ARTICLE OPEN ACCESS Practical guidelines to manage discordant situations of SMN2 copy number in patients with spinal muscular atrophy Ivon Cuscó, PhD,* Sara Bernal, PhD,* Laura Blasco-Pérez, MSc, Maite Calucho, MSc, Laura Alias, PhD, Correspondence Dr. Tizzano Pablo Fuentes-Prior, PhD, and Eduardo F. Tizzano, MD, PhD etizzano@vhebron.net Neurol Genet 2020;6:e530. doi:10.1212/NXG.0000000000000530 Abstract Objective Assessment 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. Methods We 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 rec- ommend how to proceed in each case. Results We 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. Conclusions Our 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. *These authors are equal contributors with a major role in drafting and revising the manuscript and analyzing the data. From the Medicine Genetics Group (I.C., L.B.-P., M.C., E.F.T.), Vall dHebron Research Institute (VHIR), Barcelona; Department of Clinical and Molecular Genetics (I.C., L.B.-P., M.C., E.F.T.), Hospital Vall dHebron, Barcelona; Department of Genetics (S.B., L.A.), Hospital de la Santa Creu i Sant Pau, Barcelona; Biomedical Research Institute Sant Pau (IIB Sant Pau) (S.B., L.A.), Hospital de la Santa Creu i Sant Pau, Barcelona; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII, U-705 Barcelona) (S.B., L.A.), Madrid; Molecular Bases of Disease (P.F.-P.), Biomedical Research Institute Sant Pau (IIB Sant Pau), Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. Go to Neurology.org/NG for full disclosures. Funding information is provided at the end of the article. The Article Processing Charge was funded by Fundació Hospital Universitari Vall d’Hebron Institut de Recerca (VHIR) from funds of grant no. FIS PI18/000687. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary FL-SMN = full-length SMN; MLPA = multiplex ligation-dependent probe amplification; NGS = next-generation sequencing; SMA = spinal muscular atrophy; SMN = survival motor neuron; SMN-del7 = SMN2 transcripts lacking exon 7; SNV = single nucleotide variant. Spinal muscular atrophy (SMA) is a neuromuscular disorder type I or II (reviewed in Calucho et al., 2018).4 Thus, accurate with a global incidence of approximately 1:11,000 live births and estimation of SMN2 copy number is essential in the present a worldwide carrier frequency of 1:51.1 According to age at scenario of therapeutic advances with 3 specific SMA therapies onset and achieved motor abilities, patients with SMA are already approved—nusinersen, onasemnogene abeparvovec, and usually classified into type I (never sit), II (never walk unaided), risdiplam—and with the perspective of SMA neonatal screening or III (achieve independent walking abilities). Independent of and early diagnosis to initiate treatment.8,9 We propose a the clinical severity, all forms of SMA are caused by loss or practical guide for the management of discordant SMA cases homozygous loss-of-function pathogenic variants of the SMN1 based on systematic specified actions once SMN2 copy number gene, located at 5q13.2,3 The number of copies of SMN2, the has been determined for a given patient. Our approach is in- highly homologous paralog of SMN1, is currently the most dependent of the method used to estimate SMN2 copy number important modifier of disease phenotype; in most patients with and focuses on the manifestations of the patient to decide how SMA, this number varies between 1 and 5.4 In fact, both SMN1 to proceed in each case. and SMN2 encode, in principle, the same survival motor neuron (SMN) protein. However, a single C→T transition in exon 7 disrupts an exon splicing enhancer and/or creates a splicing Methods silencer, and as a consequence, SMN2 works as a hypomorphic allele that produces mainly transcripts lacking exon 7 (SMN- This guideline can be applied to the vast majority of genetically del7).5 The SMN-del7 protein is functionally compromised and confirmed SMA cases with biallelic deletion of SMN1 and to unstable and therefore rapidly degraded by the ubiquitin- patients who may need further analysis (e.g., those with hybrid proteasome system.6 Thus, the SMA phenotype is ultimately SMN2-SMN1 genes or pathogenic SMN1 variants). We base due to insufficient levels of full-length SMN (FL-SMN) protein. the current guideline on our previously published meta-analysis of SMA genotype-phenotype correlations and in our continued On confirmation of biallelic deletion or pathogenic variants of multidisciplinary experience with patients referred to our con- the SMN1 gene in a given patient, the number of SMN2 copies sultation, both national (Spain) and international.4 Briefly, our is usually determined and reported. In previous years, this fig- approach considers the initial report of SMN2 copy number for ure was mainly informative and mostly used to elaborate a given patient, which is in turn based on a quantitative analysis genotype-phenotype correlations rather than to predict a par- by multiplex ligation-dependent probe amplification (MLPA) ticular phenotype. However, recent advances in SMA thera- using a mixture of specific probes for the SMA locus (P021-B peutics have strengthened the importance of estimating as SMA MLPA kit, a new version of the MLPA kit that includes accurately as possible the number of SMN2 copies for all pa- probes for all exons of the SMN genes, in addition to introns 6 tients with SMA. Indeed, whereas genetic confirmation of SMA and 7).10,11 However, our proposed guide can be applied to any is relatively straightforward (95% of the patients can be di- report regardless of the method used for SMN2 analysis. agnosed with a simple qualitative test), the assessment of Starting with the estimated SMN2 copy number reported, we