International Journal of Molecular Sciences Article Deep Molecular Characterization of Milder Spinal Muscular Atrophy Patients Carrying the c.859G>C Variant in SMN2 Laura Blasco-Pérez 1,2 , Mar Costa-Roger 1,2 , Jordi Leno-Colorado 1,2 , Sara Bernal 3,4 , Laura Alias 3,4 , Marta Codina-Solà 1,2 , Desirée Martínez-Cruz 1,2 , Claudia Castiglioni 5 , Enrico Bertini 6 , Lorena Travaglini 6 , José M. Millán 4,7 , Elena Aller 4,7 , Javier Sotoca 8 , Raúl Juntas 8 , Christina Engel Hoei-Hansen 9,10 , Antonio Moreno-Escribano 11 , Encarna Guillén-Navarro 4,11 , Laura Costa-Comellas 12 , Francina Munell 12 , Susana Boronat 13 , Ricardo Rojas-García 4,14 , Mónica Povedano 15 , Ivon Cuscó 1,2,4 and Eduardo F. Tizzano 1,2, * 1 Medicine Genetics Group, Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona (UAB), 08035 Barcelona, Spain; lblasco@vhebron.net (L.B.-P.); mar.costa@vhebron.net (M.C.-R.); jleno@vhebron.net (J.L.-C.); mcodina@vhebron.net (M.C.-S.); desiree.martinez@vhebron.net (D.M.-C.); icusco@santpau.cat (I.C.) 2 Department of Clinical and Molecular Genetics, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona (UAB), 08035 Barcelona, Spain 3 Genetics Department and Sant Pau Biomedical Research Institute, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain; sbernal@santpau.cat (S.B.); lalias@santpau.cat (L.A.) 4 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28029 Madrid, Spain; jose_millan@iislafe.es (J.M.M.); aller_ele@gva.es (E.A.); guillen.encarna@gmail.com (E.G.-N.); rrojas@santpau.cat (R.R.-G.) 5 Departamento de Neurología Pediátrica, Clínica Las Condes, 7591047 Santiago de Chile, Chile; castiglionic@gmail.com 6 Unit of Neuromuscular and Neurodegenerative Disease, Ospedale Pediatrico Bambino Gesu, IRCCS, Citation: Blasco-Pérez, L.; 00165 Rome, Italy; ebertini@gmail.com (E.B.); lorena.travaglini@opbg.net (L.T.) Costa-Roger, M.; Leno-Colorado, J.; 7 Unidad de Genética, Hospital La Fe and IIS La Fe, 46026 Valencia, Spain 8 Neuromuscular Diseases Unit, Neurology Department, Hospital Universitari Vall d’Hebron, Bernal, S.; Alias, L.; Codina-Solà, M.; Martínez-Cruz, D.; Castiglioni, C.; 08035 Barcelona, Spain; jsotoca@vhebron.net (J.S.); rjuntas@vhebron.net (R.J.) 9 Department of Paediatrics, Copenhagen University Hospital, 2100 Copenhagen, Denmark; Bertini, E.; Travaglini, L.; et al. Deep christina.hoei-hansen@regionh.dk Molecular Characterization of Milder 10 Department of Clinical Medicine, University of Copenhagen, 1165 Copenhagen, Denmark Spinal Muscular Atrophy Patients 11 Neurology Service and Medical Genetics Section, Hospital Clínico Universitario Virgen de la Arrixaca, Carrying the c.859G>C Variant in IMIB-Arrixaca, Universidad de Murcia, 30120 Murcia, Spain; antonio_moreno9@yahoo.es SMN2. Int. J. Mol. Sci. 2022, 23, 8289. 12 Pediatric Neurology Section, Vall d’Hebron Research Institute (VHIR), Hospital Universitari Vall d’Hebron, https://doi.org/10.3390/ Universitat Autònoma de Barcelona (UAB), 08035 Barcelona, Spain; laura.costa@vhebron.net (L.C.-C.); ijms23158289 fmunell@vhebron.net (F.M.) 13 Pediatrics Department, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain; sboronat@santpau.cat Academic Editor: Giuseppe Novelli 14 MND Clinic, Department of Neurology, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Received: 22 June 2022 Barcelona, 08025 Barcelona, Spain 15 Unidad Funcional de Enfermedad de Motoneurona, Servicio de Neurología, Hospital Universitario de Accepted: 22 July 2022 Bellvitge, 08907 Barcelona, Spain; 30058mpp@gmail.com Published: 27 July 2022 * Correspondence: etizzano@vhebron.net; Tel.: +34-93274600 (ext. 6945) Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Abstract: Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by biallelic published maps and institutional affil- loss or pathogenic variants in the SMN1 gene. Copy number and modifier intragenic variants in iations. SMN2, an almost identical paralog gene of SMN1, are known to influence the amount of complete SMN proteins. Therefore, SMN2 is considered the main phenotypic modifier of SMA, although genotype–phenotype correlation is not absolute. We present eleven unrelated SMA patients with milder phenotypes carrying the c.859G>C-positive modifier variant in SMN2. All were studied Copyright: © 2022 by the authors. by a specific NGS method to allow a deep characterization of the entire SMN region. Analysis of Licensee MDPI, Basel, Switzerland. two homozygous cases for the variant allowed us to identify a specific haplotype, Smn2-859C.1, in This article is an open access article distributed under the terms and association with c.859G>C. Two other cases with the c.859G>C variant in their two SMN2 copies conditions of the Creative Commons showed a second haplotype, Smn2-859C.2, in cis with Smn2-859C.1, assembling a more complex allele. Attribution (CC BY) license (https:// We also identified a previously unreported variant in intron 2a exclusively linked to the Smn2-859C.1 creativecommons.org/licenses/by/ haplotype (c.154-1141G>A), further suggesting that this region has been ancestrally conserved. The 4.0/). deep molecular characterization of SMN2 in our cohort highlights the importance of testing c.859G>C, Int. J. Mol. Sci. 2022, 23, 8289. https://doi.org/10.3390/ijms23158289 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 8289 2 of 13 as well as accurately assessing the SMN2 region in SMA patients to gain insight into the complex genotype–phenotype