Newborn Screening for Spinal Muscular Atrophy by
Calibrated Short-Amplicon Melt Profiling
Steven F. Dobrowolski,1*Ha T. Pham,2Frances Pouch Downes,3Thomas W. Prior,4
Edwin W. Naylor,5and Kathy J. Swoboda6,7
BACKGROUND: The management options for the auto-
somal recessive neurodegenerative disorder spinal
muscular atrophy (SMA) are evolving; however, their
efficacy may require presymptom diagnosis and con-
tinuous 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.
METHODS: 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 amplifica-
tion product, and we used a unique means of melt cal-
ibration to normalize profiles. Samples that we had
SMN2 copies established genotypes associated with
particular profiles. The system was evaluated with ap-
proximately 1000 purified DNA samples, 100 self-
created dried blood spots, and ?1200 dried blood
spots from newborn screening tests.
RESULTS: Homozygous deletion of SMN1 exon 7 pro-
duced a distinctive melt profile that identified SMA pa-
tients. Samples with different numbers of SMN1 and
SMN2 copies were resolved by their profiles. All samples
with homozygous deletions were unambiguously recog-
nized, and no normal sample was misidentified as a
population-based screening. A reliable screening test
will facilitate the identification of an SMA-affected co-
efit 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
© 2012 American Association for Clinical Chemistry
The most recent expansion of newborn screening
(NBS)8was technology driven through the application
of tandem mass spectrometry (1, 2). NBS has ex-
panded and will continue to expand as the natural his-
tories of additional disorders are understood and as
new treatment options are developed. Traditionally,
NBS testing methods have detected metabolic, endo-
crine, and hemoglobin disorders via measurements of
proteins, enzymes, hormones, and small molecules.
Recently, severe combined immunodeficiency syn-
dromes have been recommended for inclusion in NBS
programs. This category of diseases is the first instance
in which DNA-based testing has been broadly applied
as the primary screening tool (3).
Spinal muscular atrophy (SMA) is an autosomal
recessive neurodegenerative disorder that affects ap-
proximately 1 in 10 000 newborns (4). Defects in the
duce a deficiency in or an absence of survival motor
degeneration (5, 6). Deletions that involve the seventh
exon of SMN1 are observed in 95%–98% of SMA-
affected patients (7). The telomeric SMN1 gene is
ent at position c.840 of the SMN2 gene, whereas a cy-
tosine is present at the homologous site in SMN1. The
thymine at c.840 in SMN2 disrupts an exon-splicing
enhancer motif, which causes the mRNA-processing
machinery to missplice ?90% of SMN2 transcripts
little functional product. The proximity of the homol-
1Department of Pathology, Children’s Hospital of Pittsburgh, Pittsburgh, PA;
2ARUP Laboratories, Salt Lake City, UT;3Michigan Department of Community
Health, Lansing, MI;4Department of Pathology, Ohio State University, Colum-
bus, OH;5BioChem Genetics LLC, Isle of Palms, SC; Departments of6Neurology
and7Pediatrics, University of Utah School of Medicine, Salt Lake City, UT.
* Address correspondence to this author at: Children’s Hospital of Pittsburgh,
Department of Pathology, 4401 Penn Ave., Pittsburgh, PA 15201. Fax 412-692-
6550; e-mail firstname.lastname@example.org.
Received January 21, 2012; accepted March 15, 2012.
Previously published online at DOI: 10.1373/clinchem.2012.183038
8Nonstandard abbreviations: NBS, newborn screening; SMA, spinal muscular
atrophy; DBS, dried blood spots on filter paper; IRB, Institutional Review Board.
9Human genes: SMN1, survival of motor neuron 1, telomeric; SMN2, survival
motor neuron 2, centromeric.
Clinical Chemistry 58:6
Molecular Diagnostics and Genetics
PCR, analysis) is approximately 2.5 h, whereas the
turnaround time reported for the Luminex-based test
dye, and so forth) is approximately $1.40/sample,
whereas the reagent cost for the Luminex system is
approximately $2.70/sample. We believe our system
has the qualities (robust, inexpensive, fast turn-
around) for facilitating prospective screening of
SMA. A prospective SMA-screening pilot project to
be initiated in 2012 will assess 400 000 newborns. In
the course of that project, we will accurately deter-
mine sensitivity, specificity, positive predictive
value, and negative predictive value.
the intellectual content of this paper and have met the following 3 re-
quirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
uscript submission, all authors completed the author disclosure form.
Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: F.P. Downes, American College of
Stock Ownership: None declared.
Honoraria: None declared.
subcontract from sponsoring NIH contract HHSN275201000018C;
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the
of data, or preparation or approval of manuscript.
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Screening for Spinal Muscular Atrophy
Clinical Chemistry 58:6 (2012)