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. 2021 Jul 21;7(3):47.
doi: 10.3390/ijns7030047.

Novel Modification of a Confirmatory SMA Sequencing Assay that Can Be Used to Determine SMN2 Copy Number

Binod Kumar  1   2 , Samantha Barton  1 , Jolanta Kordowska  1 , Roger B Eaton  1   2 , Anne M Counihan  1 , Jaime E Hale  1 , Anne Marie Comeau  1   2
Affiliations

Novel Modification of a Confirmatory SMA Sequencing Assay that Can Be Used to Determine SMN2 Copy Number

Binod Kumar et al. Int J Neonatal Screen. .

Abstract

Promising treatments for spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, prompted calls for inclusion in newborn screening (NBS). In January 2018, the New England Newborn Screening Program (NENSP) began statewide screening for SMA using a tiered algorithm looking for the absence of SMN1 Exon 7. When results from the first and second tier needed reconciliation, we developed and validated a third tier DNA sequencing assay to ensure the presence or absence of SMN1 Exon 7. All nine infants referred to specialty centers through NBS showed single base substitution of c.840C>T, and were confirmed to have SMA. Further, a minor sequencing protocol modification allowed the estimation of SMN2 copy number in SMA affected patients; we developed and validated a copy-number assay yielding 100% match with seven previously characterized specimens of SMA patients. All nine SMA-affected infants found through NBS were also assayed for SMN2 copy number. Results were comparable but not 100% matched with those that were reported by independent diagnostic laboratories. In conclusion, a sequencing protocol confirms NBS findings from real-time qPCR, and its modified application allows NBS programs that have sequencing capabilities to provide SMN2 copy numbers without the need for additional instrumentation.

Keywords: SMA; SMN2; copy number; newborn screening; sequencing.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Flow chart of current SMA screening algorithm at New England Newborn Screening program. (B) Primer locus targeting SMN1 gene during SMA sequencing. Primers were tagged with M13, and indicated in bold. SMA seq-F- 5′-TGTAAAACGACGGCCAGT-AACCTTAACTGCAGCCTAATAATTG-3′ and SMA seq-R- 5′-CAGGAAACAGCTATGACC-GCTGGCAGACTTACTCCTTAAT-3′ were used for sequencing.
Figure 2
Figure 2
Typical examples of SMA sequencing assay on reference samples from SMA affected patients and their parents.
Figure 3
Figure 3
Typical example of SMA sequencing of specimens from nine SMA-affected infants who we referred to specialty centers through screening; all showed single base substitution of c.840C>T.
Figure 4
Figure 4
Flow diagram for determination of SMN2 copy number of SMA affected specimen. DNAs were measured using QUBIT, and equal amount of spike DNA and test DNA were mixed, followed by bidirectional sequencing.
Figure 5
Figure 5
Validation of use of modified sequencing for “determination of SMN2 copy number” using eight previously characterized SMA affected specimens. DNA from SMA affected specimens, and a Coriell DNA (positive control) were mixed in equal concentration with spike DNA, and bidirectional sequencing was performed. The SMN1 gene sequence was used as the reference sequence, and data were analyzed to determine the SMN2 copy number in the test specimen as described in the methods section.
See this image and copyright information in PMC

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