SMN2 copy number requires a quantitative methodology that then focus on the manifestations of the patient and how to is not easily implemented in most laboratories. Issues of DNA proceed in case of an unexpected discordance. An unambiguous sample quality, calibration controls, and expertise to resolve assignment of the SMA type by motor milestones criteria (0 ambiguous cases have been previously discussed.4 Along these “congenital,” I “never sit,” II “never walk,” or III “walker”) was lines, around 40% of samples recently studied by the same initially widely established for simplicity. However, when nec- methodology in different laboratories yielded discordant re- essary, these categories were further refined into subtypes Ia, b, sults.7 Furthermore, intrinsic biological factors are also a source and c, IIa and b, IIIa and b, and the milder type IV SMA and of discrepancies and add complexity to understanding how a even with minimal manifestations, as previously defined.9,12 specific SMN2 genotype influences the final phenotype in a Altogether, we distinguish up to 10 different clinical diagnostic given patient.4 categories to which genetically confirmed cases may be ascribed (table 1) to establish genotype-phenotype correlations and Numerous studies have shown that the higher the number of define possible discrepancies. copies of SMN2, the larger the amount of FL-SMN protein produced, and thus the milder the associated SMA phenotype. Data availability However, this correlation is not absolute, and some patients All data and scripts used to generate the analyses of this article with 2 copies of SMN2 have mild SMA phenotypes, whereas are available on request unless the type of request compro- some with 4 or more copies of the gene have been described as mises ethical standards or legal requirements. 2 Neurology: Genetics | Volume 6, Number 6 | December 2020 Neurology.org/NG Guidelines in a symptomatic context Table 1 Spinal muscular atrophy (SMA) major clinical The different situations of symptomatic patients, the number of diagnostic categories in genetically confirmed SMN2 copies, the observed phenotypes and the rationale, and cases actions suggested in each case are summarized in table 3. Un- Clinical categories/ expected discordant cases include patients having (1) a single SMA type Main clinical description SMN2 copy but noncongenital disease forms (types Ib, II, or PS Presymptomatic cases (identified at birth by even III), (2) 2 SMN2 copies with type II or III SMA, (3) 3 newborn screening or previous affected sibling) copies of the gene with severe disease forms (type Ia and b), and 0/Ia Congenital cases/patients with early (4) at least 4 SMN2 copies but more severe SMA (types I or II). manifestations within the first weeks of life Ib Patients with manifestations within first 3 mo of life Ic Children capable of head control, nonsitters Discussion IIa Sitters who are not able to stand up We have developed a practical guide for management and IIb Sitters who are able to stand up, but not to walk advice to help in the interpretation and resolution of discordant independently SMA cases according to the number of SMN2 copies and phenotype. Our approach applies to virtually all genetically IIIa Onset before age 3 y, short-term walkers confirmed cases and is independent of the method used to IIIb Onset after age 3 y, long-term walkers determine SMN2 copy number (table 4), but focuses instead IV Walkers with weakness initiated in adult life on the manifestations of the patient. We suggest several rec- ommendations to rapidly define the course of actions for a MM Patients with only MMs (include also asymptomatics) given SMA patient. SMN2 copy number estimation is essential to establish accurate genotype-phenotype correlations, to pre- Abbreviations: MM = minimal manifestation; PS = presymptomatic. dict disease evolution, to stratify patients for clinical trials, and Based on references 4, 9, and 12. to define those eligible for a given treatment. However, in some patients, this information may be insufficient to correlate with the observed phenotype. So far, the number of copies of the SMN2 gene and the presence of rare SMN2 variants (e.g., NM_ Results 017411.3:c.859G>C and NM_017411.3:c.835-44A>G) re- We defined several discordant situations according to SMN2 main the major modifiers of SMA disease phenotype.14–17 copy number in patients with a specific phenotype in 2 dif- ferent scenarios: (1) presymptomatic diagnosis of a case The main characteristics of methods currently used to quan- detected in a newborn screening program or because of a titate SMN2 copies (TaqMan, LightCycler, MLPA, PCR-CE, previous SMA family history and (2) when a symptomatic and digital PCR) are given in table 4.10,11,18–29 In a meta- patient is genetically confirmed. The spectrum of possible analysis of 33 studies published from 1999 to 2017, in which situations includes from 1 to 4 or more SMN2 copies. A SMN2 copy number was reported for a total of 3,393 patients genetically confirmed neonate is considered presymptomatic with SMA, MLPA was used in 54% of patients (n = 1870) based mainly on the absence of hypotonia, weakness, hypo- or followed by LightCycler in 21.4% (n = 741) and TaqMan in areflexia, or fasciculations. Other manifestations may be more 6.5% (n = 228) and fewer patients with the remaining subtle and therefore not clearly noticeable.9,13 In the second methodologies.4,22,27 All these different methodologies have scenario, according to the patient’s phenotype, different dis- advantages and disadvantages, and there are technical aspects crepancies are discussed. We defined recommendations beyond the method itself that have to be considered such as according to the reported literature