SMCHD1 genetic variants in type 2 FacioScapuloHumeral dystrophy and challenges in predicting pathogenicity and disease penetrance.

SMCHD1 genetic variants in type 2 FacioScapuloHumeral dystrophy and challenges in predicting pathogenicity and disease penetrance. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SMCHD1 genetic variants in type 2 FacioScapuloHumeral dystrophy and challenges in predicting pathogenicity and disease penetrance. Frederique Magdinier, Laurene Gerard, Megane Delourme, Benjamin Ganne, and 36 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3881525/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Dec, 2024 Read the published version in European Journal of Human Genetics → Version 1 posted 12 You are reading this latest preprint version Abstract The molecular diagnosis of type 1 FacioScapuloHumeral Dystrophy (FSHD1) relies on the detection of a shortened D4Z4 array at the 4q35 locus while until recently, the diagnosis of FSHD2 relied on the absence of a shortened D4Z4 allele in clinically affected patients. The vast majority of FSHD2 patients carry a heterozygous variant in the SMCHD1 gene. In addition, a decreased in D4Z4 DNA methylation is consistently associated with FSHD1 and FSHD2. In molecular genetic diagnostics, predicting the pathogenicity of SMCHD 1 variants remains challenging, as many are classified as variants of unknown significance or likely pathogenic. To refine the diagnosis of FSHD2, define 4q-associated molecular features and validate the pathogenicity of SMCHD1 variants, we explored a cohort of 54 FSHD2 patients carrying a variant in SMCHD1 or hemizygosity of the 18p32 locus encompassing the gene. Genetic and epigenetic analyses together with a clinical description of patients were combined to confirm the pathogenicity of new SMCHD1 variants and previously reported ones initially classified as likely pathogenic. We defined a threshold of 40% of methylation at the D4Z4 DR1 site as associated with SMCHD1 pathogenic variants. We also showed that the number of D4Z4 units on the shortest 4qA allele ranges from 11 up to 35 units in patients clinically affected with FSHD2. Using prediction tools, our study further highlighted the difficulty in interpretating the impact of pathogenic variants on the severity of the disease. Our study further emphasizes the complex relationship between D4Z4 methylation, SMCHD1 variants, and disease penetrance in FSHD. Health sciences/Diseases/Neurological disorders/Neuromuscular disease Biological sciences/Genetics/Epigenetics/DNA methylation FacioScapuloHumeral Dystrophy DNA methylation D4Z4 SMCHD1 Diagnosis pathogenicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction With a prevalence of 1:8 000–1:12 000, FacioScapuloHumeral Dystrophy (FSHD) is one of the most common muscular dystrophies 1 , 2 . The clinical symptoms involve asymmetric weakness followed by atrophy of specific facial, shoulder and upper arm muscles with progression to the lower extremities 3 . The most prevalent form of FSHD (FSHD1, 95% of cases) is associated to the monoallelic contraction of the D4Z4 macrosatellite repeat array on chromosome 4q. At this 4q35 locus, individuals suffering of FSHD carry between 1 and 10 copies of the repetitive DNA element while this number is higher than 11 units in healthy individuals. In approximately 5% of individuals displaying the typical clinical FSHD features, referred to as type 2 (FSHD2), the number of D4Z4 is similar to healthy individuals. Approximately 80% of these patients carry a heterozygous variant in the SMCHD1 gene encoding the Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 protein 4 . In rare cases, heterozygous variants in DNMT3B 5 , homozygous LRIF 1 variants 6 , or hemizygosity of the p18 locus encompassing SMCHD1 7–9 were reported. As most of repetitive DNA elements 10 , D4Z4 is enriched in CpG dinucleotides with > 65% of them methylated in healthy individuals 11 , 12 but hypomethylated in FSHD patients 11 , 13 , 14 . Besides FSHD, SMCHD1 hemizygosity or sequence variants are associated with at least three other syndromes with very distinct phenotypical features, such as 18p deletion syndrome 15 , 16 , Bosma Arhinia and Microphtalmia Syndrome (BAMS) 17 , 18 or Isolated Hypogonadotrophic Hypogonadism (IHH) with Combined Pituitary Hormone Deficiency (CHPD) and Septo-Optic Dysplasia (SOD) 19 . Reduced D4Z4 DNA methylation in blood DNA is consistently associated with SMCHD1 variants in FSHD as well as in the other