Newborn Screening for Spinal Muscular Atrophy by Calibrated Short-Amplicon Melt Profiling

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 58; no. 6; pp. 1033 - 1039
• Main Authors: Dobrowolski, Steven F, Pham, Ha T, Pouch Downes, Frances, Prior, Thomas W, Naylor, Edwin W, Swoboda, Kathy J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Association for Clinical Chemistry 01.06.2012; Oxford University Press
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Blood; Colleges & universities; Deoxyribonucleic acid; DNA; Exons; Fundamental and applied biological sciences. Psychology; Gene Deletion; Gene Dosage; Genes; Genetic engineering; Genetic testing; Genotypes; Homozygote; Humans; Infant, Newborn; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Medical screening; Molecular biophysics; Neonatal Screening - methods; Polymerase Chain Reaction; Prospective Studies; Spinal Muscular Atrophies of Childhood - diagnosis; Spinal Muscular Atrophies of Childhood - genetics; Survival of Motor Neuron 1 Protein - blood; Survival of Motor Neuron 1 Protein - genetics; Survival of Motor Neuron 2 Protein - blood; Survival of Motor Neuron 2 Protein - genetics; Technological change; Human; Nervous system diseases; Neuromuscular diseases; Spinal amyotrophy; Biochemistry; Medical screening; Gene amplification; Newborn; Central nervous system disease; Clinical biology; Degenerative disease; Molecular biology; Spinal cord disease
• ISSN: 0009-9147; 1530-8561; 1530-8561
• DOI: 10.1373/clinchem.2012.183038

The management options for the autosomal recessive neurodegenerative disorder spinal muscular atrophy (SMA) are evolving; however, their efficacy may require presymptom diagnosis and continuous treatment. To identify presymptomatic SMA patients, we created a DNA-based newborn screening assay to identify the homozygous deletions of the SMN1 (survival of motor neuron 1, telomeric) gene observed in 95%-98% of affected patients. We developed primers that amplify a 52-bp PCR product from homologous regions in the SMN1 and SMN2 (survival of motor neuron 2, centromeric) genes that flank a divergent site at site c.840. Post-PCR high-resolution melt profiling assessed the amplification product, and we used a unique means of melt calibration to normalize profiles. Samples that we had previously characterized for the numbers of SMN1 and SMN2 copies established genotypes associated with particular profiles. The system was evaluated with approximately 1000 purified DNA samples, 100 self-created dried blood spots, and >1200 dried blood spots from newborn screening tests. Homozygous deletion of SMN1 exon 7 produced a distinctive melt profile that identified SMA patients. Samples with different numbers of SMN1 and SMN2 copies were resolved by their profiles. All samples with homozygous deletions were unambiguously recognized, and no normal sample was misidentified as a positive. This assay has characteristics suitable for population-based screening. A reliable screening test will facilitate the identification of an SMA-affected cohort to receive early intervention to maximize the benefit from treatment. A prospective screening trial will allow the efficacy of treatment options to be assessed, which may justify the inclusion of SMA as a target for population screening.