10981004, 2021, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/humu.24200 by Readcube (Labtiva Inc.), Wiley Online Library on [07/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Received: 4 September 2020 | Revised: 13 January 2021 | Accepted: 11 March 2021 DOI: 10.1002/humu.24200 METHODS Beyond copy number: A new, rapid, and versatile method for sequencing the entire SMN2 gene in SMA patients Laura Blasco‐Pérez1,2 | Ida Paramonov1,2 | Jordi Leno1,2 | Sara Bernal3,4 | Laura Alias3,4 | Pablo Fuentes‐Prior5 | Ivon Cuscó1,2,4 | Eduardo F. Tizzano1,2 1 Medicine Genetics Group, Vall d'Hebron Research Institute (VHIR), Barcelona, Spain Abstract 2 Department of Clinical and Molecular Spinal muscular atrophy (SMA) is caused by bi‐allelic loss or pathogenic variants in the Genetics, Hospital Vall d'Hebron, Barcelona, Spain SMN1 gene. SMN2, the highly homologous copy of SMN1, is considered the major phe- 3 Genetics Department, Hospital de la Santa notypic modifier of the disease. Determination of SMN2 copy number is essential to Creu i Sant Pau, Barcelona, Spain establish robust genotype–phenotype correlations and predict disease evolution, to 4 Centro de Investigación Biomédica en Red de stratify patients for clinical trials, as well as to define those eligible for treatment. Dis- Enfermedades Raras (CIBERER) 5 cordant genotype–phenotype correlations are not uncommon in SMA, some of which are Molecular Bases of Disease, Biomedical Research Institute Sant Pau (IIB Sant Pau), due to intragenic SMN2 variants that may influence the amount of complete SMN Hospital de la Santa Creu i Sant Pau, transcripts and, therefore, of full‐length SMN protein. Detection of these variants is Barcelona, Spain crucial to predict SMA phenotypes in the present scenario of therapeutic advances and Correspondence with the perspective of SMA neonatal screening and early diagnosis to start treatments. Ivon Cuscó and Eduardo F. Tizzano, Department of Clinical and Molecular Here, we present a novel, affordable, and versatile method for complete sequencing of Genetics, Hospital Vall d'Hebron, 08035, the SMN2 gene based on long‐range polymerase chain reaction and next‐generation Barcelona, Spain. Email: icusco@vhebron.net and sequencing. The method was validated by analyzing samples from 53 SMA patients who etizzano@vhebron.net lack SMN1, allowing to characterize paralogous, rare variants, and single‐nucleotide polymorphisms of SMN2 as well as SMN2–SMN1 hybrid genes. The method identifies Funding information Fundació Daniel Bravo Andreu, partial deletions and can be adapted to determine rare pathogenic variants in patients Grant/Award Number: 2016; Spanish with at least one SMN1 copy. Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias and cofunded with ERDF, Grant/Award Number: FIS PI18/ KEYWORDS 000687; Galicia AME, Grant/Award Number: next‐generation sequencing, paralogous variants, phenotype–genotype correlations, SMN2 2019; Biogen, Grant/Award Number: copies, spinal muscular atrophy ESP‐SMG‐17‐11256; SMA Europe, Grant/Award Number: 2014‐2016 1 | INTRODUCTION (Sugarman et al., 2012). SMA patients are classified into different clinical groups based on the age of onset, clinical severity, and achieved motor Spinal muscular atrophy (SMA) is the second most common recessive milestones. In the most severe form, type I SMA, patients are never able genetic disease of infancy and early childhood, with an incidence of 1 in to sit and generally die of respiratory failure before the age of 2 years. 5000–10,000 live births and a worldwide carrier frequency of 1:51 Intermediate type II SMA patients are able to sit but never walk, thus This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. © 2021 The Authors. Human Mutation published by Wiley Periodicals LLC. Human Mutation. 