diseases 4 , 11 , 14 , 17 , 18 . Thus, this epigenetic mark appears as a good standard for validating the pathogenicity of SMCHD1 variants, in particular for variants of unknown significance (VUS) 20 , or to aid in the diagnosis of a patient showing neuromuscular symptoms evocative of FSHD in the absence of a short D4Z4 array. We explored here a cohort of 54 patients clinically diagnosed with FSHD and carrying a non-pathogenic 4qA allele. Among these 54 cases, 48 are carriers of a variant in SMCHD1 and 6 of a deletion of the 18p32.1 locus encompassing the gene. In all patients, we analyzed D4Z4 methylation level by sodium bisulfite sequencing (BSS). In SMCHD1 variant carriers, we defined that the D4Z4 methylation level is below a threshold of 40% with the shortest D4Z4 allele ranging from 10 to 35 units. Combining the identification of SMCHD1 variants by whole exome sequencing (WES), DNA methylation analysis with information of patient’s clinical phenotype led us to define a threshold of FSHD2-associated methylation level, together with the classification of several VUS and propose a workflow in the diagnosis process of FSHD2 now applicable in molecular diagnosis. Results Identification of new and recurrent SMCHD1 variants in 48 type 2 FSHD patients. Using Molecular Combing (MC), WES and BSS, we molecularly explored 48 patients presenting with clinical symptoms specific to FSHD. These patients were classified as FSHD2 in the absence of a short D4Z4 allele (Sup table 1). Seven patients out of 48 are carriers of a cis -duplication of the D4Z4 array at the 4q35 locus 21 . Interpretation of variants was done following the American College of Medical Genetics (ACMG) guidelines 20 (Sup Table 1). Thirty-one were not reported before and 10 are listed in the LOVD database ( https://databases.lovd.nl/shared/genes/SMCHD1 ). Among previously identified variants, we identified three unrelated cases (17073; 24045; 26871) carrying a missense variant in the region encoding the ATPase domain of the protein and reported as pathogenic (c.1580C > T; p.T527M). Three other cases were reported 22 . The first one is a female carrying a 50RU 4qA-type allele (methylation level of 24% at the Fse I site as determined by Southern blotting (SB)). The two other cases are the affected father (9RU-4qA; Fse I site, 18%) and affected son (9RU-4qA; Fse I site, 11%). Given the different genetic features between patients, we concluded of six independent cases. This recurrent variant associated with D4Z4 hypomethylation (11–24% at the Fse I site, 8–27% by BSS) confirming its pathogenicity. Another recurrent variant at position c.2088_2138del was previously reported for a father and daughter carrying a 28 D4Z4 4qA allele and 1 female patient carrying a 16 D4Z4 4qA allele ( Fse I site, 10%). As all three patients were diagnosed in France, it is likely that the female patient described in our cohort was reported previously (carrier of a 16RU) 23 . It is interesting to note that the level of methylation defined at different positions by BSS or SB 23 yield similar results (8% versus 11%, respectively). Besides our #11440 patient who carries a c.2338 + 4A > G insertion reported as likely pathogenic, three other cases with an insertion at the same position were also found 23 . We report one patient carrying a c.3048 + 1G > C splicing variant likely associated with exon skipping. The exact same variant was not reported before but two other variants near the same position are listed in LOVD. We also report here three unrelated individuals carrying insertion, deletion or substitution at position c.3274. These variants classified as likely pathogenic occur in a highly repetitive region and cause deletion of Lysine 1092 together with the creation of a cryptic splice site possibly causing the skipping of exon 25 and disruption of the open reading frame, leading to haploinsufficiency. Eight identical variants were also reported at position c.3274 4,24–27 together with eight individuals carrying a variant at position c.3276 24,27–30 . Among them, two were identified in large scale WES of patients phenotypically diagnosed as suffering of Limb Girdle Muscular Dystrophy (LGMD) 28 , 29 . All variants are associated with D4Z4 hypomethylation and 4qA alleles of different sizes (19 to 47 RU) 23 , 24 . We also report two patients in which multiple SMCHD1 variants were detected. The first one (18267) carries three variants. The first variant (c.1436G > A), predicted as pathogenic as