and our own experience, DNA sample quality and interpretation and control issues. as follows. Digital PCR approaches28 and novel protocols using next- generation sequencing (NGS) may help with the resolution of Guideline in a neonatal screening: particularly difficult cases. Noteworthy, NGS methodologies asymptomatic context allow a thorough analysis of SMN2 copies at the genomic level The different situations that could be encountered when including also introns and allowing a better investigation of the facing a presymptomatic patient, the number of SMN2 equivalency and quality of the SMN2 copies. In addition, NGS copies, the predicted phenotypes and suggested actions in provides valuable information that may be validated to establish each situation, and their rationale are given in table 2. Pa- more comprehensive genotype-phenotype correlations.19–21 tients with 1 SMN2 copy usually present a congenital SMA form, and the discordance refers to their presenting without A virtually asymptomatic neonate with a single SMN2 copy is symptoms in the neonatal period. On the other hand, an an obviously unexpected situation. As indicated in table 2, apparently normal neonate should be expected to have at congenital type 0 cases have only 1 SMN2 copy, which is least 2 SMN2 copies, and different predictions and actions insufficient to rescue the phenotype of the disease at the are endorsed. prenatal stage. In these patients, SMA manifests usually at Neurology.org/NG Neurology: Genetics | Volume 6, Number 6 | December 2020 3 Table 2 Suggested course of actions in SMA cases identified during newborn screening SMN2 Manifestations copy at birth (clinical Expected Recommended actions and expected number category) correlation Rationale for recommended actions phenotype 1 Not observed Noa Presence of only 1 SMN2 copy is usually associated Retest for SMN2 copy number with a new sample (PS) with congenital SMA. If a child is asymptomatic at and/or consider another method/laboratory. If the birth and remains so for the first weeks of life, this presence of a single SMN2 copy is confirmed, test would suggest an error in the previous SMN2 for rare positive variants associated with better- quantitation or the presence of a positive modifier than-expected phenotypes (e.g., c.859G>Cb and single nucleotide variant in the single gene copy. c.835-44A>G), e.g., by Sanger sequencing, or perform next-generation sequencing (NGS) analysis.19–21 2 Not observed Yes Neonates with 2 SMN2 copies usually have a normal Test for rare positive variants in SMN2 associated (PS) appearance. There is a latency period in which SMA with better-than-expected phenotypes (e.g., symptoms may not be detectable. However, subtle c.859G>Cb and c.835-44A>G). If negative, the manifestations of the disease might appear shortly patient has >90% probability of developing severe, after birth. type I SMA.4 If positive, the patient will be virtually a sitter or walker later in their life4,14 3 Not observed Yes Neonates with 3 SMN2 copies have a normal Test for rare positive variants in SMN2 associated (PS) appearance and usually without manifestations at with better-than-expected phenotypes (e.g., least for the first 3 mo of life. c.859G>Cb and c.835-44A>G). If negative, the patient has about 60% probability of developing type II disease, 35% type III, and 5% type Ic. Similar Bayesian estimations can also be calculated.4,25 If positive, the patient will be virtually a walker later in their life4,14 ≥4 Not observed Yes Neonates with 4 SMN2 copies have a normal Retest for SMN2 copy number with a new sample (PS) appearance. About 14% of SMA cases worldwide and/or consider another method/laboratory. If have 4 SMN2 copies.4 copy number is confirmed, the patient has >90% probability of being a walker later in their life (SMA types III or IV).4 Test for the rare positive variants is an option. Abbreviations: PS = presymptomatic; SMA = spinal muscular atrophy. a In this case, the expected category would be congenital (see text for further details and discussion). Percentages are calculated according to reference 4. b A commercial test is available for the c.859G>C variant (table 4). birth with at least marked hypotonia and weakness, but more phenotype-genotype discrepancies are due to technical or commonly with a complex clinical picture that includes in biological reasons. addition respiratory problems, contractures, cardiac malfor- mation, vascular necrosis,30 and diffuse and progressive brain Genetically confirmed SMA cases of newborns with 2 SMN2 abnormalities.31 If the patient does not manifest any of these copies have a high probability (>90%) of developing type I symptoms, the most likely explanation is an erroneous de- disease, but they usually have a normal appearance at birth. termination of SMN2 copy number, which should be ex- There is a latency period—from 1 to several weeks—in which cluded. Retesting with a new DNA sample, eventually using a clear symptoms of weakness and hypotonia may not be de- different method or performing the analysis in a different tectable. However, subtle or less evident manifestations may laboratory, might solve the issue. However, if the presence of appear early after birth such as hypo- or areflexia, weak cry, only 1 SMN2 copy is confirmed, it is possible that single diaphragmatic breathing, feeding problems, and dysauto- nucleotide variants (SNVs) of this single gene copy or a po- nomic manifestations (i.e., increase of sweating and irregular tential SMN2-SMN1 hybrid structure32 make it functionally skin responses to temperature changes).9 On the other hand, superactive, i.e., capable of generating more full-length mRNA exceptional cases with 2 SMN2 copies may manifest overt transcripts and FL-SMN