2021;42:787–795. wileyonlinelibrary.com/journal/humu | 787 10981004, 2021, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/humu.24200 by Readcube (Labtiva Inc.), Wiley Online Library on [07/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 788 | BLASCO‐PÉREZ ET AL. being confined to wheel‐chair. Type III patients walk unassisted but may SMN2 gene copies (n = 51), but the cohort included one patient each lose this ability during infancy or adolescence (Wang et al., 2007; Zerres with two and four SMN2 copies, respectively. In addition, samples & Rudnik‐Schöneborn, 1995). from three patients who carry one SMN1 copy were studied to assess Bi‐allelic absence or pathogenic variants of the Survival of Motor the versatility of the method to detect SMN1‐specific pathogenic Neuron 1 (SMN1) gene cause SMA (Lefebvre et al., 1995). A cen- variants. These patients were: patient SMA54, who had a hetero- tromeric and nearly identical paralog, SMN2, encodes in principle the zygous deletion of SMN1, the pathogenic variant c.399_402del same protein as SMN1 (Lefebvre et al., 1995; Monani et al., 1999; (p.(Glu134Serfs*14)) in the other SMN1 allele, and three SMN2 co- Rochette et al., 2001). However, a silent transition within exon 7 of pies; patient SMA55, who had a heterozygous deletion of SMN1, the the SMN2 gene causes exon skipping and results in a truncated, pathogenic variant c.815A>G (p.(Tyr272Cys)) in the other SMN1 al- nonfunctional variant (SMN‐Δ7) (Lorson et al., 1999). It has been es- lele, one complete SMN2 copy, and two partial SMN copies com- timated that each SMN2 copy can produce only around 10% to 15% of prising exons 1 to 6 (also known as SMN1/2Δ7‐8 deletion, Arkblad functional SMN protein, depending on the cells and tissues studied et al., 2006), and SMA55F, father of SMA55 harboring 2 SMN1 copies (Boza‐Morán et al., 2015; Soler‐Botija et al., 2005; Wirth et al., 2013). (one with the variant c.815A>G), and two SMN2 copies. The number of SMN2 copies and the presence of intragenic SMN2 Genetic confirmation of SMA by bi‐allelic defects in SMN1 (Alías variants are known modifiers of SMA disease severity (Bernal et al., 2009) as well as SMN2 copy number determination by multi- et al., 2010; Prior et al., 2009; Ruhno et al., 2019). Indeed, numerous plex ligation‐dependent probe amplification (MLPA) were carried out studies show that the higher the SMN2 copy number, producing larger as previously described (Alías et al., 2011). Patients were classified as amount of full‐length SMN protein, the milder the associated SMA I, II, or III according to their severity and motor milestone achieve- phenotype and vice versa. However, this inverse correlation is not ab- ments. One patient presenting with type 0 (congenital) SMA was also solute (Calucho et al., 2018). Whereas the determination of SMN2 copy studied. All patients were unrelated with the exception of the number is widely implemented to study SMA patients, the actual struc- SMA55/55F pair mentioned above (a child with type 0 SMA and his tures and genomic sequences of SMN2 copies are usually not included in father) and two pairs of siblings, SMA17/SMA18 (both type II) and the characterization of SMA patients. SMA27/SMA51 (types II and III, respectively). DNA samples were The current scenario of SMA therapy is rapidly evolving due to the obtained from peripheral blood. All participants or their legal guar- approval in the last years of nusinersen/Spinraza, an antisense‐tailored dians signed written informed consent. The study was approved by therapy (Finkel et al., 2017), AVXS101/Zolgensma, an adeno‐associated the Ethical Committee of our Hospital (PR(AG)229/2018). viral‐based gene therapy (Mendell et al., 2017), as well as the recent approval of the first oral drug to treat SMA, risdiplam/Evrysdi (http:// www.fda.gov). However, these disease‐modifying therapies are expensive 2.2 | PCR design and library preparation treatments, and their efficacy needs to be periodically assessed. Although responses to treatment vary in SMA patients, it is not yet known whe- We studied the complete genomic SMN2 sequence, including pro- ther specific features of SMN2 are correlated with these responses moter, 5′‐UTR and 3′‐UTR regions. To this end, we designed three (Cuscó et al., 2020). Thus, it becomes crucial to investigate genomic overlapping PCRs (~12‐kb each) to amplify a target region of ap- SMN2 data to better characterize SMA patients and accurately predict proximately 31.5 Kb (chr5:69,342,511‐69,374,064). These long‐ disease evolution. range PCRs were successfully set up using TaKaRa LA Taq® DNA Here, we report a novel method for sequencing the whole SMN2 polymerase (#RR002A; Takara Bio). Given the high homology be- gene based on long‐range polymerase chain reaction (PCR) and