it may alter the ATPase domain was found in another patient carrying 23 RUs and 2% of methylation at the most proximal Fse I site 23 . The second variant (c.3801 + 2T > C), likely associated with a frameshift and premature stop was also reported in another patient (14 RU; 10% at Fse I) 24 . The third variant was not described before. The second patient (25897; 21 RU, 16.7% of DR1 methylation) carries a c.5281C > T variant in the region encoding the Hinge domain, never reported before and predicted as a VUS. The other variant, predicted as likely pathogenic (c.5705del) is also located in the region encoding the Hinge domain and was not reported either. Characterization of 6 cases carrying a 18p deletion. Deletion of the 18p locus is associated with a multisystemic syndrome with a variable phenotype depending on the breakpoint and size of the deleted region. This syndrome associated with deletion of 1 to 101 genes in the short arm of chromosome 18 involves cognitive impairment, congenital heart disease, small stature and minor facial dysmorphism 15 , 16 . Based on the prevalence of 4qA alleles in the population, approximately 12% of patients carrying a deletion encompassing SMCHD1 might be at risk for FSHD 9 . We analyzed here 6 patients carrying a heterozygous deletion of a variable size of the 18p32 locus encompassing SMCHD1 and referred to our center for a suspicion of FSHD (Sup Table 2). All of them carry a type A allele with a number of D4Z4 units ranging from 10 to 25 units, on their shortest 4q chromosome. Mapping of D4Z4 unit counts in SMCHD1 variant-carriers. In order to determine the distribution of the shortest D4Z4 4qA array in patients carrying a SMCHD1 variant, we plotted the number of D4Z4 units determined by MC for 40 SMCHD1 variants carriers (Fig. 1A). We excluded the eight cases carrying a cis -duplication and the six patients carrying a deletion of the 18p locus. The number of D4Z4 units ranges from 10 to 35 RUs with a mean size of 16 RUs. 27/40 patients (67.5%) carry less than 16 RUs, 5/40 patients (12.5%) carry 16–20 RUs and 8/40 patients (20%) carry more than 20 units considered as the threshold for which SMCHD1 variants are associated with FSHD 23 , 31 (Fig. 1A). For patients carrying an 18p deletion, the mean size of the array is around 15 D4Z4 with 1/6 patients carrying more than 20 D4Z4 units (Sup Fig. 1). As a threshold of 20 D4Z4 units has been proposed as associated with the clinical signs of the disease in FSHD2 patients carrying a pathogenic SMCHD1 variant 23 , 31 , we reanalyzed the data reported in the LOVD database to determine the proportion of patients below or above this limit for the 4 listed categories of variants: benign, VUS, likely pathogenic or pathogenic (Fig. 1B). For pathogenic variants (174), we calculated a mean threshold of 16 D4Z4 units with the majority of patients carrying a shorter D4Z4 array. We noticed that 31/174 (17.8%) individuals carrying a pathogenic variant carry more than 20 D4Z4 units on their shortest 4q chromosome (Fig. 1B). This proportion is in the same range as the one determined in our cohort (17.8% versus 20%, respectively). We therefore consider that the limit of 20 repeated units of D4Z4 as permissive for FSHD2 cannot be strictly applied for the molecular diagnosis of FSHD when a variant in SMCHD1 is identified and when this variant is associated with D4Z4 hypomethylation and more importantly a clinical phenotype. Analysis of DNA methylation profile for validation of SMCHD1 variants pathogenicity. We next evaluated the impact of the different SMCHD1 variants on D4Z4 DNA methylation by BSS of the DR1 site 11 , 14 . The mean methylation level was 19.6%, with a 5–40% range for individuals carrying a variant in SMCHD1 or a deletion of the 18p locus (Fig. 2A) regardless of the type of variant (Supplementary Fig. 1B). By analyzing the distribution of the methylation percentage, we noticed one outlier (15667, 52%) who carries a deletion in exon 2 likely leading to a frameshift and premature stop codon. This patient also carries a cis -duplication of the D4Z4 array that might be pathogenic 21 . Altogether, the level of methylation slightly correlates with the number of D4Z4 units of the shortest A-type 4q35 allele (Fig. 2B) but more strongly correlates with the total number of D4Z4 units on 4q ends (Fig. 2C) or total number of D4Z4 units of the two 4q and the two 10q alleles (Fig. 2D). This is consistent with a role for SMCHD1 in regulating D4Z4 methylation, regardless