correlations and improve prognostic outcomes. Keywords: spinal muscular atrophy; SMN2 copies; phenotype–genotype correlations; positive modifiers; next-generation sequencing 1. Introduction Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by the degeneration and loss of alpha motor neurons in the spinal cord anterior horns, leading to progressive atrophy of proximal muscles, weakness, respiratory failure, and even death. It is the second most common recessive genetic disease of infancy and early childhood with an incidence around 1:11,000 live births and a carrier frequency of 1:51 worldwide [1,2]. SMA patients are mainly classified into five clinical groups on the basis of age of onset, achieved motor milestones, and clinical severity. Type 0 or congenital, the most severe, appears prenatally, and the patient’s life expectancy is very short, usually a few weeks or months. Patients with type I, or Werdnig–Hoffmann disease (onset within the first six months of life), are never able to sit unsupported and generally do not survive beyond the age of two years. In the intermediate SMA type II (onset between 6 and 18 months of life), children acquire the ability to sit unsupported, but they never walk unaided and usually reach adolescence. Type III patients (Kugelberg–Welander disease) walk independently for a long time but eventually become wheelchair-bound. They can be further subdivided into type IIIa and IIIb depending on the age of disease onset (before or after three years of age). Finally, patients with SMA type IV present an adult onset and milder disease course [3–5]. It is important to bear in mind that current SMA therapies can modify the trajectory of SMA patients; therefore, this classification is mainly applied on clinical data prior to treatment [6,7]. At the molecular level, SMA is caused by the loss or mutation of both copies of the survival of motor neuron 1 (SMN1) gene, which encodes the survival motor neuron protein (SMN). In most cases, the disease is due to the homozygous absence of SMN1 (95%), although pathogenic point variants have also been described [8–10]. Adjacent to SMN1, in a more centromeric position, lies SMN2, an almost identical paralog gene generated by a segmental duplication [11]. The fact that SMN2 is present in humans and not in any other species suggests that the duplication of SMN1 occurred recently in time. Consequently, the homology between both genes is extremely high, differing only in 16 positions called paralogous sequence variants (PSVs) [12,13]. This makes the region highly unstable, which leads to genomic instability predisposing to gene deletions, duplications, and conversions between both genes. Indeed, SMN1 and SMN2 genes can be present in multiple copies in the general population, both in cis and trans configuration [14]. Theoretically, the SMN2 gene encodes the same protein as SMN1, but one of the PSVs, a silent transition in exon 7, alters the splicing pattern in most SMN2 pre-mRNA transcripts. This causes the skipping of exon 7, resulting in a non-functional protein (SMN-∆7) instead of the full-length protein [13]. As SMN-∆7 is highly unstable and rapidly degraded, it is unable to compensate the absence or deficiency of SMN1 in SMA patients [15]. It has been reported that each copy of SMN2 can only produce about 10–15% of functional SMN proteins [16–18], being the number of SMN2 copies the main modifier of SMA disease described to date. Concretely, an inverse correlation between the number of SMN2 copies and the sever- ity of the phenotype has been widely reported, given that the higher the number of SMN2 copies producing SMN functional protein, the milder the SMA phenotype [19–21]. Never- theless, this correlation is not absolute, since discordant patients have been described in the literature, further classified as better-than-expected or worse-than-expected phenotypes Int. J. Mol. Sci. 2022, 23, 8289 3 of 13 according to their SMN2 copy number [19,21]. It is known that the presence of the c.859G>C and c.835-44A>G (-44G) variants, located in exon 7 and intron 6 of SMN2, respectively, explains some of the better-than-expected discordant phenotypes. These SNVs, considered positive modifiers of SMA disease, increase the inclusion of exon 7 and therefore generate greater amounts of functional SMN protein [21–25]. The full characterization of SMN2, including dosage and structure, will be more relevant in the current scenario where new therapies for SMA are being implemented. It is well known that SMN2 dosage is the main modifier of SMA, but it seems that this could be just the tip of the iceberg of a much more complicated framework. Indeed, all differences between SMN1 and SMN2 can be revealed by specific NGS studies [12]. It is also possible that these findings may relate to phenotype variability or to SMN2-specific treatment response [20]. In this work, we performed an in-depth characterization of the SMN region in eleven SMA patients carrying the c.859G>C modifier variant in the SMN2 gene (SMN2859C ) and presenting a milder phenotype. By defining the genetic background of SMN2859C , we discovered the existence of a common haplotype alongside the SMN2 gene in linkage disequilibrium with the variant and a second less common haplotype harboring two SMN2859C copies in cis. 2. Results 2.1. Clinical and Molecular Characterization of Patients All SMA patients described in this study (ten males and one female) presented a