Genetic screening of spinal muscular atrophy using a real-time modified COP-PCR technique with dried blood-spot DNA

Rochmah, Mawaddah Ar; Harahap, Nur Imma Fatimah; Niba, Emma Tabe Eko; Nakanishi, Kenji; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saitô, Toshio; Saito, Kayoko; Lai, Poh San; Takeshima, Yasuhiro; Takeuchi, Atsuko; Bouike, Yoshihiro; Okamoto, Maya; Nishio, Hisahide; Shinohara, Masakazu

doi:10.1016/j.braindev.2017.04.015

Cited by 12 publications

(21 citation statements)

References 34 publications

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“…2017-196967, PCT/JP2018/37732). In the previous version of our system, genomic DNA was extracted from DBS before targeted pre-amplification [18]. However, the new version used in the present study did not include the DNA extraction step.…”

Section: Smn1-deletion Detection Systemmentioning

confidence: 99%

“…An aliquot of pre-amplified PCR product was added to the reaction mixture with DNA polymerase KOD FX Neo (TOYOBO) and EvaGreen ® Dye (Biotium, Hayward, CA, USA). The primer set for SMN1-specific amplification consisted of R111 and SMN1-COP (5 -TGT CTG AAA CC-3 ) [18,21]. The PCR conditions for the reaction mixture of 50 µL were: (1) initial denaturation at 94 • C for 7 min; (2) 20 cycles for SMN1 denaturation at 94 • C for 1 min, annealing at 37 • C for 1 min, and extension at 72 • C for 1 min; and (3) melting analysis.…”

Section: Gene-specific Amplification Of Smn1 Exonmentioning

confidence: 99%

“…We have developed a rapid, accurate, and high-throughput system for detecting SMN1 deletion using a real-time modified competitive oligonucleotide priming-polymerase chain reaction (mCOP-PCR) technique combined with targeted pre-amplification of the SMN genes from dried blood spots (DBS) [18]. Here, we describe the results of a pilot study of newborn screening for SMA to validate our system and to genotype all DBS collected from newborns in Japan.…”

Section: Introductionmentioning

confidence: 99%

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A Novel System for Spinal Muscular Atrophy Screening in Newborns: Japanese Pilot Study

Shinohara

Niba

Wijaya

et al. 2019

IJNS

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Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by SMN1 gene deletion/mutation. The drug nusinersen modifies SMN2 mRNA splicing, increasing the production of the full-length SMN protein. Recent studies have demonstrated the beneficial effects of nusinersen in patients with SMA, particularly when treated in early infancy. Because nusinersen treatment can alter disease trajectory, there is a strong rationale for newborn screening. In the current study, we validated the accuracy of a new system for detecting SMN1 deletion (Japanese patent application No. 2017-196967, PCT/JP2018/37732) using dried blood spots (DBS) from 50 patients with genetically confirmed SMA and 50 controls. Our system consists of two steps: (1) targeted pre-amplification of SMN genes by direct polymerase chain reaction (PCR) and (2) detection of SMN1 deletion by real-time modified competitive oligonucleotide priming-PCR (mCOP-PCR) using the pre-amplified products. Compared with PCR analysis results of freshly collected blood samples, our system exhibited a sensitivity of 1.00 (95% confidence interval [CI] 0.96–1.00) and a specificity of 1.00 (95% CI 0.96–1.00). We also conducted a prospective SMA screening study using DBS from 4157 Japanese newborns. All DBS tested negative, and there were no screening failures. Our results indicate that the new system can be reliably used in SMA newborn screening.

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Section: Smn1-deletion Detection Systemmentioning

confidence: 99%

Section: Gene-specific Amplification Of Smn1 Exonmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A Novel System for Spinal Muscular Atrophy Screening in Newborns: Japanese Pilot Study

Shinohara

Niba

Wijaya

et al. 2019

IJNS

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“…Several methods have been tested for their suitability as newborn screening assays. Previously-described approaches include real-time quantitative PCR [21,22], droplet digital PCR [23], competitive oligonucleotide priming PCR [24,25], and next generation sequencing [26]. A major difference is that, in contrast to other assays, Melt Assay MC002 does not detect carriers.…”

Section: Discussionmentioning

confidence: 99%

Validation of a Fast, Robust, Inexpensive, Two-Tiered Neonatal Screening Test algorithm on Dried Blood Spots for Spinal Muscular Atrophy

