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

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.

Current Status of Newborn Bloodspot Screening Worldwide 2024: A Comprehensive Review of Recent Activities (2020–2023)

“…As an example, SMA analysis originally included a tiered algorithm looking for the absence of SMN1 Exon 7. When results from the first and second tier needed reconciliation, a third tier DNA sequencing assay was developed that confirms SMN2 copy numbers without the need for additional instrumentation [ 246 ]. A program report on screening 179,467 newborns confirmed the value of the screening algorithm in detecting SMA-affected infants who show absent SMN1 Exon 7 by Real-Time™ quantitative PCR (qPCR) [ 247 ].…”

Systematic Review of Newborn Screening Programmes for Spinal Muscular Atrophy

“…The review identified 37 cohort studies of newborn screening for SMA [ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ]. No RCTs of newborn screening were identified.…”

“…Cohort studies relating to newborn screening programmes for SMA were identified from the following 17 countries ( Table 1 ): the UK [ 9 ], Belgium [ 10 , 11 , 12 ], Germany [ 13 , 14 , 15 , 16 , 17 , 18 ], Italy [ 19 , 20 ], Latvia [ 21 ], Portugal [ 22 ], Poland [ 23 ], Ukraine [ 24 ], Norway [ 25 ], Australia [ 26 , 27 , 28 , 29 ], the USA, Canada [ 30 , 31 , 32 ], Brazil [ 45 ], Japan [ 46 , 47 , 48 , 49 , 50 ], Taiwan [ 51 , 52 ], China [ 53 , 54 , 55 ] and Russia [ 56 , 57 ]. The USA screening programmes were reported for several US states: California [ 33 ], Georgia [ 34 ], Kentucky [ 35 ], Massachusetts [ 36 , 37 ], New York [ 38 , 39 , 40 ], North Carolina [ 41 ], Wisconsin [ 42 ], Utah [ 43 ] and Ohio [ 44 ].…”

Newborn screening for spinal muscular atrophy in Japan: One year of experience

“…The MLPA test is performed by a different clinical laboratory, and it takes about 5 days to obtain the results (including the time required to transport the specimen). In addition to the MLPA method, measurement of the SMN2 copy number by droplet digital PCR [ 17 , 18 ] and real-time PCR [ 19 ] using DBSs has also been reported. Using these methods to measure the SMN2 copy number in the DBSs may shorten the time taken for diagnosis.…”