biallelic absence of SMN1 as the determinant of SMA and shared the presence of at least one copy of SMN2859C . Seven of these individuals carried two SMN2 copies, including five with the c.859G>C modifier variant in their two SMN2 genes (patients 1 to 5) and two with the variant in only one SMN2 (patients 6 and 7). The other four patients presented three SMN2 copies, and the variant was only present in one of their SMN2 alleles (patients 8 to 11). A summary of the clinical and molecular data of the patients is shown in Table 1. Our cohort comprised SMA patients of Spanish, Italian, Danish, and Chilean origins, and the majority were classified as SMA type IIIb (8/11) and the remaining patients as SMA type IIIa (2/11). The remaining case (Patient 6 in Table 1) was classified as type II based on his age of onset, which was prior to 18 months. Currently, at three years of age, he has not yet achieved independent ambulation. Table 1. Clinical and molecular data of patients. Information regarding general characteristics of patients, SMA phenotype, and SMN1/2 genotypes. Patient 1 2 3 4 5 6 7 8 9 10 11 Origin Spanish Spanish Chilean Italian Spanish Spanish Danish Spanish Spanish Spanish Spanish Gender Male Male Male Male Male Male Female Male Male Male Male Age (years) 49 35 21 17 28 3 8 71 54 57 47 Consanguinity Yes Yes No No No No No No No No No Bernal Bernal Blasco- Bernal Bernal et al., et al., This Pérez et al., et al., Reported This work This work This work This work This work 2010 (P2) 2010 (P3) work et al., 2021 2010 (P4) 2010 (P5) [23] [23] [12] [23] [23] SMA type IIIb IIIb IIIb IIIb IIIb II IIIa IIIb IIIb IIIa IIIb Age of 12 12 18 24 10 years 4 years 13 years 15 years 14 years 9-10 years 13 years onset years months months months Not yet Walked Yes Yes Yes Yes Yes accom- Yes Yes Yes Yes Yes unaided plished Wheelchair Not appli- 41 years 23 years No No No No 49 years No 22 years 37 years bound age cable SMN1 0 0 0 0 0 0 0 0 0 0 0 copies SMN2 2 2 2 2 2* 2 2 3 3 3 3 copies Presence of 2/2 2/2 2/2 2/2 2/2 1/2 1/2 1/3 1/3 1/3 1/3 c.859G>C * In addition, presents a partial SMN gene comprising only exons 1 to 6 (SMN1/2∆7-8) (see Figure 1). Int. J. Mol. Sci. 2022, 23, 8289 4 of 13 2.2. Haplotype Characterization by Deep Sequencing of SMN2 Genes NGS data confirmed the biallelic absence of the entire SMN1 gene in all patients, since specific nucleotides of SMN2 were found in homozygous state (AB ratio of 100%) in all PSV positions. Similarly, the NGS results corroborated the SMN2 copy number previously assigned by MLPA via the AB ratio analysis of all the different variants detected in the SMN region of each patient. In patients with two SMN2 copies (except for patient 5), all variants were detected with an approximate allele frequency of 50% or 100%, whereas in patients with three SMN2 copies, variants were found at a frequency of around 33%, 66%, or 100% (data not shown, available upon request). Patient 5 was a special case where variants were observed at a frequency of around 33–66–100% in the 50 region and around 50–100% frequency in the 30 region. This phenomenon was due to the presence of two complete SMN2 genes and a partial SMN gene comprising exons 1 to 6 (SMN1/2∆7-8) (see Table 2). In addition, the AB analysis of all patients confirmed the copy number of the c.859G>C modifier in each case. Overall, our 11 patients represented 16 alleles with the c.859G>C variant, including five cases with two SMN2859C and the remainder with just one allele with the variant (Table 2). 2.2.1. Establishment of Two Haplotypes Associated with the c.859G>C Modifier Variant We initially performed an in-depth analysis of the complete SMN2 region in patients 1 and 2, who carried two SMN2859C genes and had consanguineous parents. The studies revealed that both patients were completely homozygous for the entire studied region and identical between them. Thus, we were able to determine the specific SMN2 sequence associated with the c.859G>C modifier in their alleles, establishing a haplotype called Smn2- 859C.1 (Table 2). Similarly, sequencing results in patient 3 revealed an almost identical sequence to Smn2-859C.1 in his two SMN2 genes, with the exception of one rare variant (69356349-A-G) with an allele frequency of ~50%. In contrast, patients 4 and 5, who also presented two SMN2859C copies, showed several variants in only one of their SMN2859C along the studied region. Nonetheless, it was possible to infer that one of their SMN2 genes matched the sequence of the Smn2-859C.1 haplotype. Interestingly, in both patients, it was possible to assume a second haplotype associated with the c.859G>C variant that we defined as Smn2-859C.2 (Table 2). Applying this preliminary information, the Smn2-859C.1 haplotype was also inferred in one of the SMN2 copies of the remaining patients (patients 6 to 11), with few discrepant positions in patients 7, 10, and 11 (see Table 2). To explore deeper into the structure of the SMN2 genes, co-segregation studies from patients with two SMN2859C were carried out through MLPA together with NGS or allele- specific PCR. These investigations showed that patient 2 carried his two Smn2-859C.1 hap- lotypes in trans, inheriting one from each progenitor (Figure 1B). Patient 10 s co-segregation was incomplete, as a sample from his father was not available, but this family was consan- guineous, and the mother only presented one Smn2-859C.1 haplotype. Therefore, we could assume that