next‐ tween SMN1 and SMN2 sequences, primers were not specific to generation sequencing (NGS). The method allows determining all variants SMN2, but the 53 patients analyzed have zero SMN1 copies, allowing described so far as disease modifiers in SMA patients without SMN1 as in principle an SMN2 exclusive analysis. In addition, samples from well as to identify new variants and structural changes. Furthermore, the three patients with at least one SMN1 copy were analyzed. Primer technique can be adapted to determine rare pathogenic variants in sequences and PCR conditions are given in Table S1. heterozygous patients with at least one SMN1 copy. Inclusion of this After amplification, the concentration of the three PCR products technique in the routine diagnosis of SMA patients is expected to im- was measured using Qubit 2.0 Fluorometer (Thermo Fisher Scientific) prove individual genotype–phenotype correlations and, therefore, to help and mixed equimolarly. One thousand nanogram of the obtained predict more accurately the evolution of the disease. mixture was fragmented with NEBNext® dsDNA Fragmentase® (New England Biolabs) to generate DNA fragments of ~200 bp. Then, the NEBNext Ultra DNA library prep kit for Illumina and NEBNext® 2 | M A TE R I A L S AN D ME TH O D S MultiplexOligos for Illumina® Dual Index Primers Set 1 (New England Biolabs) were used to generate the libraries. The necessary purifica- 2.1 | Patients tions and size selections were performed using AMPure XP beads (Beckman Coulter). We studied 53 genetically confirmed SMA patients with homozygous The quality and size of the libraries were assessed using absence of SMN1. The vast majority of studied patients had three QIAxcel (Qiagen) and were quantified with Qubit. Finally, the 10981004, 2021, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/humu.24200 by Readcube (Labtiva Inc.), Wiley Online Library on [07/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License BLASCO‐PÉREZ ET AL. | 789 libraries of all patients were equimolarly mixed and sequenced 3 | RES ULTS using a 500‐cycle MiSeq reagent kit v2 with a paired‐end run of 2 × 251 bp reads in a MiSeq instrument (Illumina). The number of In all analyzed samples, a mean depth of coverage of 1720× patients included in each run was calculated to ensure a minimum (435×–7478×) was obtained. This high coverage allows to accurately coverage of ×400. All procedures were performed following the determine the allelic frequency of the detected variants in each sample manufacturer's instructions. (AB ratio). Given that the copy number of SMN2 genes had been already In samples harboring at least one copy of SMN1, pathogenic studied by MLPA, we could use the AB ratio to calculate the number of variants were ascribed to SMN1 using a long‐range PCR and Sanger copies in which the detected variant was present. sequencing of SMN1 as previously described (Kubo et al., 2015), with To assess the specificity of our method to identify genetic variants in slight modifications. the 5q13 complex region, we first analyzed PSVs that are specific for the SMN2 gene. To this end, a list of SMN1 and SMN2 PSVs was created based initially on a literature review (Monani et al., 1999), and expanded 2.3 | Bioinformatics analysis with the information obtained from a basic local alignment search tool‐ like alignment tool (BLAT) between the two genes using the reference The data analysis pipeline included the quality trimming of Illumina genome Hg19. The detailed information obtained from sequencing the sequences using Trimmomatic (Bolger et al., 2014). Sequences were SMN2 gene in samples from 53 patients was used to determine mapped to an artificial genome reference that contains only the and eventually confirm which positions should be considered real PSVs SMN2 coordinates, based on the reference genome (UCSC hg 19 or SNPs or SNVs. Based on previous reports and BLAT data, we initially version, build 37.1). This strategy avoids arbitrarily reporting only considered 22 PSVs (Table 1). Four of these variants exhibited high one of the possible alignments produced by most mapping algo- variability between samples, indicating that they should not be con- rithms and therefore avoids dispersion of the read‐depth signal from sidered genuine PSVs (by