinal muscular atrophy (SMA)Human Genetics, 2002
- Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1Nature Genetics, 2002
- Quantitative Analyses of SMN1 and SMN2 Based on Real-Time LightCycler PCR: Fast and Highly Reliable Carrier Testing and Prediction of Severity of Spinal Muscular AtrophyAmerican Journal of Human Genetics, 2002
- Genotype determination at the survival motor neuron locus in a normal population and SMA carriers using competitive PCR and primer extensionHuman Mutation, 2000
- Duplications and de novo deletions of theSMNt gene demonstrated by fluorescence-based carrier testing for spinal muscular atrophyAmerican Journal of Medical Genetics, 1999
- Identification of Proximal Spinal Muscular Atrophy Carriers and Patients by Analysis of SMNT and SMNC Gene Copy NumberAmerican Journal of Human Genetics, 1997
- Identification and characterization of a spinal muscular atrophy-determining geneCell, 1995