his father also presented one Smn2-859C.1 haplotype, and he should harbor both Smn2-859C.1 haplotypes in trans (Figure 1A). In contrast, the co-segregation study in patient 3 revealed that both Smn2-859C.1 haplotypes were in cis, forming a complex allele inherited from the mother (Figure 1C). Co-segregation in patients 4 and 5 indicated that the two SMN2859C genes (Smn2-859C.1 and Smn2-859C.2 haplotypes) were located in cis. Specifically, patient 4 inherited this complex allele from his father and a null allele (without SMN1 and SMN2) from his mother (Figure 1D), while patient 5 inherited the complex allele from his mother and the other allele with a partial non-functional SMN1/2∆7-8 gene from his father (Figure 1E). Int. J. Mol. Sci. 2022, 23, 8289 5 of 13 Table 2. Haplotype characterization of SMN2 genes. Detail of the 30 positions comprising the Smn2-859C.1 (green) and Smn2-859C.2 (blue) haplotypes in our patients. Punctual discrepancies are represented in red. The novel variant c.154-1141G>A (69360651-G-A, hg19/GRCh37), exclusively associated with the Smn2-859C.1 haplotype, is indicated in green in the first column. The c.859G>C modifier variant is marked in red. The remaining alleles of each patient not carrying the c.859G>C are represented in grey. Patient 1 2 3 4 5 6 7 8 9 10 11 Smn2- Smn2- Location hg19 1 1 1 1 1 1 1 2 1 2 - 1 - 1 - 1 - - 1 - - 1 - - 1 - - 859C.1 859C.2 69342881-T-C C C C C C C C C C C C C C C C C C C C C C C/T C/T C C C C C/T C/T 69343230-C-T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T 69343570-G-T T T T T T T T T T T T T T T G T T T T T T T T T T T T T/G T/G 69347007-C-T T T T T T T T T C T C C T C C C T C C T C C T C C T C C 69349821-T-C C C C C C C C C T C T T C T C T C T T C T T C T T C T T 69350284-A– - - - - - - - - - - - - - - A - - - - - - - - - - - A A A 69351711-A-G G A A A A A A A G A G A A A A A A A A A A A A A A A A A 69353192-G-A A G G G G G G G A G A A G G G G G G G G G G G G G G G G 69354973-A-G G G G G G G G G A G A G G A G G G G G G G G G G G G G/A G/A intron 1 69356085-A-G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G 69356349-A-G G A A A A A G A G A G A A A A A A A A A A A A A A A A A 69357245-C-G G C C C C C C C G C G G C C C G C G G C G G C G G C C/G C/G 69357509-G-A A A A A A A A A G A G G A G A G A G G A G G A G G A G G 69358318-A-G G A A A A A A A G A G A A A A A A A A A A A A A A A A A 69358605-A-G G G G G G G G G G G G G G G A G G G G G G G G G G G G G/A G/A 69359034-C-T T T T T T T T T C T C C T C T C T C C T C C T C C T C C 69360020-G-T T T T T T T T T G T G G T G T G T G G T G G T G G G G G intron 2a 69360651-G-A A A A A A A A A G A G G A G A G A G G A G G A G G A G G intron 2b 69362410-T-C C C C C C C C C C C C C T C T C T C T T C T T C T T C T T exon 3 69362949-A-G G G G G G G G G G G G G G G A G G G G G G G G G G G G G/A G/A 69363717-C-T T T T T T T T T T T T T T C T C T C C T C C C C C T C C intron 4 69363866-A-G G A A A A A A A G A G A A A A A A A A A A A A A A A A 69364605-A-G G G G G G G G G G G G G G A G G G G G G G G G G G G G/A G/A intron 5 69365216-G-C C C C C C C C C G C G C G C G C G G C G G C G G C G G 69368084-A-G G G G G G G G G G G G G G A G G G G/A G/A G G G G G G G G/A G/A intron 6 69368329-G-A A A A A A A A A A A A A A G A A A G/A G/A A A A A A A A G/A G/A 69371981-C-A A A A A A A A A A A A A A C A A A A A A A A A A A A A/C A/C exon 7 69372372-G-C C C C C C C C C C C C C C G C G C G G C G G C G G C G G 69373667-A-G G G G G G G G G G G G G G A G G G G G G G G G G G G G G downstream 69373682-C-G G G G G G G G G G G G G G C G C G C C G C C G C C G G/C G/C Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 2 of 14 Int. J. Mol. Sci. 2022, 23, 8289 6 of 13 Figure 1. Pedigree representation of cases with two SMN2 859C copies. According to segregation Figure 1. Pedigree representation of cases with two SMN2859C copies. According to segregation studies, studies,aacis cisor ortrans transconfiguration configurationwas wasdefined definedinineach eachpatient. patient.(A) (A)InInpatient patient1,1,the thefather’s father’ssample sam- was available, and SMN2 ple was not available, and SMN2 configuration was inferred based on the results from mother not configuration was inferred based on the results from the and the mother patient. Given that the father did not present symptoms, we can assume that he carries and patient. Given that the father did not present symptoms, we can assume that he carries at least at least one SMN1 one SMN1 gene. In In addition, addition,being beingaaconsanguineous consanguineousfamily, family,weweassumed assumedthatthatSmn2-859.C1 Smn2-859.C1 was was transmitted by both parents (untested inferred alleles are represented by a dashed line). transmitted by both parents (untested inferred alleles are represented by a dashed line). (B) In patient (B) In patient 2, 2, the Smn2-859.C1 the Smn2-859.C1 haplotypehaplotype was inherited was inherited from both from both in parents, parents, in agreement agreement with the con- with the consanguinity in the family. (C) Patient 3 had two copies of SMN2 with Smn2-859.C1 in cis, inheritedinherited sanguinity in the family. (C) Patient 3 had two copies of SMN2 with Smn2-859.C1 in cis, from his from his mother. (D) Patient 4 also had two copies of SMN2 in cis, one with Smn2-859.C1 and the mother. (D) Patient 4 also had two copies of SMN2 in cis, one with Smn2-859.C1 and the other with other with Smn2-859.C2 haplotype, forming a complex allele inherited from the father. (E) Patient Smn2-859.C2 haplotype, forming a complex allele inherited from the father. (E) Patient 5 inherited the 5 inherited the complex allele from his mother and the other allele with a partial non-functional complex allelegene SMN1/2Δ7-8 fromfrom his mother and the other allele with a partial non-functional SMN1/2∆7-8 gene his father. from his father. 