of its chromosomal position. Based on these results, we now consider a threshold of 40% of DR1 methylation as associated with a pathogenic SMCHD1 variant or hemizygosity of the 18p locus. Moreover, if the mean D4Z4 number in FSHD2 patients is of 16, approximately half of patients carries a longer D4Z4 array strongly arguing against a strict threshold in D4Z4 number for FSHD2 31 . Impact of missense variants on SMCHD1 protein conformation and function. We report here 12 different missense variants leading to amino-acid substitution affecting the different domains of the protein (Fig. 3A). However, their impact on the function of the protein remains unknown. A majority of FSHD2 variants map to the N-terminal region, which harbors an ubiquitin-like (UBL) fold (amino acid (AA) residues 25–109), required for stabilizing the ATPase dimer, the GHKL-ATPase catalytic domain (AA 110–395) and a transducer domain (TD, AA 396–577) (Fig. 3A). The crystal structure of the GHKL-ATPase and SMC hinge domains were solved, with identification of amino acids of critical function for ATPase activity, SMCHD1 binding to chromatin or homodimerization 32 – 34 . Among the missense variants reported here, two variants in the ATPase domain associated with D4Z4 hypomethylation abrogate (p.Q193P 35 ) or diminish (p.T527M 36 ) the ATPase activity. Based on the known structure of the various domains or AlphaFold2-based predictions of the structure, we further analyzed the potential impact of all missense variants on SMCHD1 function. The p.T111M variant is located at the limit between the UBL fold required for stabilization of the homodimer and chromatin localization and the ATPase domain 33 , 34 (Fig. 3B-D). This variant maps to a β-strand within the so-called “straps” that connect the UBL to the GHKL-ATPase cores and are swapped between monomers (Fig. 3B-C) 33 . By extending across to the neighboring molecule in the dimer, the strap provides an additional β-strand, in anti-parallel configuration, to the β-sheet formed by the adjacent protomer, providing extensive hydrogen bonds between the two monomers (Fig. 3D). Thr111 forms hydrogen bonds with Asn289 in the ATPase domain of the adjacent monomer (Fig. 3D). Variants mapping within the strap, including p.T111M or the previously described p.A110T, are likely to disturb this critical interface, with a potential impact on SMCHD1 dimerization and binding to DNA. The p.A110T variant associated with D4Z4 hypomethylation (13%) was reported as likely pathogenic in a patient with mild symptoms, facial sparing and late onset 27 . The patient reported here (p. T111M, 24996) has a milder decrease in D4Z4 methylation (31.4%) associated with an atypical phenotype (no scapular involvement) and a late onset (at age 58) suggesting that variants located in this part of the protein might be associated with a mild phenotype. The p.G128C variant, predicted as pathogenic is also associated with a marked D4Z4 hypomethylation (13.7%). It maps to the long segment (AA 122–134), next to the strap, that interacts with segment (AA 520–529) in the switch loop of the TD of the adjacent monomer, prolonging the interface between the two protomers (Fig. 3B,C). This variant is susceptible to disturb this interface. The p.T527M variant at same interface might similarly affect protomers interaction (Fig. 3B,C). In addition, the fact that Thr527 also maps to Motif V of the GHKL-ATPase which functions by positioning the “switch” lysine, Lys525, at the ATP binding site for a hydrogen bond with the γ-phosphate 33 , could explain why p.T527M displays a decreased ATPase activity 36 . The p.T527M variant reported here in three patients was also reported before. This variant was first described in a patient with a severe clinical phenotype diagnosed with both FSHD1 and FSHD2 while his mother, carrying only the p.T527M SMCHD1 variant had a low clinical score 22 . The three patients of our cohort carry a D4Z4 allele of > 11 units associated with a decreased D4Z4 methylation (7 to 15%). All three patients display a typical FSHD phenotype with onset during early adulthood suggesting that variants in the interface of the ATPase dimer might be associated to clinical features of variable severity but are likely causative of the disease. The GHKL-ATPase catalytic domain consists of a Bergerat ATP-binding fold defined by motif I-V and a flexible ATP lid. ATP bound to the active site is stabilized by interaction with residues from each of these motifs, whereas