Detection of spinal muscular atrophy carriers by nested polymerase chain reaction of single sperm cells.
Spinal muscular atrophy (SMA) is an autosomal recessive disorder with a carrier frequency of approximately 1 in 40. Approximately 95% of patients have homozygous deletions of exon 7 and/or 8 of the SMN1 gene. Carrier testing for SMA is relatively complex and requires quantitative polymerase chain reaction (PCR) of genomic DNA to determine SMN1 copy number. The purpose of this study was to assess the feasibility of carrier testing for SMA in males, by nested PCR analysis of SMN1 deletions in single sperm cells. A nested PCR method was developed to amplify SMN1 exon 7 in single cells. Restriction enzyme digestion with DraI was used to differentiate between the highly homologous SMN1 and SMN2 genes. Single sperm cells from five known SMA carriers and six noncarriers were analyzed. Among the five carriers, a total of 132 single sperm cells were analyzed and SMN1 exon 7 deletion was detected in 68 cells (51.5%). In contrast, among the six noncarriers, a total of 136 single sperm cells were analyzed. Of these, an apparent SMN1 exon 7 deletion was detected in four sperm cells. This was interpreted as an allele dropout (ADO) rate of 2.9%. We conclude that nested PCR of SMN1 exon 7 is an accurate and reproducible method for detection of SMA male carriers with a SMN1 deletion.
Prenatal Diagnosis Unit, Genetic Institute, Tel Aviv Sourasky Medical Center, affiliated with Sackler Faculty of Medicine, Tel Aviv University, Israel. yyaron@tasmc.health.gov.il
Yuval
Y
Yaron
Tania
T
Cohen
Nava
N
Mey-Raz
Tamar
T
Schwartz
Ami
A
Amit
Mira
M
Malcov
D001483:Base Sequence; D025063:Chromosome Disorders / Q000235:genetics*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D005091:Exons / Q000235:genetics; D005260:Female; D005808:Genes, Recessive / Q000235:genetics; D006579:Heterozygote; D006801:Humans; D008297:Male; D009134:Muscular Atrophy, Spinal / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D016601:RNA-Binding Proteins / Q000235:genetics*; D055532:SMN Complex Proteins; D012680:Sensitivity and Specificity; D017384:Sequence Deletion / Q000235:genetics*; D013094:Spermatozoa / Q000166:cytology; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein
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