2.2.2. Difference between Haplotypes and Detection of a Novel Variant Exclusively As- All together, these results indicated that the Smn2-859C.1 haplotype was consistent sociated with the Smn2-859C.1 Haplotype in our cohort, since all patients presented it in association with the c.859G>C variant, eitherAnalyzing as a single the sequence allele or as partofofboth haplotypes, a more Smn2-859C.1 complex allele formed by consists of 24 variants the Smn2-859C.1 and while Smn2-859C.2 comprises 22 variants, sharing 16 of these positions Smn2-859C.2 haplotypes in cis. Based on our 11 SMA patients, we have not observed and differinganyin the other 14. In particular, the sequence near the c.859G>C variant clinical difference between Smn2-859C.1 and Smn2-859C.2 haplotypes, although we only is shared between Smn2-859C.1 found two casesand Smn2-859C.2 carrying haplotypes the Smn2-859C.2 and spans at least 8848 bp (chr5:69365217- haplotype. 69374064). These haplotypes were not found in a total of 338 SMA patients without the c.859G>C 2.2.2. variant, Difference although between some of the Haplotypes andvariants Detection contained of a Novel in the haplotypes Variant are present Exclusively Associated in this larger the Smn2-859C.1 withcohort. Haplotype Interestingly, we noticed the presence of a novel variant, c.154- 1141G>A (69360651-G-A, Analyzing the sequence hg19/GRCh37), located of both haplotypes, in intron 2aconsists Smn2-859C.1 (Table 2). This of 24 variant variants was while detected in all Smn2-859C.2 patients22 comprises with the Smn2-859C.1 variants, sharing 16 ofhaplotype but absent these positions in the Smn2-859C.2 and differing in the other 14. In particular, haplotype. the sequence Moreover, nearwas this variant the c.859G>C not detected variant in theis 338 shared SMA patientsSmn2-859C.1 between without the and Smn2-859C.2 c.859G>C variant.haplotypes and spanschange The c.154-1141G>A at leasthas 8848 notbp (chr5:69365217-69374064). been reported in the general These popu- haplotypes were to lation according notgnomAD, found in ISB a total of 338and Kaviar3, SMA patients Bravo (as ofwithout 18 July the c.859G>C 2022) [26–28]. variant, In silico although some analysis of thisofdeep the variants introniccontained in the the variant using haplotypes softwareare present [29], SpliceAI in this larger cohort. Alamut Visual Int. J. Mol. Sci. 2022, 23, 8289 7 of 13 Interestingly, we noticed the presence of a novel variant, c.154-1141G>A (69360651-G-A, hg19/GRCh37), located in intron 2a (Table 2). This variant was detected in all patients with the Smn2-859C.1 haplotype but absent in the Smn2-859C.2 haplotype. Moreover, this variant was not detected in the 338 SMA patients without the c.859G>C variant. The c.154-1141G>A change has not been reported in the general population according to gnomAD, ISB Kaviar3, and Bravo (as of 18 July 2022) [26–28]. In silico analysis of this deep intronic variant using the software SpliceAI [29], Alamut Visual Software version 2.11 (SOPHiA GENETICS), and ESRseq [30] did not predict an effect on the splicing process. 3. Discussion Here, we present 11 patients with a clinical and molecular diagnosis of SMA caused by the biallelic absence of SMN1 and with a milder phenotype explained by at least one SMN2859C gene, given that 10 out of 11 patients were walkers. We identified a specific sequence, named Smn2-859C.1, present in all patients from our cohort in linkage disequi- librium with the c.859G>C variant. In addition, two cases showed a more complex Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 ofallele, 14 assembled by Smn2-859C.1 and Smn2-859C.2 in cis. In order to study the genetic origin of the c.859G>C variant, we expanded our cohort of Spanish cases with patients from Denmark, Italy, and Chile. We applied NGS methodologies three SMN2 copies and the variant in more than one allele and, in fact, no patient with exclusively focused on the SMN region to determine the exact sequence of SMN2 associated this genotype has been described in the literature either. This could be due to the fact that with the c.859G>C variant in each patient [12]. By studying the patients with two SMN2859C , patients we werewith three able SMN2 copies to determine two showing haplotypes SMA type II or associated III are with the not currently variant, tested for Smn2-859C.1 and theSmn2-859C.2. variant. Another reason could be that cases with this genotype perhaps The Smn2-859C.1 haplotype, with minor modifications, was present do not mani- in all fest 11clear disease patients symptoms (14/16 SMN2859C duealleles), to the higher production either in ofconfiguration, cis or trans SMN protein and therefore while the Smn2- may never be diagnosed. Similarly, it has been previously speculated 859C.2 haplotype was only found in two patients (2/16 SMN2859C alleles), alwaysthat some individ- in cis uals with zero SMN1 and four or five SMN2 copies may present minimal symptoms configuration with the Smn2-859C.1 haplotype (Figure 2). Notably, no patient was found or be to asymptomatic