definition). Two other variants, including the all SMN genes. This approach increases the power to detect small candidate position g.69371981A/C previously described as a PSV changes of coverage and variants with lower AB ratios, which are (Monani et al., 1999) and g.69367553G/A were not found neither in characteristic of heterozygous variants in multicopy regions. Results SMN1 nor SMN2 genes from our samples and were considered as very show a single location per each read that corresponds to any of the infrequent variants. The remaining 16 changes are genuine PSVs be- SMN genes. Mapping was performed using burrows‐wheeler aligner tween SMN1 and SMN2 genes, being c.835‐1606C/T in intron 6 con- (BWA)‐align and BWA‐sample with default parameters. Variant sidered here for the first time as a PSV (Table 1). These PSVs were calling was performed with Genome Analysis Toolkit (GATK) Unified present in all the reads and in more than 95% of the samples, confirming Genotyper and Haplotype Caller (McKenna et al., 2010), and variant that all these positions were specific for SMN2. However, in two patients annotation with ANNOVAR (Codina‐Solà et al., 2016; Wang (SMA04 and SMA39) we observed some discrepancies in the PSV ratio et al., 2010). compatible with the presence of hybrid genes. Both patients presented six SMN1 PSVs all located in intron 6 (Chr5: 69370451‐ 69370895). The PSV ratio indicates that in SMA04, two of the three SMN copies are 2.4 | Genetic variant types SMN2–SMN1 hybrids, while in SMA39, only one of the three copies is an SMN2–SMN1 hybrid (Figure 1). We discriminated the genetic variants identified in three categories: We also detected variants previously described as beneficial for (1) paralogous sequence variants (PSVs), which are positions differing SMN2 function (Bernal et al., 2010; Prior et al., 2009; Ruhno et al., 2019; between duplicated genes (in this context, variants differing between Wu et al., 2017). Using the AB ratio data, we were able to confirm the SMN1 and SMN2); (2) rare single‐nucleotide variants (SNVs), which are presence of the c.859G>C (p.(Gly287Arg)) variant in patient SMA52 with those present in less than 1% of the population or never de- two SMN2 copies in the heterozygous state and of c.835‐44A>G scribed; and (3) single‐nucleotide polymorphisms (SNPs), which are (g.69372304A>G, commonly known as A‐44G) in one of the three SMN2 variations in a single position present in over 1% of the population. copies of patient SMA21 (Figure 2 and Table S2). Furthermore, in two Determining whether the variants found are SNPs, SNVs, or PSVs sisters with different phenotypes (SMA27, type II and SMA51, type III) allowed us to evaluate the specificity of the method in the detection of and three SMN2 copies, we found the PSV conversion c.835‐1897C>T SMN1 versus SMN2 and to identify hybrid structures in the samples. (g.69370451C>T) in one of their three alleles. In our series, we did not This classification is not related to a possible effect of variants on identify any of the other more recently published variants considered as SMN2 expression and phenotype, a topic out of the scope of this candidates to be modifiers (Ruhno et al., 2019; Wadman et al., 2020). article. Finally, to verify whether our technique could also be extended to PSV ratios were computed as quotients of the number of reads identify SMN1 variants, we studied samples from three SMA patients belonging to a given functional copy and the total coverage. harboring at least one SMN1 copy. The PSV genotype in all three cases Mutation nomenclature refers to GenBank NC_000005.9, was in agreement with the copy numbers previously determined by RefSeq NM_000344.3 for SMN1, and RefSeq NM_017411.4 MLPA. SMA54 (1_SMN1/3_SMN2 MLPA genotype) showed the SMN1 for SMN2. PSVs at a frequency of ~25% (one of the four SMN copies of the 10981004, 2021, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/humu.24200 by Readcube (Labtiva Inc.), Wiley Online Library on [07/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 790 | BLASCO‐PÉREZ ET AL. TABLE 1 The 22 candidate positions for paralogous sequence variants (PSVs) between SMN1 and SMN2 are shown Gene Conversion nomenclature Source of SMN1 position SMN2 position location Ref SMN1 Ref SMN2 Categorization SMN2>SMN1 information 