protein than wild-type SMN2 and disease at birth as usually occurs in type 0 cases.34 Thus, and thus to at least partly rescue the phenotype. Thus, testing for considering the continuous spectrum of phenotypes in SMA, known positive variants such as NM_017411.3:c.859G>C16 it would be difficult to differentiate between congenital type and NM_017411.3:c.835-44A>G15 is recommended. If neg- 0 and type Ia disease, and both categories could be merged ative, it would be interesting to conduct an SMN2 NGS study into type 0/Ia disease.12 of the patient to unravel changes that may act as positive modifiers of disease severity. Along these lines, at least 10 To better predict the evolution of patients with 2 SMN2 SMA cases with 1 SMN2 copy and type II or even III disease copies, it would be advisable to test for the presence of rare have been reported or personally communicated to date.4,33 positive variants mentioned above. Indeed, in our experience, Unfortunately, these apparently discrepant cases have not around 40% of cases with 2 SMN2 copies and a milder phe- been further studied, and it remains to be seen whether these notype (types II or III) may harbor one of these SNVs.4,14 4 Neurology: Genetics | Volume 6, Number 6 | December 2020 Neurology.org/NG Table 3 Suggested course of actions in symptomatic SMA cases, for whom phenotypes and genotypes are not correlated (see text for further details and discussion) Observed SMN2 manifestations/ Expected copy milestones clinical clinical number category category Rationale for recommended actions Recommended actions 1 Patients with type I, 0 Patients with 1 SMN2 copy usually present Retest for SMN2 copy number with a new sample II, or III SMA congenital SMA. Patients with typical type I, II, or and/or consider another method/laboratory. If even III disease forms might point to an error in the the presence of a single SMN2 copy is confirmed, initial SMN2 copy number determination or to the test for SNVs in the gene that have been presence of a positive modifier in their single SMN2 previously associated with better-than-expected copy. phenotypes (e.g., c.859G>Ca and c.835-44A>G), e.g., by Sanger sequencing, or perform next- generation sequencing (NGS) analysis.19–21 2 Typical type II or Ia, Ib, Ic The vast majority of patients with 2 SMN2 copies Retest for SMN2 copy number with a new sample type III patients have typical type I disease. Exceptions are usually and/or consider another method/laboratory. If due to the presence of positive SMN2 modifiers. confirmed, test for rare variants in SMN2 that have been previously associated with better- than-expected phenotypes (e.g., c.859G>Ca and c.835-44A>G). If negative, perform NGS analysis to detect novel SNVs or other changes that could be positive modifiers of disease severity.19–21 3 Type I cases with Ic, IIa, IIb, Type Ic cases usually manifest disease between 3 Retest for SMN2 copy number with a new sample disease onset IIIa, IIIb and 6 mo of life, and have 3 SMN2 copies. A typical and/or consider another method/laboratory. If before the age of 3 type Ib patient has 2 copies of the gene. Three SMN2 copy number is confirmed, perform further mo (Ia; Ib) copies are also detected in type II and III patients. studies to identify SNVs or partial intragenic deletions that could act as negative phenotype modifiers (e.g., complete MLPA,11 NGS19–21). ≥4 Non-walkers, either IIIa, IIIb, IV, Most reported cases of type I or II SMA patients with Retest for SMN2 copy number with a new sample type I or II MM 4 SMN2 copies are due to pitfalls in the quantitation and/or consider another method/laboratory. If of SMN2 copy number. SMN2 copy number is confirmed, perform further studies to identify SNVs or partial intragenic deletions that could act as negative phenotype modifiers (e.g., complete MLPA,11 NGS19–21). Abbreviations: MLPA = multiplex ligation-dependent probe amplification; MM = minimal manifestation; NGS = next-generation sequencing; SMA = spinal muscular atrophy; SNV = single nucleotide variant. a A commercial test is available for the c.859G>C variant (table 4). Negative variants in SMN2 have not been discovered, but cases.4 In the light of this finding, it is rather surprising that in warrant further investigation. a recent pilot newborn screening study, 15 of 37 detected cases (40%) had 4 SMN2 copies.36 Excluding technical issues In patients with 3 SMN2 copies, our previous meta-analysis with SMN2 quantitation, if these results are reproduced in revealed that about 60% of cases develop type II disease, 35% other newborn screening studies, it would be tempting to type III, but 5% still had the more severe type Ic SMA.4 speculate that a certain number of individuals in the general Therefore, all neonates with 3 gene copies would be expected to population with 0,4 genotype (i.e., no SMN1 gene but 4 have a normal appearance and to remain essentially asymp- SMN2 copies) remain with minimal symptoms or asymp- tomatic at least for the first 3 months of life. The NURTURE tomatic throughout their lives and thus undetected. Pre- study of presymptomatic patients with 2 or 3 SMN2 copies liminary results of the SMA newborn screening program in treated with nusinersen has shown that patients with 3 gene Australia reported 9 positive cases, but none had 4 SMN2 copies treated in the neonatal period have in general a better copies.37 It is important to highlight that copy number studies evolution.13 Again, here it is advisable to check for rare positive in positive patients detected by newborn screening should be variants to better predict the expected outcomes. performed in expertise centers and with a validated method- ology. In the shared decision to immediately start treatment of The treatment