throughout their lives, remaining undetected [35]. This corroborates harbor the c.859G>C variant in association with any other haplotype, regardless of their the importance of implementing ethnic lineage, which pointsdetection towards aofcommon the c.859G>C-positive ancestral originmodifier as part of the in all cases. genetic diagnosis routine in SMA. Figure Figure 2. Expected 2. Expected SMA SMA phenotype phenotype in cases in cases withwith two two SMN2 SMN2 copies copies according according to thetopresence the presence of of c.859G>C. c.859G>C.AnAnadditive effect additive on on effect SMASMAphenotype phenotypeis observed depending is observed on whether depending on whether the the c.859G>C c.859G>C variant is found variant in one is found or both in one SMN2 or both copies. SMN2 SMN2 copies. gene is SMN2 represented gene as a rectangle, is represented and theand the as a rectangle, presence of the c.859G>C variant in exon 7 is indicated by an asterisk. Not all SMN2 genotypes presence of the c.859G>C variant in exon 7 is indicated by an asterisk. Not all SMN2 genotypes represented in this figure were detected in this study (see Figure 1 for more details). represented in this figure were detected in this study (see Figure 1 for more details). AtThe thisc.859G>C level of analysis variant and basedpreviously has been on the clinical information reported to increaseavailable for each the inclusion pa- of SMN2 tient, exonwe7 did not observe by 20%, categorical which leads to thephenotypic generation differences between of of higher amounts thefunctional Smn2-859C.1 or proteins Smn2-859C.2 haplotypes, than the wild-type SMN2nor gene the cis or trans [22,25]. configuration Patients carrying of this the Smn2-859C.1 haplotype, variant developed milder since SMA allphenotypes cases with compared two SMN2with 859C copies presented a milder phenotype (IIIb). Interest- those with the same SMN2 copy number but without the ingly, patient 2, with the exact same sequence and configuration as patient 1, also devel- oped type IIIb SMA, but his onset was noted earlier in comparison with patient 1 and the remaining cases with two SMN2859C. At present, we are unable to explain this minor dis- parity considering all the studies performed in SMN2. Thus, this fact suggests disease on- set could also be conditioned by as yet unknown factors, other than SMN2 structure. Int. J. Mol. Sci. 2022, 23, 8289 8 of 13 variant [23]. In our case, the deep characterization of the entire SMN region supports that SMN2859C is, at first sight, primarily responsible for the milder phenotype in our patients. To date, together with our six newly described patients, a total of 44 patients carrying c.859G>C have been reported worldwide, including a patient recently detected by newborn screening [21–23,25,31–34]. In general population databases, the c.859G>C variant is re- ported at a frequency of approximately 0.3% with 132 homozygotes detected [26]. However, it is possible that the data are not accurate given the high homology between SMN1 and SMN2 and their copy number variability, which poses a challenge in the analysis and proper annotation of the SMN region with non-specific NGS techniques, such as exome or genome. Nevertheless, it is possible to estimate the frequency of this variant in the SMA population based on previous studies. According to the data of Calucho et al. (2018) [19], the allelic frequency of the c.859G>C variant is 1.04% (13/1250 alleles) in a series of 625 Spanish SMA patients. In fact, in this cohort, approximately 25% of better-than-expected cases with two SMN2 copies carried the variant [19]. Although c.859G>C appears to be relatively uncommon, at present it is not routinely tested in SMA patients, deserving more studies to clearly establish its incidence. Concerning clinical classification, patients with two SMN2 genes usually debut in the first six months of life and are classified as SMA type I [19]. In our series, patients with two SMN2 copies and the positive modifier presented at least type II or type III disease (Table 1). Furthermore, an additive effect was observed since patients with the c.859G>C change in both SMN2 genes had a better phenotype than patients carrying the variant only in one SMN2, confirming previous observations [23] (Figure 2). For instance, patient 3 (with two SMN2859C copies) developed the first SMA symptoms at 12 years of age, being classified as type IIIb, whereas patient 7 (with the variant in one of his SMN2 genes) had manifestations at 18 months of life with a clinical diagnosis of type IIIa. Regarding cases with three SMN2 copies, all patients presented the c.859G>C variant in only one of their alleles, developing a type III phenotype. Interestingly, we did not find any patient with three SMN2 copies and the variant in more than one allele and, in fact, no patient with this genotype has been described in the literature either. This could be due to the fact that patients with three SMN2 copies showing SMA type II or III are not currently tested for the variant. Another reason could be that cases with this genotype perhaps do not manifest clear disease symptoms due to the higher production of SMN protein and therefore may never be diagnosed. Similarly, it has been previously speculated that some individuals with zero SMN1 and four or five SMN2 copies may present minimal symptoms or be asymptomatic throughout their lives, remaining undetected [35]. This corroborates the importance of implementing detection of the c.859G>C-positive modifier as part of the genetic diagnosis routine in SMA. At this level of analysis and based on the clinical information available for each patient, we did not observe categorical phenotypic differences between the Smn2-859C.1 or Smn2- 859C.2 haplotypes, nor the cis or trans configuration of the Smn2-859C.1 haplotype, since all cases with two SMN2859C copies presented a milder phenotype (IIIb). Interestingly, patient 2, with the exact same sequence and configuration as patient 1, also developed type IIIb SMA, but his onset was noted earlier in comparison with patient 1 and the remaining cases with two SMN2859C . At present, we are unable to explain this minor disparity considering all the studies performed in SMN2. Thus, this fact suggests disease onset could also be conditioned by as yet unknown factors, other than SMN2 structure. As mentioned above, the Smn2-859C.2 haplotype was detected in cis configura- tion with respect to Smn2-859C.1, assembling a complex allele containing two different SMN2859C genes (Figure 1). These two haplotypes differ in several positions, but an identi- cal block of at least 8848 bp around c.859G>C is present in both (Table 2 and Figure 3B). This observation, together with the fact that we also detected an allele formed by two Smn2- 859C.1 haplotypes in cis, points towards a possible origin of the complex allele through homologous recombination, implicating a double cross-over event [36]. In this event, two alleles would be involved (Figure 3A): allele A, consisting of two SMN2 genes with the Smn2-859C.1 haplotype, and allele B, formed by at least one SMN2 with an unknown Int. J. Mol. Sci. 2022, 23, 8289 9 of 13 haplotype containing part of Smn2-859C.2 but without the c.859G>C variant. In the double Int. J. Mol. Sci. 2022, 23, x FOR PEERhomologous REVIEW recombination process, allele A would maintain both c.859G>C variants5 ofas 14 well as gain the part of the Smn2-859C.2 haplotype from allele B, generating the complex allele that we detected in our patients (Figure 3B). Figure3.3.Proposed Figure Proposedmechanism mechanismofoforigin originand andstructure structureofofSmn2-859C.1 Smn2-859C.1andandSmn2-859C.2 Smn2-859C.2haplotypes. haplo- types. (A) Hypothetic origin of the Smn2-859C.2 haplotype through homologous recombination (A) Hypothetic origin of the Smn2-859C.2 haplotype through homologous recombination involving a involving double a double cross-over cross-over event betweenevent between allele A (withallele two A (with two Smn2-859C.1 Smn2-859C.1 haplotypes repre- haplotypes represented in green) sented in green) and allele B (containing 0 the 5′ region of the Smn2-859C.2 haplotype, in blue, and and allele B (containing the 5 region of the Smn2-859C.2 haplotype, in blue, and the 3’ end with the 3’ end with an unknown sequence, in grey). (B) SMN2 structure details (representing exons an unknown sequence, in grey). (B) SMN2 structure details (representing exons and introns from and introns from top to bottom) and location of variants of the Smn2-859C.1 and Smn2-859C.2 hap- top to bottom) lotypes, as welland location as the of variants unknown the Smn2-859C.1 originalofhaplotype and Smn2-859C.2 that presumably originatedhaplotypes, as well the Smn2-859C.2 as the unknown haplotype. original haplotype The c.154-1141G>A that presumably (69360651-G-A, originated hg19/GRCh37) the Smn2-859C.2 variant haplotype. is indicated in green, The c.154-1141G>A (69360651-G-A, whereas the c.859G>C modifierhg19/GRCh37) is shown in redvariant is explanation (further indicated inin green, whereas the text the c.859G>C and Table 2). modifier is shown in red (further explanation in the text and Table 2). Finally, it should be noted that the Smn2-859C.1 haplotype contains the novel variant Finally, it should c.154-1141G>A, be noted located that2a. in intron Smn2-859C.1 theAccording haplotype to our results,contains the novel this variant is in variant linkage c.154-1141G>A, disequilibrium with the c.859G>C modifier given that, in our larger cohort ofin349 located in intron 2a. According to our results, this variant is linkage SMA disequilibrium patients, it was with only the c.859G>C detected modifier in those given carrying that, in our the c.859G>C largerand variant, cohort of 349 it was SMA not found patients, it wasdatabases. in population only detected Thisinobservation those carrying the c.859G>C suggests variant, and that the sequence it wasthis between not variant found in population databases. This observation suggests that the sequence between and the c.859G>C modifier has been ancestrally conserved. In silico splicing tools did not this variant and the c.859G>C modifier has been ancestrally conserved. In silico splicing tools did not predict any specific effect of this deep intronic variant. However, we could not rule out predict any specific effect of this deep intronic variant. However, we could not rule out some influence of this change, given the limitations of splicing predictors; thus, it deserves some influence of this change, given the limitations of splicing predictors; thus, it deserves further investigation. further investigation. Our NGS approach to characterize these patients revealed new information that Our NGS approach to characterize these patients revealed new information that could could be relevant for the different functions and/or alterations of SMN2. It is important to be relevant for the different functions and/or alterations of SMN2. It is important to consider whether the function and expression of SMN2 is not only modified depending consider whether the function and expression of SMN2 is not only modified depending on on the cis or trans configuration of SMN2859C but also on the presence of the Smn2-859C.1 the cis or trans configuration of SMN2859C but also on the presence of the Smn2-859C.1 or or Smn2-859C.2 haplotype. Long regulators, cis- or trans-acting elements, may distinc- tively influence its function and/or expression according to the topography of the region. 