ATP lid closing is required for hydrolysis. Gln193 is located within the α-helix between motif II and this ATP lid. It is involved in water-mediated hydrogen bond-based interactions with ATP (Fig. 3B, E). The p.Q193P variant identified in a patient with a typical and severe FSHD phenotype might thus interfere directly or indirectly with SMCHD1 catalytic activity. In agreement, this variant associated with a marked hypomethylation (14.7%) but with an increased D4Z4 chromatin binding is devoid of ATPase activity suggesting that abrogation of the ATPase activity might inhibit the release of the homodimer from DNA 35 . Mutation of the adjacent Leu194 in an SMCHD1 ATPase construct similarly impedes ATPase activity and diminishes homodimerization 33 . We identified two variants in the TD, whose effect on SMCHD1 structure is less obvious. The outcome of the p.P440L variant, predicted as likely pathogenic but never described before is unclear. It is located next to a highly anionic region, flanking a cavity formed between the TD (Fig. 3B) that might be important to accommodate potential positively charged clients. The variant, which might perturb the positioning of the disordered Asp/Glu loop and the binding to a potential polypeptide client is associated with hypomethylation in a patient with a typical phenotype and onset at the age of 25 and showing 16% of methylation, confirming its probable pathogenicity. The p.R479Q predicted as pathogenic was identified in a severely affected patient. Several other previously reported variants at Arg479 are associated with hypomethylation 4 , 23 , 24 . According to the crystal structure, Arg479 is localized within a β-hairpin at the interface with the ATPase domain Motif I, Arg479 forming a hydrogen bond with Asp150, between the key catalytic residues Glu147 and Asn151. As mutation of the adjacent Gly478 leads to a defective ATPase activity and dimerization 33 , 36 , it would thus be interesting to also evaluate how this variant impacts the ATPase activity. Very little is known on the function of the middle region (AA 578–1615) that occupies more than half of the protein size in SMCHD1 function as it does not contain any recognizable domains. We identified three missense variants mapped to this region associated with D4Z4 hypomethylation. In the monomer, AlphaFold2 prediction indicates that the linker region is organized as a succession of nine modules, formed mostly by β-sheets, connected by flexible loops (Fig. 3F). This type of structure is a unique feature among the SMC proteins, which are entirely α-helical in the corresponding region. The organization of this region within the SMCHD1 dimer and its function are unknown. However, negative stain electron microscopy of full-length SMCHD1 indicates that, within the dimer, the protomers are aligned head-to-head, to form an extended rod that connect the SMC hinge to the GHKL-type ATPase domain 37 . A recent model proposed that these central linker regions open up upon SMC hinge DNA binding, allowing DNA to reel through the dimer 34 . However, it is not possible to clearly predict how these variants might impact this function. The p.R585H variant maps into a loop within the first module contributes to stabilize the relative orientation of the β-sheets, via numerous hydrogen bonds with proximal and distant amino-acid residues. This variant was described in a mother and daughter carrying 11 D4Z4 units. The daughter, clinically diagnosed at the age of 14 with myopathic features displays a D4Z4 methylation level of 31%. Her mother, clinically diagnosed with a typical FSHD at the age of 30 displays a methylation level of 19% suggesting that the variant is pathogenic. Similarly, Tyr774 connects two distinct β-sheets within the second module via multiple interactions, including hydrogen bonds, which are likely to be disturbed within the p.Y774C variant. At the opposite ends of the linker, the p.G1384V variant also maps to a long flexible loop. In the absence of SMCHD1 dimer structure, we cannot exclude that these variants also disturb the interaction between SMCHD1 protomers and/or interaction with other proteins. We report two novel FSHD variants of the core SMC hinge. This domain is an obligate homodimer that forms the principal interface for SMCHD1 dimer and can interact with chromatin (Fig. 3G). A recent crystal structure resolution permitted the characterization of the homodimer interface and identified two positively charged clusters