recommendations for presymptomatic cases neonates with 4 SMN2 copies or delay the initiation of with 4 SMN2 copies are still an evolving issue.8,35,36 Based on treatment, several alternatives—each with advantages and available evidence, and in the absence of a reliable biomarker disadvantages—have to be considered (outlined in table 5). of disease evolution, in the United States, it has been recently Whatever decision is taken, it is important to recall that dis- recommended to initiate treatment of all infants with 4 copies ease onset in these patients before the first year of life is rather of SMN2.35 In our meta-analysis of 3,393 cases, patients with unlikely, giving the health care team and the parents more 4 copies accounted for less than 14% of all reported SMA time to weigh advantages and disadvantages of each Neurology.org/NG Neurology: Genetics | Volume 6, Number 6 | December 2020 5 Table 4 Major features of the more commonly used methods to determine SMN2 copy number Method Main characteristics Advantages Disadvantages Real-time PCR Multiplex TaqMan real-time quantitative Fast, robust, and sensitive technique. Low Assays are performed in triplicate and (TaqMan PCR assay. cost. Requires only small amounts of DNA. an internal control is necessary to platform)22,23 Easy interpretation of results by normalize results, as the method is automated variant reporting software. based on relative quantitation. Only the DNA quality important, but not as limiting number of copies of SMN2 exon 7 is as in other techniques. Neither a standard determined. Real-time PCR technology curve nor control samples are necessary. is not always available in a routine laboratory. Real-time PCR Quantitative assay on the basis of real- Low cost. Easy interpretation of results by As a relative quantitative assay, it (LightCycler time PCR, performed with a LightCycler automated variant reporting software. requires standard curves to normalize platform)25,26 instrument (Roche Diagnostics, Basel, results. DNA quality is limiting, large Switzerland) by using the fluorescence amounts of DNA are necessary, and the resonance energy transfer technique (PCR process is laborious. Only the number of products are based on the use of SYBR copies of SMN2 exon 7 is determined. Green). Multiple ligation- Variation of the multiplex PCR assay that Low cost. Robust and sensitive. Requires As a relative quantitative assay, dependent probe permits amplification of multiple target only small amounts of DNA and its quality normalization of results is necessary, for amplification, genes with a single primer pair. is not as limiting as in other techniques. which control samples are required. version B110,11,27 Quantitates gene doses. Does not require sophisticated logistic (a Long assay time (24 h minimum). therrmocycler and a vertical electrophoresis sequencer). Easy interpretation of results by automated variant reporting, free software (Coffalyser). SMN1 and SMN2 exons analyzed in the same experiment, detection of exonic hybrid genes, and partial intragenic deletions. False positive deletions may result from mutations located in regions of hybridization of the probes AmplideX PCR-CE Quantitative method based on a multiplex Robust and sensitive technique. Requires As a relative quantitative assay, SMN1/SMN2 PCR and separation by capillary only small amounts of DNA. Simple and normalization is necessary, which (asuragen.com) electrophoresis to calculate copy number rapid workflow (<4 h). Does not require requires calibration samples. Analysis of exon 7 in SMN1 and SMN2 genes, using sophisticated technology or structural limited to exon 7 of SMN1 and SMN2 specific fluorescently labeled primers. logistics (a thermocycler and a vertical genes, therefore unable to detect partial Quantitation is based on the peak area electrophoresis sequencer). Easy deletions. ratio of the target gene to an endogenous interpretation of results by automated control, normalized to a calibration variant reporting software. Scalable sample. design that allows the study of additional variants, such as hybrid genes, silent carriers, and 1 SMN2 modifier variant (PLUS KIT). Droplet digital DNA is partitioned into thousands of Eliminates the need for standard curves by The special technology necessary to PCR (ddPCR) droplets that are subsequently amplified, using references or endogenous controls. perform ddPCR is not available in most 18,28,29 and fluorescently labeled probe signals As an absolute quantitation method of laboratories. High costs limit within each droplet are recorded as either exons 7 and 8 in SMN1 or SMN2, there is no determination of the copy number of all positive or negative, depending on the need for normalization. Requires SMN2 exons. Therefore, partial presence or absence of a nucleotide extremely low DNA concentrations (e.g., intragenic SMN2 deletions are not target. from dried blood spots). detected. Next-generation Non–Sanger-based high-throughput DNA Allows analysis of complete genes (exons Not available in several diagnostic sequencing (NGS) sequencing technologies (several and introns) and to detect laboratories. Laborious process and 19–21 platforms are currently available). rearrangements and point mutations. longer assay times. Interpretation of results requires specialized bioinformatics tools and usually a bioinformatician. Quantitative studies using NGS are not very robust when SMN1 and SMN2 genes coexist due to their extremely high homology. therapeutic alternative. Some parents may want to move Different approaches should be considered when dealing with forward without further testing, but it is crucial that an expert symptomatic cases. Here, most of the discrepancies should be team adequately communicates about