Spinal muscular atrophy diagnosis and carrier screening from genome sequencing data

Chen, Xiao; Sanchis‐Juan, Alba; French, Courtney; Connell, Andrew J.; Delon, Isabelle; Kingsbury, Zoya; Chawla, Aditi; Halpern, Aaron L.; Taft, Ryan J.; Bentley, David; Butchbach, Matthew E.R.; Raymond, F. Lucy; Eberle, Michael A.

doi:10.1038/s41436-020-0754-0

Cited by 85 publications

(104 citation statements)

References 39 publications

(52 reference statements)

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“…[14][15][16][17] The main characteristics of methods currently used to quantitate SMN2 copies (TaqMan, LightCycler, MLPA, PCR-CE, and digital PCR) are given in table 4. 10,11,[18][19][20][21][22][23][24][25][26][27][28][29] In a metaanalysis of 33 studies published from 1999 to 2017, in which SMN2 copy number was reported for a total of 3,393 patients with SMA, MLPA was used in 54% of patients (n = 1870) followed by LightCycler in 21.4% (n = 741) and TaqMan in 6.5% (n = 228) and fewer patients with the remaining methodologies. 4,22,27 All these different methodologies have advantages and disadvantages, and there are technical aspects beyond the method itself that have to be considered such as DNA sample quality and interpretation and control issues.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, NGS provides valuable information that may be validated to establish more comprehensive genotype-phenotype correlations. [19][20][21] A virtually asymptomatic neonate with a single SMN2 copy is an obviously unexpected situation. As indicated in table 2, congenital type 0 cases have only 1 SMN2 copy, which is insufficient to rescue the phenotype of the disease at the prenatal stage.…”

Section: Discussionmentioning

confidence: 99%

“…In the shared decision to immediately start treatment of neonates with 4 SMN2 copies or delay the initiation of treatment, several alternatives-each with advantages and disadvantages-have to be considered (outlined in table 5). Whatever decision is taken, it is important to recall that disease onset in these patients before the first year of life is rather unlikely, giving the health care team and the parents more time to weigh advantages and disadvantages of each [19][20][21] ).…”

Section: Yesmentioning

confidence: 99%

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Practical guidelines to manage discordant situations of SMN2 copy number in patients with spinal muscular atrophy

et al. 2020

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ObjectiveAssessment of SMN2 copy number in patients with spinal muscular atrophy (SMA) is essential to establish careful genotype-phenotype correlations and predict disease evolution. This issue is becoming crucial in the present scenario of therapeutic advances with the perspective of SMA neonatal screening and early diagnosis to initiate treatment, as this value is critical to stratify patients for clinical trials and to define those eligible to receive medication. Several technical pitfalls and interindividual variations may account for reported discrepancies in the estimation of SMN2 copy number and establishment of phenotype-genotype correlations.MethodsWe propose a management guide based on a sequence of specified actions once SMN2 copy number is determined for a given patient. Regardless of the method used to estimate the number of SMN2 copies, our approach focuses on the manifestations of the patient to recommend how to proceed in each case.ResultsWe defined situations according to SMN2 copy number in a presymptomatic scenario of screening, in which we predict the possible evolution, and when a symptomatic patient is genetically confirmed. Unexpected discordant cases include patients having a single SMN2 copy but noncongenital disease forms, 2 SMN2 copies compatible with type II or III SMA, and 3 or 4 copies of the gene showing more severe disease than expected.ConclusionsOur proposed guideline would help to systematically identify discordant SMA cases that warrant further genetic investigation. The SMN2 gene, as the main modifier of SMA phenotype, deserves a more in-depth study to provide more accurate genotype-phenotype correlations.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Yesmentioning

confidence: 99%

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Practical guidelines to manage discordant situations of SMN2 copy number in patients with spinal muscular atrophy

et al. 2020

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“…The Absolute Q dPCR System provides reliable measurements of SMN1 and SMN2 copy numbers with a workflow that is similar to qPCR, reducing hands-on time and time-to-results when compared to other currently available dPCR platforms. Some dPCR copy number variation assays rely on digestion of the template genomic DNA with a restriction endonuclease 23 . This is because sequence repeats of two or more copies can be in close proximity to each other, increasing the probability that they will be partitioned in the same microchamber and result in a miscalculation of the copy number.…”

