Alberta Spinal Muscular Atrophy Newborn Screening-Results from Year 1 Pilot Project

Abstract

Spinal muscular atrophy (SMA) is a progressive neuromuscular disease caused by biallelic pathogenic/likely pathogenic variants of the survival motor neuron 1 (SMN1) gene. Early diagnosis via newborn screening (NBS) and pre-symptomatic treatment are essential to optimize health outcomes for affected individuals. We developed a multiplex quantitative polymerase chain reaction (qPCR) assay using dried blood spot (DBS) samples for the detection of homozygous absence of exon 7 of the SMN1 gene. Newborns who screened positive were seen urgently for clinical evaluation. Confirmatory testing by multiplex ligation-dependent probe amplification (MLPA) revealed SMN1 and SMN2 gene copy numbers. Six newborns had abnormal screen results among 47,005 newborns screened during the first year and five were subsequently confirmed to have SMA. Four of the infants received SMN1 gene replacement therapy under 30 days of age. One infant received an SMN2 splicing modulator due to high maternally transferred AAV9 neutralizing antibodies (NAb), followed by gene therapy at 3 months of age when the NAb returned negative in the infant. Early data show that all five infants made excellent developmental progress. Based on one year of data, the incidence of SMA in Alberta was estimated to be 1 per 9401 live births.

Keywords: SMA; SMN1; gene therapy; multiplex qPCR; newborn screening.

Figure 1

Representative Amplification Plots Generated using Taqman Probes and the CT Method of Relative Quantification Developed for Newborn Screening of SMA/SCID. Plots are shown for a normal sample (A), a sample with a positive SMA screen (B), and a no-template control (C). Panel (D) compares the SMN1 amplification curves in a heterozygous deletion, a homozygous deletion, and a normal sample. No SMN1 amplification was detected in the positive SMA screen as expected. Plots show the CT value of three targets (blue: SMN1, red: RPP30 and green: TREC). ΔRn = the reporter signal normalized to the fluorescence signal of Applied Biosystems ROX Dye minus the baseline; ΔRn is plotted against PCR cycle number. Amplification threshold level was manually determined to be 0.04 for SMN1.

Figure 2

Timeline–diagnosis algorithm established in Alberta Newborn Screening Program. All DBS samples collected from across the province and received at the newborn laboratory in Edmonton undergo screening for SMA at the Molecular Genetics Lab (MGL). All samples with zero-copy of the SMN1 gene are subjected to duplicate testing, and labeled as screen positive only if both tests yield positive results. This process typically requires 6–8 days. Upon obtaining a positive screening result, a genetic counselor immediately contacts the pediatric neurologist, who arranges a meeting with the proband’s family to collect new blood samples for confirmatory diagnostic testing. The diagnostic confirmation test is conducted on the new blood samples from the proband, using the MLPA kit at MGL. This test not only confirms the results of the screening test but also determines the copy number of the SMN2 gene. On average, this diagnostic testing process takes 6 to 10 business days. Treatment is selected based on the copy numbers of SMN2 and is initiated as soon as possible.