the disease and man- initially faced with a retesting of the patient with a new sample, ages their expectations.8 The implementation of neonatal a different method or even in a second laboratory. According screening in different regions will help to better define to the results of this second test, it might be advisable to protocols of follow-up and validate biomarkers of disease continue testing for known variants in SMN2. A recent SMA progression such as levels of plasma phosphorylated neuro- test that includes testing for the NM_017411.3:c.859G>C filament heavy chain in these patients.38 variant has been made commercially available (table 4) 6 Neurology: Genetics | Volume 6, Number 6 | December 2020 Neurology.org/NG Table 5 Factors to be considered when deciding to treat neonates with genetically confirmed spinal muscular atrophy with 4 SMN2 copies Factors to consider Treat presymptomatically Treat when symptoms appear Disease appearance and Avoid possible long-term disease complications Risk of long-term disease complications complications Opportunity of treatment Some patients might be unnecessarily treated Depends on rescue of disease manifestations for a long time Time to initiate therapy Predicted large therapeutic window Therapeutic window might be too short or lost Adverse events when Risk of treatment complications Reduced risk of treatment complications continuous therapy Economic aspects Higher cost of therapy Higher cost of managing morbidity Quality of life (QoL) issues Effect of years of treatment on QoL Effect of disease on QoL Parent and family expectations Unease of treating a healthy baby Increased stress during follow-up waiting for the imminent onset of manifestations (asuragen.com). Furthermore, a new version of the SMA be adopted in line with the current scenario of early genetic MLPA kit including all exons of the SMN1 and SMN2 genes diagnosis, therapeutic intervention, and evolving phenotypes.41 has been reported. This new version of the kit would allow In this context, time to development of different manifestations detection of some intragenic or 59 terminal deletions that were and age at treatment initiation are becoming crucial as pre- previously extremely difficult to detect.11 However, not all cases dictors of the trajectory of the disease.9,42 In this envisaged might be resolved with an accurate SMN2 copy number as- perspective, a better and clearer definition of the SMN2 ge- sessment or checking for known variants by Sanger sequencing. notype (copies and sequence) in each patient would be ex- If the results of all these studies are not categorical, SMN2 NGS tremely relevant. Along these lines, our proposed guideline studies should be considered to determine whether the SMN2 would help to systematically and rigorously identify discordant copies are functionally identical (table 4).19–21 SMA cases that warrant further genetic investigation. Certainly, the SMN2 gene, as the main modifier of SMA phe- Acknowledgment notype, deserves a more in-depth study beyond the current The authors thank all colleagues for their confidence, information, standard copy number determination. We believe that in terms and reference of patients for study. of its impact on SMA phenotype, SMN2 copy number might be considered as the tip of an iceberg of which other genetic and epigenetic features, most notably SNVs, represent the sub- Figure The iceberg representation of the genetic factors merged part with relevant effects to phenotype of the patients that influence SMA phenotype with SMA (figure). A number of other genes have been pro- posed as candidate modifiers of the SMA phenotype including methylation status of SMN2 (reviewed in Maretina et al., 2018),39 although none of them are yet validated in clinical practice. Given that SMN2 variants modify the disease phe- notype and that transcripts derived from SMN2 are targets for splicing modifiers in the therapeutic scenario, it is essential to gain a thorough insight into the complete SMN2 sequences of discordant patients. Furthermore, we need to unveil possible linkages between specific SMN2 variants and factors involved in SMN2 splicing, on the one hand, and responses to treatment, on the other hand. In patients receiving expensive treatments, their efficacy should be periodically assessed to decide whether to continue treatment or to look for alternatives. Responses to treatment may vary in patients with SMA (from responders to slow responders to nonresponders),40 but it is currently un- SMN2 copy number might be considered as the tip of an iceberg of which known whether specific features of their SMN2 genes are di- other SMN2 genetic and epigenetic features, most notably SMN2 SNVs, rectly correlated with these responses. Discovery and validation represent the submerged part. Other modifier genes and whole genomic data may complete possible influences. SMA = spinal muscular atrophy; SNV of positive and negative genetic markers remain thus an urgent = single nucleotide variant. matter in SMA research. New SMA classifications may need to Neurology.org/NG Neurology: Genetics | Volume 6, Number 6 | December 2020 7 Study funding This work was partially supported by grants from Fundación Appendix (continued) Daniel Bravo Andreu (to E.F.T. and P.F.-P. and supporting Name Location Contribution M.C.), SMA Europe (to E.F.T. and P.F.