Detection of heterozygousSMN1 deletions in SMA families using a simple fluorescent multiplex PCR method

Detection of heterozygous SMN1 deletions in SMA families using a simple fluorescent multiplex PCR method EDITOR-With a prevalence of 1/6000 live births, spinal muscular atrophy (SMA) represents the second most common fatal autosomal recessive disorder after cystic fibrosis. 1 2SMA is characterised by the degeneration of anterior horn cells of the spinal cord, resulting in progressive, symmetrical limb and trunk paralysis associated with muscular atrophy.This condition is clinically heterogeneous and has been subdivided into three types according to age of onset and clinical course 3 : type I (Werdnig-HoVmann disease, MIM 253300), type II (intermediate form, MIM 253550), and type III SMA (Kugelberg-Welander disease, MIM 253400).The SMA locus has been mapped to chromosome 5q11.2-q13.35][6] The survival motor neurone (SMN) gene, which lies within this element, is duplicated and both copies are expressed.The telomeric gene (SMN1) has been shown to be deleted or mutated in all three types of SMA. 4 SMN1 encodes almost the full length transcript whereas the centromeric copy Figure 1 Quantification of SMN1 and SMN2 copies using a non-competitive fluorescent multiplex PCR assay.Genomic fragments were PCR amplified using dye labelled primers (table 1).After DraI digestion, fragments were separated by electrophoresis on an ABI 377 DNA automated sequencer and electropherograms from two subjects were superimposed.In each electropherogram, the y axis displays fluorescence intensity in arbitrary units and the x axis indicates the size (in bp) of the fragments.The expected sizes are indicated.The superimposition of two distinct samples, a control sample with two copies of SMN1 and two of SMN2 (blue) and the analysed sample (red), indicates the number of SMN1 and SMN2 copies.The result of this quantification for one normal subject and five relatives of a SMA family (fig 2) are presented.(A) Normal subject with two copies of SMN1 and two copies of SMN2.(B) Index case's father (I.1) with one copy of SMN1 and one copy of SMN2.(C) Mother (I.2) with one copy of SMN1 and two copies of SMN2.(D) UnaVected sib (II.1) with one copy of SMN1 and no copy of SMN2.(E) AVected sib (II.2) with no copy of SMN1 and two copies of SMN2.(F), UnaVected sib (II.4) with two copies of SMN1 and one copy of SMN2.