Section: Discussionmentioning

confidence: 99%

Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument

Jiang¹,

Lin²,

Gallagher³

et al. 2020

Sci Rep

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Digital PCR (dPCR) technology has been proven to be highly sensitive and accurate in detecting copy number variations (CNV). However, a higher-order multiplexing dPCR assay for measuring SMN1 and SMN2 copy numbers in spinal muscular atrophy (SMA) samples has not been reported. Described here is a rapid multiplex SMA dPCR genotyping assay run on a fully integrated dPCR instrument with five optical channels. The hydrolysis probe-based multiplex dPCR assay quantifies SMN1, SMN2, and the total SMN (SMN1 + SMN2) while using RPPH1 gene as an internal reference control. The quadruplex assay was evaluated with characterized control DNA samples and validated with 15 blinded clinical samples from a previously published study. SMN1 and SMN2 copy numbers were completely concordant with previous results for both the control and blinded samples. The dPCR-based SMA copy number determination was accomplished in 90 min with a walk-away workflow identical to real-time quantitative PCR (qPCR). In summary, presented here is a simple higher-order multiplexing solution on a novel digital PCR platform to meet the growing demand for SMA genotyping and prognostics.

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“…Next-generation sequencing (NGS) has greatly improved the ability to simultaneously analyze multiple genetic loci for sequence and copy number variants; however, there are limitations to this application, particularly for genes with homology, like SMN1 and SMN2 (Mandelker et al, 2016). In recent years, the feasibility of using NGS analysis for SMA carrier screening and diagnostic purposes has been described (Larson et al, 2015;Feng et al, 2017;Chen et al, 2020;Liu et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Incorporating Spinal Muscular Atrophy Analysis by Next-Generation Sequencing into a Comprehensive Multigene Panel for Neuromuscular Disorders

Tan

Westbrook

Truty

et al. 2020

Genetic Testing and Molecular Biomarkers

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Background: Spinal muscular atrophy (SMA) is traditionally molecularly diagnosed by multiplex ligationdependent probe amplification or quantitative polymerase chain reaction (qPCR). SMA analyses are not routinely incorporated into gene panel analyses for individuals with suspected SMA or broader neuromuscular indications. Aim: We sought to determine whether a next-generation sequencing (NGS) approach that integrates SMA analyses into a multigene neuromuscular disorders panel could detect undiagnosed SMA. Materials and Methods: Sequence and copy number variants of the SMN1/SMN2 genes were simultaneously analyzed in samples from 5304 unselected individuals referred for testing using an NGS-based 122-gene neuromuscular panel. This diagnostic approach was validated using DNA from 68 individuals who had been previously diagnosed with SMA via quantitative PCR for SMN1/SMN2. Results: Homozygous loss of SMN1 was detected in 47 unselected individuals. Heterozygous loss of SMN1 was detected in 118 individuals; 8 had an indeterminate variant in ''SMN1 or SMN2'' that supported an SMA diagnosis but required additional disambiguation. Of the remaining SMA carriers, 44 had pathogenic variants in other genes. Concordance rates between NGS and qPCR were 100% and 93% for SMN1 and SMN2 copy numbers, respectively. Where there was disagreement, phenotypes were more consistent with the SMN2 results from NGS. Conclusion: Integrating NGS-based SMA testing into a multigene neuromuscular panel allows a single assay to diagnose SMA while comprehensively assessing the spectrum of variants that can occur in individuals with broad differential diagnoses or nonspecific/overlapping neuromuscular features.

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Spinal muscular atrophy diagnosis and carrier screening from genome sequencing data

Cited by 85 publications

References 39 publications

Practical guidelines to manage discordant situations of SMN2 copy number in patients with spinal muscular atrophy

Practical guidelines to manage discordant situations of SMN2 copy number in patients with spinal muscular atrophy

Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument

Incorporating Spinal Muscular Atrophy Analysis by Next-Generation Sequencing into a Comprehensive Multigene Panel for Neuromuscular Disorders

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