-P.), Biogen, and Spanish Instituto de Salud Carlos III, Fondo de Inves- Pablo Molecular Bases of Disease, Drafting/revision of the Fuentes- Biomedical Research manuscript for content, tigaciones Sanitarias and cofunded with ERDF funds (grant Prior, PhD Institute Sant Pau (IIB Sant including medical writing no. FIS PI18/000687) to E.F.T. and L.A. Pau), Hospital de la Santa for content, and major role Creu i Sant Pau, Barcelona, in the acquisition of data Spain Disclosure Eduardo F. Medicine Genetics Group, Drafting/revision of the E.F. Tizzano discloses grant support to conduct CTs on Tizzano, Vall dHebron Research manuscript for content, MD, PhD Institute (VHIR); Department including medical writing SMA from Ionis/Biogen and serves as a consultant to of Clinical and Molecular for content; major role in AveXis, Novartis, Biogen, Biologix, Cytokinetics, and Genetics, Hospital Vall the acquisition of data; and Roche. The other authors report no disclosures relevant dHebron, Barcelona, Spain study concept or design to the manuscript. Go to Neurology.org/NG for full disclosures. Publication history References Received by Neurology: Genetics June 23, 2020. Accepted in final form 1. Sugarman EA, Nagan N, Zhu H, et al. Pan-ethnic carrier screening and prenatal diagnosis for spinal muscular atrophy: clinical laboratory analysis of >72,400 speci- September 29, 2020. mens. Eur J Hum Genet 2012;20:27–32. 2. Wirth B. An update of the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA). Hum Mutat 2000; 15:228–237. 3. Alı́as L, Bernal S, Fuentes-Prior P, et al. Mutation update of spinal muscular atrophy in Spain: molecular characterization of 745 unrelated patients and identification of four Appendix Authors novel mutations in the SMN1 gene. Hum Genet 2009;125:29–39. 4. Calucho M, Bernal S, Alı́as L, et al. Correlation between SMA type and SMN2 copy Name Location Contribution number revisited: an analysis of 625 unrelated Spanish patients and a compilation of 2834 reported cases. Neuromuscul Disord 2018;28:208–215. Ivon Medicine Genetics Group, Drafting/revision of the 5. Monani UR, Lorson CL, Parsons DW, et al. A single nucleotide difference that alters Cuscó, Vall d’Hebron Research manuscript for content, splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. Hum PhD Institute (VHIR); Department including medical writing Mol Genet 1999;8:1177–1183. of Clinical and Molecular for content, and major role 6. Vitte J, Fassier C, Tiziano FD, et al. Refined characterization of the expression and Genetics, Hospital Vall in the acquisition of data stability of the SMN gene products. Am J Pathol 2007;171:1269–1280. dHebron, Barcelona, Spain 7. Schorling DC, Becker J, Pechmann A, Langer T, Wirth B, Kirschner J. Discrepancy in redetermination of SMN2 copy numbers in children with SMA [online]. Neurology Sara Department of Genetics, Drafting/revision of the 2019;93:267–269. Bernal, Biomedical Research manuscript for content, 8. Dangouloff T, Burghes A, Tizzano EF, et al. 244th ENMC international workshop: PhD Institute Sant Pau (IIB Sant including medical writing newborn screening in spinal muscular atrophy May 10–12, 2019, Hoofddorp, the Pau), Hospital de la Santa for content, and major role Netherlands. Neuromuscul Disord 2020;30:93–103. Creu i Sant Pau, Barcelona; in the acquisition of data 9. Tizzano EF. Treating neonatal spinal muscular atrophy: a 21st century success story? Centro de Investigación Early Hum Dev 2019;138:104851. Biomédica en Red de 10. Alı́as L, Bernal S, Barceló MJ, et al. Accuracy of marker analysis, quantitative real-time Enfermedades Raras polymerase chain reaction, and multiple ligation-dependent probe amplification to (CIBERER-ISCIII, U-705 determine SMN2 copy number in patients with spinal muscular atrophy. Genet Test Barcelona), Madrid, Spain Mol Biomarkers 2011;15:587–594. 11. Vijzelaar R, Snetselaar R, Clausen M, et al. The frequency of SMN gene variants Laura Medicine Genetics Group, Drafting/revision of the lacking exon 7 and 8 is highly population dependent. PLoS One 2019;14:e0220211. Blasco- Vall dHebron Research manuscript for content, 12. Talbot K, Tizzano EF. The clinical landscape for SMA in a new therapeutic era. Gene Pérez, MSc Institute (VHIR); including medical writing Ther 2017;24:529–533. Department of Clinical for content 13. De Vivo DC, Bertini E, Swoboda KJ, et al. Nusinersen initiated in infants during the and Molecular Genetics, presymptomatic stage of spinal muscular atrophy: interim efficacy and safety results Hospital Vall dHebron, from the Phase 2 NURTURE study. Neuromuscul Disord 2019;29:842–856. Barcelona, Spain 14. Bernal S, Alı́as L, Barceló MJ, et al. The c.859G>C variant in the SMN2 gene is associated with types II and III SMA and originates from a common ancestor. J Med Maite Medicine Genetics Group, Drafting/revision of the Genet 2010;47:640. Calucho, Vall dHebron Research manuscript for content, 15. Wu X, Wang SH, Sun J, Krainer AR, Hua Y, Prior TW. A-44G transition in SMN2 intron MSc Institute (VHIR) 08035 including medical writing 6 protects patients with spinal muscular atrophy. Hum Mol Genet 2017;26:2768–2780. Barcelona, Spain. for content 16. Prior TW, Krainer AR, Hua Y, et al. A positive modifier of spinal muscular atrophy in Department of Clinical the SMN2 gene. Am J Hum Genet 2009;85:408–413. and Molecular Genetics, 17. Vezain M, Saugier-Veber P, Goina E, et al. A rare SMN2 variant in a previously Hospital Vall dHebron, unrecognized composite splicing regulatory element induces exon 7 inclusion and 08035 Barcelona, Spain reduces the clinical severity of spinal muscular atrophy. Hum Mutat 2010;31: E1110–E1125. Laura Department of Genetics, Analysis or interpretation of 18. Vidal-Folch N, Gavrilov D, Raymond K, et al. Multiplex droplet digital PCR method Alias, PhD Biomedical Research data applicable to newborn screening, carrier status, and assessment of spinal muscular Institute Sant Pau (IIB Sant atrophy. Clin Chem 2018;64:1753–1761. Pau), Hospital de la Santa 19. Chen X, Sanchis-Juan A, French CE, et al. Spinal muscular atrophy diagnosis and Creu i Sant Pau, Barcelona; carrier screening from genome