Validation of a Fast, Robust, Inexpensive, Two-Tiered Neonatal Screening Test algorithm on Dried Blood Spots for Spinal Muscular Atrophy

Abstract

Spinal muscular atrophy (SMA) is one of the leading genetic causes of infant mortality with an incidence of 1:10,000. The recently-introduced antisense oligonucleotide treatment improves the outcome of this disease, in particular when applied at an early stage of progression. The genetic cause of SMA is, in >95% of cases, a homozygous deletion of the survival motor neuron 1 (SMN1) gene, which makes the low-cost detection of SMA cases as part of newborn screening programs feasible. We developed and validated a new SALSA MC002 melting curve assay that detects the absence of the SMN1 exon 7 DNA sequence without detecting asymptomatic carriers and reliably discriminates SMN1 from its genetic homolog SMN2 using crude extracts from newborn screening cards. Melting curve analysis shows peaks specific for both the SMN1 gene and the disease modifying SMN2 homolog. The detection of the SMN2 homolog, of which the only clinically relevant difference from the SMN1 gene is a single nucleotide in exon 7, was only used to confirm a correct reaction in samples that lacked the SMN1 gene, and not for SMN2 quantification. We retrieved 47 DBS samples from children with genetically-confirmed SMA, after informed consent from parents, and 375 controls from the national archive of the Dutch National Institute for Public Health and the Environment (RIVM). The assay correctly identified all anonymized and randomized SMA and control samples (i.e., sensitivity and specificity of 100%), without the detection of carriers, on the three most commonly-used PCR platforms with melting curve analysis. This test's concordance with the second-tier 'golden standard' P021 SMA MLPA test was 100%. Using the new P021-B1 version, crude extracts from DBS cards could also be used to determine the SMN2 copy number of SMA patients with a high level of accuracy. The MC002 test showed the feasibility and accuracy of SMA screening in a neonatal screening program.

Newborn Screening for 5q Spinal Muscular Atrophy: Comparisons between Real-Time PCR Methodologies and Cost Estimations for Future Implementation Programs

“…MLPA was the second-tier test used, performed on blood samples collected in EDTA tubes, as the already established routine in our center. The use of DBS on MLPA was investigated by Strunk et al [32] and could facilitate the workflow implementation. The authors reported 100% sensitivity and specificity of the MLPA test (P021, MRC-Holland) using crude lysates from DBS (NaOH protocol).…”

“…Dried blood spots (DBS) were stored in low-permeability zip-lock bags with silica gel desiccant packs at 4 • C (up to six months) and at −20 • C if longer prior to DNA extraction. The DNA was obtained from DBS, as described in Strunk et al [32]. Briefly, a single 3.2-mm punch was incubated at room temperature for 15 min in 100 uL of 10 mM of NaOH.…”

A Novel System for Spinal Muscular Atrophy Screening in Newborns: Japanese Pilot Study

“…Our real-time mCOP-PCR system is specific for detecting SMN1 deletions and does not provide any information about SMN2 copy number. If required for prognostic purposes, SMN2 copy number should be determined during a subsequent confirmatory assay or a second-tier assay [29,30]. However, SMN2 copy number does not always correspond to disease severity, and factors other than SMN2 copy number are also related to the severity of SMA [31].…”

Assessment of Spinal Muscular Atrophy Carrier Status by Determining SMN1 Copy Number Using Dried Blood Spots

“…Many methods for SMN1-specific detection have been reported [23,[25][26][27][28][29][30] that can be broadly divided into two methodological groups, A and B. Methods in group A are based on the co-amplification of SMN1 and SMN2 with common primers, and the presence of SMN1 is determined by an SMN1-specific binding of a fluorescence-labeled oligonucleotide probe [23,[25][26][27][28][29]. Methods in group B are based on amplification with SMN1-specific primers and the detection of the amplified SMN1 by a fluorescence-labeled oligonucleotide probe that commonly binds to SMN1/SMN2 [30].…”