Newborn Blood Spot Screening Test Using Multiplexed Real-Time PCR to Simultaneously Screen for Spinal Muscular Atrophy and Severe Combined Immunodeficiency

Abstract

BACKGROUND Spinal muscular atrophy (SMA) is a motor neuron disorder caused by the absence of a functional survival of motor neuron 1, telomeric (SMN1) gene. Type I SMA, a lethal disease of infancy, accounts for the majority of cases. Newborn blood spot screening (NBS) to detect severe combined immunodeficiency (SCID) has been implemented in public health laboratories in the last 5 years. SCID detection is based on real-time PCR assays to measure T-cell receptor excision circles (TREC), a byproduct of T-cell development. We modified a multiplexed real-time PCR TREC assay to simultaneously determine the presence or absence of the SMN1 gene from a dried blood spot (DBS) punch in a single reaction well. METHOD An SMN1 assay using a locked nucleic acid probe was initially developed with cell culture and umbilical cord blood (UCB) DNA extracts, and then integrated into the TREC assay. DBS punches were placed in 96-well arrays, washed, and amplified directly using reagents specific for TREC, a reference gene [ribonuclease P/MRP 30kDa subunit (RPP30)], and the SMN1 gene. The assay was tested on DBS made from UCB units and from peripheral blood samples of SMA-affected individuals and their family members. RESULTS DBS made from SMA-affected individuals showed no SMN1-specific amplification, whereas DBS made from all unaffected carriers and UCB showed SMN1 amplification above a well-defined threshold. TREC and RPP30 content in all DBS were within the age-adjusted expected range. CONCLUSIONS SMA caused by the absence of SMN1 can be detected from the same DBS punch used to screen newborns for SCID.

Delay in Diagnosis of Spinal Muscular Atrophy: A Systematic Literature Review

“…[29][30][31] Regular newborn screening is currently not standard practice in the United States, although the SMAdetermining gene was identified in 1995 and the test is available. 11,31,32 The idea of newborn screening has a high level of support among parents of children who have SMA and among expecting parents. 33,34 A long delay to diagnosis has been noted in other pediatric diseases as well.…”

Newborn Screening for Severe Combined Immunodeficiency in Taiwan

“…Using real-time PCR, we identified several infants with less acute SCID syndromes (variant SCID), SCID-like syndrome (22q11.2 deletion), and many other cases of secondary T-cell lymphopenia. In addition, real-time PCR has the potential to be multiplexed immediately to detect spinal muscular atrophy (SMA) [29] or X-linked agammaglobulinemia (XLA) [30]. Therefore, more than two-thirds of Taiwan newborns were screened using the real-time PCR method.…”

Current status of newborn screening worldwide: 2015

“…[43][44][45] Laboratory quality control X-ALD NBS materials are under development. 46 Research is also ongoing to develop laboratory methods and assess public perceptions for other conditions including Fragile-X, 47,48 spinal muscular atrophy (SMA), 49,50 Wilson's disease, 51 and guanidinoacetate methyltransferase (GAMT) deficiency. 52 With increasing interest in NBS, and the possibility of extracting DNA from residual dried blood spot (DBS) specimens, has come an increasing awareness of privacy issues, particularly since NBS in the U.S. is legally required and consent is usually not included as part of U.S. screening protocols.…”