Genotype determination at the survival motor neuron locus in a normal population and SMA carriers using competitive PCR and primer extension

Precise quantitation of SMN1 copy number is of great interest in many clini cal applications such as direct detection of SMA carriers or detection of a n SMA-affected patient with a hemizygous deletion of the SMN1 gene, We desc ribe a method that combines two independent nonradioactive PCR assays: dete rmination of the relative ratio of the SMN1 and SMN2 genes using a primer e xtension assay and of the total SMN copy number using competitive PCR. Cons istency of the results of two independent approaches ensures the reliabilit y of the deduced genotype and thus avoids false interpretation of borderlin e results that can occur in quantitative assays. In all, 135 subjects were tested, including 91 normal controls and 44 SMA-affected children or SMA ca rriers. Two main genotypes were observed in controls: 2T/2C (45%) and 2T/1C (32%). A wide variability at the SMN locus is observed with nine different genotypes and up to six SMN genes. SMA carriers showed three frequent geno types, 1T/2C (50%), 1T/3C (29%), and 1T/1C (18%). Normal chromosomes with t wo SMN1 genes per chromosome are not infrequent and thus, about 3% of SMA c arriers are not detected using SMN1 copy number quantitation. Finally, as t his method does not detect point mutations (similar to 4% of SMN1 gene muta tions), reliability ranges from 93% to 100% depending on data available fro m the propositus. Hum Mutat 16:253-263, 2000. (C) 2000 Wiley-Liss, Inc.