Strunk

Abbes

Stuitje

et al. 2019

IJNS

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Spinal muscular atrophy (SMA) is one of the leading genetic causes of infant mortality with an incidence of 1:10,000. The recently-introduced antisense oligonucleotide treatment improves the outcome of this disease, in particular when applied at an early stage of progression. The genetic cause of SMA is, in >95% of cases, a homozygous deletion of the survival motor neuron 1 (SMN1) gene, which makes the low-cost detection of SMA cases as part of newborn screening programs feasible. We developed and validated a new SALSA MC002 melting curve assay that detects the absence of the SMN1 exon 7 DNA sequence without detecting asymptomatic carriers and reliably discriminates SMN1 from its genetic homolog SMN2 using crude extracts from newborn screening cards. Melting curve analysis shows peaks specific for both the SMN1 gene and the disease modifying SMN2 homolog. The detection of the SMN2 homolog, of which the only clinically relevant difference from the SMN1 gene is a single nucleotide in exon 7, was only used to confirm a correct reaction in samples that lacked the SMN1 gene, and not for SMN2 quantification. We retrieved 47 DBS samples from children with genetically-confirmed SMA, after informed consent from parents, and 375 controls from the national archive of the Dutch National Institute for Public Health and the Environment (RIVM). The assay correctly identified all anonymized and randomized SMA and control samples (i.e., sensitivity and specificity of 100%), without the detection of carriers, on the three most commonly-used PCR platforms with melting curve analysis. This test's concordance with the second-tier 'golden standard' P021 SMA MLPA test was 100%. Using the new P021-B1 version, crude extracts from DBS cards could also be used to determine the SMN2 copy number of SMA patients with a high level of accuracy. The MC002 test showed the feasibility and accuracy of SMA screening in a neonatal screening program.

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“…There are currently numerous tests that can be used to diagnose SMA, including restriction fragment length polymorphism, multiplex ligation‐dependent probe amplification, and quantitative PCR (Ar Rochmah et al., ; Kato et al., ; Kesari, Mukherjee, & Mittal, ; Ogino, Leonard, Rennert, & Wilson, ; Ogino & Wilson, ; Xu, Ogino, Lip, Fang, & Wu, ). In addition, there is now a noninvasive prenatal diagnostic analysis that can diagnose SMA using blood from expecting mothers, although due to the nature of the analysis, this test can only be offered to mothers who already have a child diagnosed with SMA, making it an unsuitable screening tool (Parks et al., ).…”

Section: Introductionmentioning

confidence: 99%

Newborn genetic screening for spinal muscular atrophy in the UK: The views of the general population

Boardman

Sadler

Young

2017

Molec Gen & Gen Med

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BackgroundSpinal muscular atrophy (SMA) is an inherited neuromuscular disorder and a leading genetic cause of infant death worldwide. However, there is no routine screening program for SMA in the UK. Lack of treatments and the inability of screening tests to accurately predict disease severity are among the key reasons implementation of screening has faltered in the UK. With the recent release of the first therapy for SMA (Nusinersen), calls are being made for a reconsideration of this stance; however, very little is known about the views of the general public.MethodsAn online survey was administered to 232 individuals with no prior relationship with SMA to assess their attitudes toward a newborn screening program for it. Results are compared with previously gathered data on the views of SMA‐affected families toward screening.ResultsEighty‐four percent of participants were in favor of newborn screening. Key reasons for support were a belief that it would lead to better healthcare and life expectancy for affected infants and facilitate informed decision‐making for future pregnancies. Key reasons for nonsupport were a belief in the potential for significant negative impact on the family unit in terms of bonding and stress.ConclusionsPublic acceptability is a key component in the evaluation of any potential screening program in the UK. This study demonstrates that newborn screening for SMA is viewed largely positively by people unfamiliar with the condition. The importance of early identification overrode all other social and ethical concerns about screening for the majority of participants.

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Genetic screening of spinal muscular atrophy using a real-time modified COP-PCR technique with dried blood-spot DNA

Cited by 12 publications

References 34 publications

A Novel System for Spinal Muscular Atrophy Screening in Newborns: Japanese Pilot Study

A Novel System for Spinal Muscular Atrophy Screening in Newborns: Japanese Pilot Study

Validation of a Fast, Robust, Inexpensive, Two-Tiered Neonatal Screening Test algorithm on Dried Blood Spots for Spinal Muscular Atrophy

Newborn genetic screening for spinal muscular atrophy in the UK: The views of the general population

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