Validation of a Fast, Robust, Inexpensive, Two-Tiered Neonatal Screening Test algorithm on Dried Blood Spots for
Annuska Strunk1, Andre Abbes1, Antoine R Stuitje2
1Department of Clinical Chemistry and Neonatal Screening, Isala Hospital, Dokter van Heesweg 2, 8025 AB Zwolle, The Netherlands.
Insights
A new assay accurately detects spinal muscular atrophy (SMA) in newborns by identifying the absence of the SMN1 gene. This breakthrough enables early diagnosis and treatment for this leading genetic cause of infant mortality.
Area of Science:
- Genetics and Molecular Biology
- Neonatal Screening
- Medical Diagnostics
Background:
- Spinal muscular atrophy (SMA) is a primary genetic cause of infant mortality, affecting 1 in 10,000 births.
- Early intervention with antisense oligonucleotide treatments significantly improves outcomes for SMA patients.
- The genetic basis of SMA (>95% of cases) involves the homozygous deletion of the survival motor neuron 1 (SMN1) gene, making genetic screening feasible.
Purpose of the Study:
- To develop and validate a novel melting curve assay (SALSA MC002) for the accurate detection of SMA in newborn screening.
- To differentiate between the SMN1 gene and its homolog SMN2, crucial for diagnosing SMA without identifying asymptomatic carriers.
Main Methods:
- Development of the SALSA MC002 melting curve assay utilizing crude extracts from newborn screening cards.
- Validation using 47 DNA samples from confirmed SMA patients and 375 control samples.
- Testing across three common PCR platforms with melting curve analysis and comparison with the P021 SMA MLPA test.
Main Results:
- The SALSA MC002 assay demonstrated 100% sensitivity and specificity in identifying SMA and control samples.
- The assay reliably distinguished SMN1 from SMN2 without detecting carriers and showed 100% concordance with the P021 SMA MLPA test.
- The P021-B1 assay version enabled accurate SMN2 copy number determination from crude DBS extracts.
Conclusions:
- The SALSA MC002 assay is a feasible, accurate, and reliable tool for SMA screening in neonatal programs.
- Early detection through this assay facilitates timely treatment, improving outcomes for infants with SMA.
- The assay's ability to use crude extracts from DBS cards simplifies the screening process.
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.