critically required for DNA binding 32 . A number of FSHD2-associated variants were associated with a reduced thermal stability of the protein or reduced stability of the SMCHD1 dimer 32 . We identified two missense variants located at the dimer interface (D1833V and G1864E) that likely affect the function of the SMC hinge in different ways. Asp1833 maps to the lumen of the hinge domain dimer donut (Fig. 3G). Its possible impact on SMCHD1 function is unknown, since it is apparently not directly involved in dimer formation, nor nucleic acid binding. The D1833V is the only FSHD2 variant that maps within the homodimer pore, whose contribution to SMCHD1 function is unknown. Other FSHD2 hinge variants map either to the dimer interface or to charged clusters at the surface of the molecule 34 . This variant was identified in a patient carrying a 11RU, with a low methylation level but presenting with a mild form and a late onset at the age of 65. The novel p.G1864E variant maps to the first position of a triglycine motif conserved in SMC hinge domains (Fig. 3G-H). Residues in SMCHD1 motif GX 6 GX 2 G contribute both to the dimer interface and to a patch of positively charged residues called cluster 3 (Fig. 3G-H), involved in DNA binding 32 . It was observed that mutation of the FSHD2-related Arg1866, Gly1871 or Phe1873 within the conserved motif reduced thermal stability of the hinge dimer 32 , 38 . Moreover, mutation of the same Arg1866, but also charged residues Arg1868 and Lys1872, exhibited a strong reduction in the hinge domain affinity for nucleic acids 32 , 38 , supporting the two critical functions of this conserved triglycine motif. Thus, like the recurrent p.R1866G/p.R1866Q variants, substitution of Gly1864 by a bulkier and negatively charged Glu in the p.G1864E variant, is likely to compromise SMCHD1 motif GX 6 GX 2 G function in both dimer formation and nuclei acid binding. Finally, the p.D1631V variant does not map to the hinge per se , but to the N-terminal coiled-coil region preceding the SMC hinge. Contrary to what occurs in other SMC proteins, the short coiled-coils flanking the hinge domain extend in opposite directions to form intermolecular coiled-coils contributing to SMCHD1 homodimerization 38 . Other variants in this region were identified in LGMD patients 29 . In our cohort, this variant was identified in a patient carrying a cis -duplication and a level of methylation around 30%. As this is the first FSHD2 variant mapping to the coiled-coil regions, it will be of interest to investigate whether it affects SMCHD1 function and whether it is associated with the disease. Discussion Pathogenicity prediction of SMCHD1 variants by available algorithms remains limited and numerous variants are classified as VUS or likely pathogenic according to the ACMG classification 20 . With the goal of refining the diagnosis of patients affected with FSHD, we report here a cohort of 48 patients initially categorized as FSHD2 and carrying a variant in SMCHD1 together with 6 cases with hemizygosity of the 18p32 locus, encompassing SMCHD1 . By combining genetic and epigenetic analyses in light of information on the clinical phenotype, we confirm the pathogenicity of 12 novel variants and of 2 previously reported ones. Using BSS, we observed a significant decrease in D4Z4 DNA methylation for all reported patients with a methylation level lower than 40% at the DR1 site 11 , 14 . In our cohort, the number of D4Z4 units on the shortest 4qA allele ranges from 10 to up to 35 repeats suggesting a variable range in size for the shortest 4qA allele among FSHD2 patients. We further concluded that the threshold of 16–20 units proposed for the classification of FSHD2 patients 31 cannot be strictly applied in the molecular diagnosis of FSHD2. We showed here that for all patients combined, the methylation of the DR1 site is below 40%, a threshold that we now consistently apply this threshold in the validation of variants pathogenicity for the molecular diagnosis of FSHD2, regardless of the number of D4Z4 units. We did not find clear evidence that amino acid substitutions are associated with a more marked D4Z4 hypomethylation compared to frameshift variants. Heterozygous germline SMCHD1 mutations are associated with at least three distinct rare human genetic diseases, FSHD2 4,39,40 , BAMS 17 , 18 and observed in rare cases of Isolated IHH/CHPD/SOD 19 . In addition, hemizygosity of the 18p32 locus encompassing SMCHD1 is associated with the 18p deletion