4. Materials and Methods 4.1. Study Participants Int. J. Mol. Sci. 2022, 23, 8289 10 of 13 Smn2-859C.2 haplotype. Long regulators, cis- or trans-acting elements, may distinctively influence its function and/or expression according to the topography of the region. 4. Materials and Methods 4.1. Study Participants We studied eleven unrelated SMA patients from different international centers with the presence of at least one SMN2859C gene. Patients were classified into SMA type according to age of onset, clinical severity, and achieved motor milestones, prior to receiving any modifying therapies. Criteria for correlating phenotype with SMN2 dosage were type I (non-sitters) with two SMN2 copies, type II (sitters) with three SMN2 copies, and type III (walkers) patients with three–four SMN2 copies [19]. Based on this model, our patients with two SMN2 copies were considered discordant, as none presented a type I SMA phenotype (Table 1). All patients were selected from a larger cohort of 349 SMA patients, undergoing an NGS study of the SMN region [12], based on the presence of the c.859G>C variant. Four of the patients were previously described as carriers of this variant (patients 1, 8, and 11 [23] and patient 7 [12]). DNA samples were extracted from peripheral blood using standard methods. Ethics approval was granted by the Clinical Research Ethics Committee of Hospital Vall d’Hebron (Comité de Ética de Investigación con Medicamentos del Hospital Universitari Vall d’Hebron (PR(AG)229/2018)). Written informed consent was obtained from all participants or their parents/legal caregivers. 4.2. SMN2 Genotyping and Haplotype Characterization All patients were genetically confirmed as SMA cases via previously described meth- ods that also included testing SMN2 modifier variants [10,23,37]. A detailed molecular characterization of SMN2 was carried out in all patients by a specific NGS sequencing method [12]. In addition, to detect the presence of the c.859G>C variant in some progenitor samples, two specific PCRs were designed to amplify exons 7 and 8 of genes SMN1 and SMN2. The allele-specific PCR technique [38] was used to amplify both genes separately to ascertain in which gene the variant was present. Standard Sanger sequencing was performed with the PCR products, allowing us to detect the c.859G>C variant. These primers are also designed to study the c.835-44A>G variant. Primer sequences and PCR conditions are provided in Table S1. 5. Conclusions This series of patients with milder phenotypes demonstrates the relevance of testing the c.859G>C variant in all SMA patients, with special consideration in cases with two or three SMN2 copies in the context of neonatal screening. Indeed, the presence of this rare variant in an asymptomatic neonate may help to predict a better phenotype by natural history per se, regardless of the therapeutic option chosen. This is crucial in order to evaluate the effects of the approved therapies to unmask long-term benefits in treated patients. Given that not all discordant cases can be explained by this positive variant, it is necessary to further analyze the SMN2 region by NGS to detect other reported candidate variants [24] and the presence of hybrid SMN1-SMN2 structures [20], as well as to unravel novel phenotypic modifier variants. In the current therapeutic context, genetic studies in patients confirmed with biallelic SMN1 absence or pathogenic variants should consider not only testing for SMN2 copies but also investigating SMN2 variants and structures as part of the integral characterization of patients receiving expensive and sometimes lifelong therapies. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms23158289/s1. Int. J. Mol. Sci. 2022, 23, 8289 11 of 13 Author Contributions: Conceptualization, L.B.-P., M.C.-R., I.C. and E.F.T.; methodology, L.B.-P., M.C.-R., S.B. (Sara Bernal), L.A. and D.M.-C.; formal analysis, J.L.-C., L.B.-P., M.C.-R., I.C. and E.F.T.; resources, C.C., E.B., L.T., J.M.M., E.A., J.S., R.J., C.E.H.-H., A.M.-E., E.G.-N., L.C.-C., F.M., S.B. (Susana Boronat), R.R.-G. and M.P.; data curation, L.B.-P., M.C.-R., M.C.-S., I.C. and E.F.T.; writing—original draft preparation, L.B.-P., M.C.-R., I.C. and E.F.T.; writing—review and editing, L.B.-P., M.C.-R., I.C., S.B. (Sara Bernal), M.C.-S. and E.F.T.; supervision, E.F.T. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by grants from Biogen (ESP-SMG-17-11256), Roche, GaliciAME and Spanish Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias and co-funded with ERDF funds (grant no. FIS PI18/000687). A grant from Horizon 2020 IMI2 Screen4Care is acknowl- edged by E.B., and L.T., E.F.T., R.J., J.S., L.C.-C., F.M., E.B., and L.T. are members of the ERN NMD Network for Rare Diseases. 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