Duplications and de novo deletions of theSMNt gene demonstrated by fluorescence-based carrier testing for spinal muscular atrophy

Abstract

Approximately 95% of individuals with spinal muscular atrophy (SMA) lack both copies of the SMNt gene at 5q13. The presence of a nearly identical centromeric homolog of the SMNt gene, SMNc, necessitates a quantitative polymerase chain reaction approach to direct carrier testing. Adapting a radioactivity-based method described previously, multiplex polymerase chain reaction was performed using fluorescently labeled primers followed by analysis on an ABI 373a DNA sequencer. The SMNt copy number was calculated from ratios of peak areas using both internal and genomic standards. Samples from 60 presumed carriers (50 parents of affected individuals and 10 relatives implicated by linkage analysis) and 40 normal control individuals were tested. Normalized results (to the mean of five or more control samples harboring two copies of the SMNt gene) were consistently within the ranges of 0.4 to 0.6 for carriers (one copy) and 0.8 to 1.2 for normal controls (two copies), without overlap. Combining linkage analyses with direct carrier test results demonstrated de novo deletions associated with crossovers, unaffected individuals carrying two SMNt gene copies on one chromosome and zero SMNt gene copies on the other chromosome, and unaffected individuals with three copies of the SMNt gene. This report demonstrates that fluorescence-based carrier testing for SMA is accurate, reproducible, and useful for genetic risk assessment, and that carrier testing may need to be combined with linkage analysis in certain circumstances.

Detection of male 2+0 and 1+0 carriers for spinal muscular atrophy by digital PCR

“…More than two decades have passed since the 2+0 genotype of SMN1 was first identified 20 . Although significant progress has been achieved in recent years, the detection of 2+0 carriers in the SMA field remains challenging.…”

“…Currently, various methods are available to detect 1+0 carriers, such as quantitative PCR, MLPA and dPCR. It has been more than 20 years since the 2+0 genotype was discovered 3,20–25 . Interestingly, researchers have used SNP haplotypes to indirectly identify 2+0 carriers, 26,27 yielding inaccurate results, highlighting the need for a novel approach to identify 2+0 carriers.…”

“…It has been more than 20 years since the 2+0 genotype was discovered. 3,[20][21][22][23][24][25] Interestingly, researchers have used SNP haplotypes to indirectly identify 2+0 carriers, 26,27 yielding inaccurate results, highlighting the need for a novel approach to identify 2+0 carriers.…”

Spinal Muscular Atrophy: Molecular Mechanisms

“…The number of SMN1 copies may be determined with an allelic specific quantitative PCR assay, utilizing the single nucleotide difference between the exon 7 of SMN1 and SMN2 genes [33,34]. Copy number testing may incorrectly determine carrier status in about 2% of the population, due to duplication of SMN1 on one chromosome and deletion on the other (2+0) [35,36]. If one SMN1 gene copy is identified in an affected case, SMN1 gene sequencing may then be subsequently undertaken to potentially identify compound heterozygous patients with an SMN1 deletion on one chromosome and SMN1 mutation on the other.…”

D5S351 and D5S1414 located at the spinal muscular atrophy critical region represent novel informative markers in the Iranian population

“…Spinal muscular atrophy (SMA) is a severe neuromuscular disease characterized by degeneration of the alpha motor neurons of the spinal cord associated with progressive symmetric weakness and atrophy of the limb muscles ( Dubowitz, 2009 ). Different phenotypes identified in this disease were usually classified into three groups according to the age of onset and severity of the clinical symptoms, i) acute Werdnig–Hoffmann (type I), ii) intermediate Werdnig–Hoffmann (type II), and iii) Kugelberg–Welander disease (type III) ( Chen et al, 1999 ). Molecular investigations had indicated that all these three types of disease were linked to the SMA critical region on chromosome 5q13 ( Brzustowicz et al, 1990 , Melki et al, 1990a , Melki et al, 1990b ).…”