syndrome 15 , 16 but also FSHD 9 . In BAMS, SMCHD1 variants span the GHKL-type ATPase domain and the region immediately C-terminal to it 17 , 18 . In this syndrome, several variants are missense mutations that cause a gain of function of SMCHD1 ATPase activity 17 , 36 . In FSHD2, missense, splice and truncating mutations dispersed across the whole coding region lead to a loss-of-function or haploinsufficiency 4 , 17 , 35 , 36 . In our cohort, the 12 different missense variants causing amino-acid substitution are predicted to affect the functional domains of the protein, either by altering the function of the catalytic domain or formation of the homodimer. The pleiotropic impact of SMCHD1 variants or haploinsufficiency remains a key question to understand how variants in the same gene might lead to at least four different syndromes, 4 , 15 , 17 – 19 in which D4Z4 hypomethylation but also DUX4 expression are consistently observed 17 , 18 , 35 , 41 – 43 . In FSHD2, SMCHD1 variants were previously located around the ATP binding site or on a loop adjacent to the ATP binding pocket 23 . On the other hand, the majority of BAMS variants were positioned at the dimer interface and proposed to alter the dimerization properties 23 . We report three variants that may also affect the formation of the SMCHD1 homodimer (T111, G128, Q193) suggesting that altered SMCHD1 dimerization is not be the main cause of the marked phenotypical differences between FSHD and BAMS. Understanding the consequences of SMCHD1 variants at the protein level thus offer an extraordinary platform for investigating DNA methylation dynamics at D4Z4 and for understanding the impact of the different SMCHD1 variants on chromatin structure or manipulation of the epigenetic machinery to treat FSHD. It is worth noting that multiple SMCHD1 variants listed in the LOVD database were also reported in large cohorts of patients clinically diagnosed LGMD 26 , 28 , 29 . This further highlights the importance of integrating all molecular data with clinical features with the goal of improving the diagnosis rate and FSHD patients’ classification. Overall, determining the pathogenicity remains a challenge for many of them, in particular in the diagnosis of FSHD as it requires other features. Hence our diagnostics workflow for FSHD2 (Fig. 4) involves sizing of all 4 D4Z4 regions (4q and 10q alleles), identification of associated A or B haplotypes and D4Z4 methylation analysis together with SMCHD1 variant screening. Importantly, this workflow also considers the importance of having access to information on the clinical features of patients provided by prescribers to refine the classification of variants in particular in case of borderline alleles (8–10 units) or complex rearrangements 21 , 44 , 45 . In this scenario, pre-natal and pre-symptomatic testing and genetic counselling in FSHD2 genetic pedigrees remain a challenge to evaluate the recurrence risk in developing the disease as clinical information is crucial in the interpretation of molecular findings. Declarations Ethical approval statement. All individuals have provided written informed consent for the use of DNA sample for medical research and the study was done in accordance with the Declaration of Helsinki. Samples were provided by the Center for biological Resources (Department of Medical Genetics, La Timone Children’s hospital) with the AC 2011-1312 and N°IE-2013-710 accreditation numbers. Acknowledgements. We are indebted and thank all patients for participating in this study. Fundings This study was funded by “Association Française contre les Myopathies” (AFM Téléthon; TRIM-RD; MoThARD grants) and Agence Nationale pour la Recherche, ANR-21-CE45-0001-01. The project leading to this publication has received funding from the Excellence Initiative of Aix-Marseille University-A*Midex, a French “investissement d’avenir programme” AMX-19-IET-007. CL and MD are the recipient of a fellowship from the French Ministry of Higher Education and Research. Conflict of interest: No conflict of interest declared. Author contributions: LG and CC performed Molecular Combing for the diagnosis of patients. MD, BG, PP, NE, CL and JPT conducted the DNA methylation experiments and analyzed the data. AB performed the protein prediction analyses and edited the manuscript. RB, KB, CM, CT and KN provided patients’ samples and clinical data, analyzed the data and edited the manuscript. GB, AB, PC, FC, ADLC, ED, TE, MF, NH, LK, PL, CL, AM, VM, JN, AP, GS, MS, TS, JS, CT, CT, CV, ESC, SA provided patients’ samples and clinical expertise. FM designed and supervised the study, obtained funding, analyzed the data, wrote and edited the manuscript. 