70231509 69356085 Intron 1 G A SNPa c.82‐3157A>G BLAT a 70240028 69364605 Intron 4 G A SNP c.628‐457A>G BLAT a 70242435 69367010 Intron 6 T C SNP c.834+432C>T BLAT b 70242978 69367553 Intron 6 A G SNV c.834+975G>A BLAT a 70244142 69368717 Intron 6 A G SNP c.834+2139G>A Monani et al. /BLAT 70245876 69370451 Intron 6 T C PSV c.835‐1897C>T Monani et al. /BLAT 70246016 69370591 Intron 6 G A PSV c.835‐1757A>G Monani et al. /BLAT 70246019 69370594 Intron 6 T C PSV c.835‐1754C>T Monani et al. /BLAT 70246156 69370731 Intron 6 G A PSV c.835‐1617A>G Monani et al. /BLAT 70246167 69370742 Intron 6 T C PSV c.835‐1606C>T BLAT 70246320 69370895 Intron 6 G A PSV c.835‐1453A>G Monani et al. /BLAT c 70246793 69371368 Intron 6 G A PSV c.835‐980A>G Monani et al. /BLAT 70246872 69371448 Intron 6 ‐ AGGCA PSV c c.835‐900_835‐896del Monani et al. /BLAT c 70246919 69371499 Intron 6 A C PSV c.835‐849C>A Monani et al. /BLAT c 70247219 69371799 Intron 6 G A PSV c.835‐549A>G Monani et al. /BLAT 70247290 69371870 Intron 6 T C PSV c.835‐478C>T Monani et al. /BLAT b,c 70247401 69371981 Intron 6 C A SNV c.835‐367A>C Monani et al. /BLAT 70247724 69372304 Intron 6 G A PSVc c.835‐44A>G Monani et al. /BLAT 70247773 69372353 Exon 7 C T PSVc c.840T>C Monani et al. /BLAT 70247921 69372501 Intron 7 A G PSVc c.*3+100G>A Monani et al. /BLAT c 70248036 69372616 Intron 7 A G PSV c.*3+215G>A Monani et al. /BLAT c 70248501 69373081 Exon 8 G A PSV c.*239A>G Monani et al. /BLAT Note: These positions were obtained from a previous bibliographic compilation (Monani et al., 1999) and were complemented with a BLAT between the two genes, as deposited in the reference genome Hg19. Repetitive regions (polyA, polyT, and polyGT) were discarded. The candidate positions were genotyped in the patients studied in this study (n = 53) and in samples with at least one SMN1 (n = 3) to check for consistency. From the 22 candidates, six were discarded as PSVs, four of them are listed as SNPs instead, and the remaining two as rare SNV. Therefore, a total of 16 nucleotides (in bold in the Table) differentiate SMN1 and SMN2 genes, 10 of which had been previously described and validated, 5 had been described but not validated, and 1 is considered here as a PSV for the first time. Abbreviations: BLAT, BLAST‐like alignment tool; SNP, single‐nucleotide polymorphism; SNV, single‐nucleotide variant. a These positions show high variability between samples and were therefore classified as SNPs (including position c.835‐367C/A previously validated by Monani et al., 1999). b In these positions, the same nucleotide has always been found in both SMN1 and SMN2. Thus, G>A and A>C exchanges appears to be very rare SNV found in the reference genome. c These 11 positions were previously classified as bona fide PSVs, after being tested in a control population of 15 individuals (Monani et al., 1999). patient). The SMN1 PSV frequency in SMA55 and SMA55F was ~50% In addition, in patient SMA55, it was also possible to corro- in line with their genotypes (1_SMN1/1_SMN2 and 2_SMN1/2_SMN2, borate the presence of the common partial SMN1/2Δ7‐8 deletion respectively). A similar consistency was obtained with SMN1 patho- of the 3′ region previously detected by MLPA (Figure 4). Indeed, genic variants. Thus, c.399_402del (p.(Glu134Serfs*14)) in SMA54 analysis of the AB ratios of all SNVs revealed a discrepancy be- showed a frequency of ~25%, while c.815A>G (p.(Tyr272Cys)) showed tween the 5′ and 3′ SMN region, showing ratios compatibles with a frequency of ~50% in SMA55 and of 25% in SMA55F. SMN1‐specific four copies (25%–75%) in the 5′ region (from promoter to exon Sanger sequencing (Kubo et al., 2015) revealed that patients SMA54 6), but compatible with two copies (50%–100%) in the 3′ region and SMA55 present this variant in hemizygous state, whereas in confirming the presence of the two alleles with the SMN1/2Δ7‐8 SMA55F it was detected in heterozygous state (Figure 3). deletion (further explanation in Figure 4). 