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.
"Numerous assays have been developed to quantify SMN2 copy number in DNA samples from SMA patients. These assays include radioactive PCR (Coovert et al., 1997), quantitative—or real-time PCR (qPCR)––(Feldk€ otter et al., 2002; Anhuf et al., 2003; G omez-Curet et al., 2007), competitive PCR/primer extension (G erard et al., 2004), denaturing high-performance liquid chromatography (Su et al., 2005), multiplex ligation-dependent probe amplification (Huang et al., 2007), quantitative capillary electrophoresis fragment analysis (QCEFA, Kirwin et al., 2013) and short-amplicon melt profiling (Dobrowolski et al., 2012). An important limitation of these established PCR-based copy number assays is the requirement for a parallel-run calibration curve to assign a breakpoint necessary that identifies placement of an ordinal SMN2 value. "
[Show abstract][Hide abstract] ABSTRACT: Proximal spinal muscular atrophy (SMA) is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutation of survival motor neuron 1 (SMN1). In the human genome, a large duplication of the SMN-containing region gives rise to a second copy of this gene (SMN2) that is distinguishable by a single nucleotide change in exon 7. Within the SMA population, there is substantial variation in SMN2 copy number; in general, those individuals with SMA who have a high SMN2 copy number have a milder disease. Because SMN2 functions as a disease modifier, its accurate copy number determination may have clinical relevance. In this study, we describe the development of an assay to assess SMN1 and SMN2 copy numbers in DNA samples using an array-based digital PCR (dPCR) system. This dPCR assay can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 copy number and disease severity. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 copy numbers from SMA samples.
[Show abstract][Hide abstract] ABSTRACT: High-resolution melting (HRM) analysis is a feasible and powerful method for mutation scanning of sequence variants. Denatured doubled-stranded DNA can be detected in fluorescence changes by increasing the melting temperature and wild-type and heterozygous samples can be easily differentiated in the melting plots. HRM analysis represents the next generation of mutation-scanning technology and offers considerable time and cost savings compared to other screening methods. HRM analysis is a closed-tube method, indicating that polymerase chain reaction amplification and subsequent analysis are sequentially performed in the well, making HRM analysis more convenient than other scanning methodologies. Taken together, HRM analysis can be used for high-throughput mutation screening for research, as well as for molecular diagnostic and clinical purposes. This review summarizes the effectiveness of HRM analysis in the diagnosis of autosomal recessive, dominant, and X-linked genetic disorders. Notably, we will also discuss the limitations of HRM analysis and how to overcome them.
Clinica chimica acta; international journal of clinical chemistry 09/2012; 414C:197-201. DOI:10.1016/j.cca.2012.09.012 · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A term infant, at familial risk for spinal muscular atrophy (SMA), had the diagnosis genetically confirmed on day 3 of life. Clinical evaluation, the CHOP INTEND motor scale and the CMAP amplitude were obtained on days 5 (pre-symptomatic), 20 (mildly weak), 34 (moderately weak) and 63 (severely weak). Palliative care was provided and he expired of an acute pulmonary infection on day 81. The CMAP amplitude and INTEND scores were initially in the normal range, then followed a corresponding decline to a nadir at day 34 and remained so at the 4th assessment. A log-transformed plot of CMAP amplitude from days 5-34 was linear. These data suggest that early motor neuron loss in SMA type I may be logarithmic and demonstrates that the INTEND motor scale closely follows the CMAP electrophysiological biomarker. This single case report supports the consideration that early intervention with a potential therapy is necessary, before the pool of functional motor neurons has plummeted. Further study of these parameters in pre-symptomatic infants with SMA type I will help guide the design of future intervention studies.
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