sequencing data. Genet Med 2020;22:945–953. Centro de Investigación 20. Ruhno C, McGovern VL, Avenarius MR, et al. Complete sequencing of the SMN2 Biomédica en Red de gene in SMA patients detects SMN gene deletion junctions and variants in SMN2 that Enfermedades Raras modify the SMA phenotype. Hum Genet 2019;138:241–256. (CIBERER-ISCIII, U-705 21. Blasco-Pérez L, Paramonov I, Iranzo L, et al. SMN2 genes study by NGS in spinal Barcelona), Madrid, Spain muscular atrophy:towards identification of new SMA modifiers. Eur J Hum Genet ESHG Congr 2020. Interactive e-poster session P10.53.C. 8 Neurology: Genetics | Volume 6, Number 6 | December 2020 Neurology.org/NG 22. Anhuf D, Eggermann T, Rudnik-Schöneborn S, Zerres K. Determination of SMN1 32. Cuscó I, Barceló MJ, Del Rio E, et al. Characterisation of SMN hybrid genes in and SMN2 copy number using TaqMan technology. Hum Mutat 2003;22:74–78. Spanish SMA patients: de novo, homozygous and compound heterozygous cases. 23. Gómez-Curet I, Robinson KG, Funanage VL, Crawford TO, Scavina M, Wang W. Hum Genet 2001;108:222–229. Robust quantification of the SMN gene copy number by real-time TaqMan PCR. 33. Sun Y, Grimmler M, Schwarzer V, Schoenen F, Fischer U, Wirth B. Molecular and Neurogenetics 2007;8:271–278. functional analysis of intragenic SMN1 mutations in patients with spinal muscular 24. Kraszewski JN, Kay DM, Stevens CF, et al. Pilot study of population-based newborn atrophy. Hum Mutat 2005;25:64–71. screening for spinal muscular atrophy in New York state. Genet Med 2018;20: 34. Kitaoka H, Shitara Y, Uchida Y, Kondo U, Omori I. Case of spinal muscular atrophy 608–613. type 0 with mild prognosis. Pediatr Int 2020;62:106–107. 25. Feldkötter M, Schwarzer V, Wirth R, Wienker TF, Wirth B. Quantitative analyses of 35. Glascock J, Sampson J, Connolly AM, et al. Revised recommendations for the SMN1 and SMN2 based on real-time lightCycler PCR: fast and highly reliable carrier treatment of infants diagnosed with spinal muscular atrophy via newborn screening testing and prediction of severity of spinal muscular atrophy. Am J Hum Genet 2002; who have 4 copies of SMN2. J Neuromuscul Dis 2020;7:97–100. 70:358–368. 36. Müller-Felber W, Vill K, Schwartz O, et al. Infants diagnosed with spinal muscular 26. Cuscó I, Barceló MJ, Rojas-Garcı́a R, et al. SMN2 copy number predicts acute or atrophy and 4 SMN2 copies through newborn screening: opportunity or burden? chronic spinal muscular atrophy but does not account for intrafamilial variability in J Neuromuscul Dis 2020;7:109–117. siblings. J Neurol 2006;253:21–25. 37. Kariyawasam DST, Russell JS, Wiley V, Alexander IE, Farrar MA. The implementa- 27. Arkblad EL, Darin N, Berg K, et al. Multiplex ligation-dependent probe amplification tion of newborn screening for spinal muscular atrophy: the Australian experience. improves diagnostics in spinal muscular atrophy. Neuromuscul Disord 2006;16: Genet Med 2020;22:557–565. 830–838. 38. Darras BT, Crawford TO, Finkel RS, et al. Neurofilament as a potential biomarker for 28. Stabley DL, Harris AW, Holbrook J, et al. SMN1 and SMN2 copy numbers in cell spinal muscular atrophy. Ann Clin Transl Neurol 2019;6:932–944. lines derived from patients with spinal muscular atrophy as measured by array digital 39. Maretina MA, Zheleznyakova GY, Lanko KM, Egorova AA, Baranov VS, Kiselev AV. PCR. Mol Genet Genomic Med 2015;3:248–257. Molecular factors involved in spinal muscular atrophy pathways as possible disease- 29. Zhong Q, Bhattacharya S, Kotsopoulos S, et al. Multiplex digital PCR: breaking modifying candidates. Curr Genomics 2018;19:339–355. the one target per color barrier of quantitative PCR. Lab Chip 2011;11: 40. Finkel RS, Mercuri E, Darras BT, et al. Nusinersen versus sham control in infantile- 2167–2174. onset spinal muscular atrophy. N Engl J Med 2017;377:1723–1732. 30. Grotto S, Cuisset J-M, Marret S, et al. Type 0 spinal muscular atrophy: further 41. Tizzano EF, Finkel RS. Spinal muscular atrophy: a changing phenotype beyond the delineation of prenatal and postnatal features in 16 patients. J Neuromuscul Dis 2016; clinical trials. Neuromuscul Disord 2017;27:883–889. 3:487–495. 42. Dangouloff T, Servais L. Clinical evidence supporting early treatment of patients with 31. Mendonça RH, Rocha AJ, Lozano-Arango A, et al. Severe brain involvement in 5q spinal muscular atrophy: current perspectives. Ther Clin Risk Manag 2019;15: spinal muscular atrophy type 0. Ann Neurol 2019;86:458–462. 1153–1161. Neurology.org/NG Neurology: Genetics | Volume 6, Number 6 | December 2020 9 Practical guidelines to manage discordant situations of SMN2 copy number in patients with spinal muscular atrophy Ivon Cuscó, Sara Bernal, Laura Blasco-Pérez, et al. Neurol Genet 2020;6; DOI 10.1212/NXG.0000000000000530 This information is current as of November 11, 2020 Updated Information & including high resolution figures, can be found at: Services http://ng.neurology.org/content/6/6/e530.full.html References This article cites 41 articles, 2 of which you can access for free at: http://ng.neurology.org/content/6/6/e530.full.html##ref-list-1 Citations This article has been cited by 3 HighWire-hosted articles: http://ng.neurology.org/content/6/6/e530.full.html##otherarticles Permissions & Licensing Information about reproducing this article in parts (figures,tables) or in its entirety can be found online at: http://ng.neurology.org/misc/about.xhtml#permissions Reprints Information about ordering reprints can be found online: http://ng.neurology.org/misc/addir.xhtml#reprintsus Neurol Genet is an official journal of the American Academy of Neurology. Published since April 2015, it is an open-access, online-only, continuous publication journal. 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