10981004, 2021, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/humu.24200 by Readcube (Labtiva Inc.), Wiley Online Library on [07/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License BLASCO‐PÉREZ ET AL. | 791 F I G U R E 1 Structure of the SMN2–SMN1 hybrid detected in patients SMA4 and SMA39. PSVs were genotyped in all patients. In SMA4 and SMA39, six SMN1 PSVs located in intron 6 (Chr5: 69370451‐69370895) were detected, which indicates the presence of hybrid genes. The AB ratio indicates that in SMA04, two of the three copies are SMN2–SMN1 hybrids (SMN1 PSVs in 66%) while in SMA39, only one of the three copies is an SMN2–SMN1 hybrid (SMN1 PSVs in 33%). PSV, paralogous sequence variant 4 | DI SCUSSION The main advantages of our method, specifically designed to deal with the complexity of the SMA region, include accessible cost, re- We have developed a novel, practical method for the genomic ana- lative simplicity, and speedy results (usually obtained in around lysis of both SMN1 and SMN2 regions. The technique focuses on the 3 days). This practical protocol can easily genotype both PSVs and genomic characterization of each SMN2 copy in SMA patients re- rare variants already described in SMA patients, but it might also gardless of the gene copy number and can also be applied to detect help to identify new variants and SMN2–SMN1 hybrid genes as part pathogenic variants in heterozygous SMA patients with at least one of more investigative and personalized approaches. The high cover- SMN1 copy. age obtained at the nucleotide level with the new method allows the Currently published methods to study the complete sequence of the determination of the number of copies in which a specific variant is SMN2 gene include whole genome sequencing (WGS) and multiplexed present, which, in turn, is essential to characterize the genomic ar- direct genomic selection (MDiGS) sequencing (Chen et al., 2020; Ruhno chitecture of each SMN2 copy. Indeed, although the method does not et al., 2019). WGS is a rather expensive and laborious technique and directly quantify SMN2 copy numbers, calculation of the number of usually needs a complex bioinformatics analysis. In MDiGS, whole DNA is copies and the presence of partial SMN genes is straightforward prepared in libraries, target regions are captured by bacterial artificial using the allelic frequencies of the variants and might be used to, for chromosomes (BAC) probes of SMN (not specific for SMN1 or SMN2), as example, confirm results reported by other methods, such as MLPA. well as of CFTR and PLS3, for quantitative comparison. Thus, the hy- Employing this method to samples from SMA patients, we elabo- bridization step is rather large and complex. By contrast, the new method rated an updated list of 16 PSVs between SMN1 and SMN2, including described here based on long‐range PCR and NGS can be easily im- one previously never described. PSV genotyping would help to detect plemented in any genetics laboratory performing NGS applications. hybrid genes and to discover new, potentially relevant conversions that F I G U R E 2 Utility of AB ratios to calculate the number of the copies in which variants are present. Patient SMA52 has two SMN2 copies (determined by MLPA) and the variant NM_017411.4:c.859G>C (p.(Gly287Arg)) was detected with a frequency of 56%, in agreement with the AB ratio expected for the variant in one over two alleles. Patient SMA21 has three SMN2 copies (determined by MLPA), and the variant c.835‐44A>G (NC_000005.9: g.69372304A>G) was detected with a frequency of 36%, in agreement with the AB ratio expected for the variant in one over three alleles. MLPA, multiplex ligation‐dependent probe amplification 10981004, 2021, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/humu.24200 by Readcube (Labtiva Inc.), Wiley Online Library on [07/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 792 | BLASCO‐PÉREZ ET AL. F I G U R E 3 Detection of the pathogenic variant NM_017411.4:c.815A>G in samples SMA55 and SMA55F. Patient SMA55 has one SMN1 copy (with the variant c.815A>G) and one SMN2 copy, while his father (SMA55F) has two SMN1 (one copy with the variant c.815A>G) and two SMN2 copies (determined by MLPA and Sanger). The pathogenic variant c.815A>G was detected in SMA55 and SMA55F through NGS with a frequency of 52% and 25%, respectively. The SMN1‐specific PCR performed confirms that the pathogenic variant c.815A>G is present in SMN1 since we observed the variant in hemizygous status in SMA55 and in heterozygous status in SMA55F. MLPA, multiplex ligation‐dependent probe amplification; NGS, next‐generation sequencing; PCR, polymerase chain reaction might act as disease modifiers (Ruhno et al., 2019; Wu et al., 2017). Our hybrid SMN genes has been previously described based mainly on the method is highly specific given that it allows detection of positive analysis of exons 7 and 8 (Cuscó et al., 2001; Hahnen et al., 1996). This modifiers, such as c.859G>C, c.835‐44A>G (g.69372304A>G), and phenomenon occurs because the complex 5q13 region contains seg- c.835‐1897C>T (g.69370451C>T). The method also determines the mental duplications prone to nonallelic homologous recombinations, de- number of SMN2 copies carrying modifier variants through analysis of letions, duplications, and gene conversion events. Usually, hybrid genes allelic frequencies (Figure 2). are detected because of the homozygous absence of SMN1 exon 7 Based on the analysis of the entire genomic SMN2 region and ac- coupled to the presence of SMN1 exon 8. Thus, hybrids upstream exon 7 cording to the PSV genotyping, we also identified specific SMN2–SMN1 cannot be detected by common methods of diagnosis, including MLPA. hybrid structures previously undetected by MLPA. The presence of In contrast, with our long PCR‐based approach, we were able to detect F I G U R E 4 Description of the utility of AB ratios to determine the presence of two partial SMN genes (SMN1/2Δ7/8). The patient (SMA55) has one SMN1 (with a pathogenic variant in exon 6*), one SMN2, and two partial SMNs from promoter to exon 6 (determined by MLPA). In the 5′ region (promoter‐ex6), the patient has a total of four SMN copies; consequently, we detected different SNPs with an allelic frequency of 25%, 50%, 75%, and 100%. By contrast, in the 3′ region (in6‐ex8), the patient has two SMN copies, and only variants with an allelic frequency of 50% are detected. Note that PSVs are located in the 3′ region; therefore, it is not possible to determine whether these partial genes are derived from SMN1 or SMN2. MLPA, multiplex ligation‐dependent probe amplification; PSV, paralogous sequence variant; SNP, single‐nucleotide polymorphisms 10981004, 2021, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/humu.24200 by Readcube (Labtiva Inc.), Wiley Online Library on [07/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License BLASCO‐PÉREZ ET AL. | 793 novel hybrid genes consisting of a SMN2 gene with a fragment of intron 6 the discovery and validation of positive and negative SMN2 variants derived from SMN1 (Figure 1). Although additional studies are needed to in each patient remain a crucial issue in SMA diagnosis and re- characterize the function of these two hybrid genes, their detection search. An additional benefit of implementing the new method might provide clues about possible functional differences between SMN2 besides the characterization of SMN2 sequences in patients with a genes. homozygous deletion of SMN1 is its application to study SMN1 in The method described here also allows a complete genomic SMA patients retaining at least one SMN1 copy. Therefore, the SMN1 analysis. In fact, we were able to detect the pathogenic var- versatile method described here is a useful tool to approach SMN1 iants c.399_402del (p.(Glu134Serfs*14)) and c.815A>G (p.(Tyr272- and SMN2 deep sequencing, which can be easily implemented in Cys)) in patients and carriers, and to determine their frequencies most SMA diagnostic laboratories. (e.g., homozygous vs. heterozygous cases). Further confirmation that a certain variant is located in SMN1 can be achieved with a specific ACKNOWLEDGM E NTS PCR of the gene (Kubo et al., 2015). Given that in our method, we This study was supported by Grants from Biogen ESP‐SMG‐17‐ analyze the whole SMN1, including promoter, 5′ and 3′ regions as 11256 and Galicia AME (to E. F. T. supporting L. B.‐P.), “Fundación well as all introns, it emerges as potentially useful to study complex Daniel Bravo Andreu” (to E. F. T. and P. F.‐P.), SMA Europe (to E.F.T SMA cases in which only one allele alteration (deletion or point and P.F.‐P.), and Spanish Instituto de Salud Carlos III, Fondo de In- mutation) has been detected using conventional techniques vestigaciones Sanitarias and cofunded with ERDF funds (Grant No. (Alías et al., 2009). FIS PI18/000687) (to E. F. T. and L. A.). Finally, we were able to confirm the presence of partial SMN copies (SMN1/2Δ7‐8) using the AB ratios. The SMN1/2Δ7‐8 deletion CO N FLI CT O F I N TER E S TS has been widely described in the literature (Arkblad et al., 2006; A patent is in preparation for this methodology and pipeline (I.C. and Calucho et al., 2018; Chen et al., 2020; Ruhno et al., 2019; Vijzelaar E. F. T.). E.F.T has received grant support to conduct clinical trials on et al., 2019) as a variant relatively common in general population SMA from Ionis/Biogen and serves as a consultant to AveXis, No- (Europeans 15.7%, Vijzelaar et al., 2019). In almost all cases, the vartis, Biogen, Biologix, Cytokinetics, Roche. The remaining authors breakpoints are consistent (3643 bp before exon 7 and 1587 bp after declare that there are no conflict of interests. exon 8 according to Ruhno et al., 2019), so it is considered that this deletion is the result of a single deletion event (Vijzelaar et al., 2019). A U T H OR C O N T R I B U T I ON S The number of SMN2 copies and specific variants of the gene The enclosed manuscript has been seen and approved by all the have been established as the main disease modifiers of the SMA authors, and they have taken care to ensure the integrity of phenotype. However, to date, studies in discordant siblings do not the work. support the hypothesis that the intra‐familiar variability is due to variants in the SMN2 locus (Calucho et al., 2018; Cuscó et al., 2006; WE B RE S O U R C E S Ruhno et al., 2019) as is the case of our sisters sharing the modifier The following web sources have been used to carry out this study: variant c.835‐1897C>T (see Table S2). Although SMN2 is the target http://www.fda.gov, for splicing modifiers in the current therapeutic scenario, thorough https://genome-euro.ucsc.edu/, sequencing of the gene is almost never performed in genetic diag- http://www.hgmd.cf.ac.uk/ac/index.php, nostic laboratories. Application of our method and thus the avail- https://mutalyzer.nl/, ability of detailed SMN2 sequences of SMA patients would help to https://gnomad.broadinstitute.org/, solve discrepancies in genotype–phenotype correlations, as well as https://arxiv.org/abs/1303.3997v2. deepen the study of intra‐familiar variability, in a prospective manner, for the analysis of presymptomatic cases detected in new- DATA A VAILABILITY STA TEMENT born screening. Indeed, being the method so specific and fast, it is All data and scripts used to generate the analyses of this paper are envisaged that it could be accommodated within the time frame for available upon request unless that the type of request compromises therapeutic decisions in SMA newborns. It is also essential to unveil ethical standards or legal requirements. All variants reported in this possible linkages between specific SMN2 variants, factors involved article are now listed in the ClinVar database (https://www.ncbi.nlm. in SMN2 splicing, and responses to therapies. SMA treatments are nih.gov/clinvar/). very expensive, and proof of their efficacy is periodically assessed in SMA patients. In particular, nusinersen is an 18‐mer oligonu- OR C ID cleotide that binds the ISNN1 region of the intron 7, and to date, Laura Blasco‐Pérez https://orcid.org/0000-0001-6483-5742 none of the modifier variants described is located in this region Jordi Leno https://orcid.org/0000-0001-6049-256X (Ruhno et al., 2019; Wadman et al., 2020; this study). However, in Laura Alias https://orcid.org/0000-0003-2193-6185 addition to the known modifier variants, other features of their Pablo Fuentes‐Prior http://orcid.org/0000-0002-6618-3204 SMN2 genes may be correlated with the level of responsiveness and Ivon Cuscó https://orcid.org/0000-0003-2104-9332 effectiveness, an issue that warrants further investigation. Thus, Eduardo F. Tizzano http://orcid.org/0000-0002-7116-6310 10981004, 2021, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/humu.24200 by Readcube (Labtiva Inc.), Wiley Online Library on [07/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 794 | BLASCO‐PÉREZ ET AL. R EF E RE N C E S Cuscó, I., Bernal, S., Blasco‐Pérez, L., Calucho, M., Alias, L., Fuentes‐Prior, Alías, L., Bernal, S., Barceló, M. J., Also‐Rallo, E., Martínez‐Hernández, R., P., & Tizzano, E. F. 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