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Laberthonniere C, Novoa-Del-Toro EM, Chevalier R et al : AKT Signaling Modifies the Balance between Cell Proliferation and Migration in Neural Crest Cells from Patients Affected with Bosma Arhinia and Microphthalmia Syndrome. Biomedicines 2021; 9. Mohassel P, Chang N, Inoue K et al : Cross-sectional Neuromuscular Phenotyping Study of Patients With Arhinia With SMCHD1 Variants. Neurology 2022; 98: e1384-e1396. Nguyen K, Broucqsault N, Chaix C et al : Deciphering the complexity of the 4q and 10q subtelomeres by molecular combing in healthy individuals and patients with facioscapulohumeral dystrophy. J Med Genet 2019. Delourme M, Charlene C, Gerard L et al : Complex 4q35 and 10q26 Rearrangements: A Challenge for Molecular Diagnosis of Patients With Facioscapulohumeral Dystrophy. Neurol Genet 2023; 9: e200076. Chen K, Dobson RC, Lucet IS et al : The epigenetic regulator Smchd1 contains a functional GHKL-type ATPase domain. Biochem J 2016; 473: 1733–1744. Additional Declarations There is no duality of interest Supplementary Files FigS1.jpg GeXXrardetal.Supplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 26 Dec, 2024 Read the published version in European Journal of Human Genetics → Version 1 posted Editorial decision: revise 11 Apr, 2024 Review # 2 received at journal 10 Apr, 2024 Review # 3 received at journal 22 Mar, 2024 Reviewer # 3 agreed at journal 11 Mar, 2024 Reviewer # 2 agreed at journal 07 Mar, 2024 Review # 1 received at journal 03 Mar, 2024 Reviewer # 1 agreed at journal 18 Feb, 2024 Reviewers invited by journal 07 Feb, 2024 Submission checks completed at journal 29 Jan, 2024 First submitted to journal 26 Jan, 2024 Unknown event 22 Jan, 2024 Editor assigned by journal 20 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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\u003cem\u003eSMCHD1\u003c/em\u003e (n=40, blue square, frameshift; violet triangle, intronic variants; red dots, missense variants). Patients with a hemizygous 18p deletion or a cis-duplication of the D4Z4 array are not represented. The mean D4Z4 array size is indicated (16 D4Z4 repeated units, RU). \u003cstrong\u003eB.\u003c/strong\u003e Scatterplot showing the distribution of D4Z4 alleles for variants reported in the LOVD database for the different categories of SMCHD1 variants: benign variants (violet dots), variants of unknown significance (VUS, blue squares), likely pathogenic (green triangles) or pathogenic variants (red triangles). For each category, the mean D4Z4 number is indicated. The dashed line corresponds to the threshold of 20 D4Z4 units. For pathogenic SMCHD1 variants, 31 patients (N=31; 17.8%) carry more than 20 D4Z4 units on their shortest D4Z4 array.\u003c/p\u003e","description":"","filename":"GeXXrardDelourmeetal.Figures1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881525/v1/b6169b857112562186ef505f.jpg"},{"id":50938012,"identity":"9a43cf15-a942-48cb-bbec-764973993068","added_by":"auto","created_at":"2024-02-09 21:22:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":307625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNA methylation levels in patients carrying a variant in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSMCHD1.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003ePercentage of methylation at the DR1 region determined by Sodium Bisulfite Sequencing for the different categories of \u003cem\u003eSMCHD1\u003c/em\u003e variants: missense (red dots), nonsense variants associated with a frameshift (blue squares), intronic (violet triangles), silent or synonymous variants (green triangles) and deletions of the 18p32 locus (orange diamond) encompassing the \u003cem\u003eSMCHD1\u003c/em\u003egene. All patients are represented. Mean +/- Standard deviation is shown. \u003cstrong\u003eB.\u003c/strong\u003ePearson correlation between the percentage of DR1 methylation and the number of D4Z4 repeats on the shortest 4qA allele, r=0.3086, pvalue=0.0232). \u003cstrong\u003eC.\u003c/strong\u003ePearson correlation between the percentage of DR1 methylation and the number of D4Z4 repeats on all 4q alleles, r=0.4615, pvalue=0.0004). \u003cstrong\u003eD. \u003c/strong\u003ePearson correlation between the percentage of DR1 methylation and the total number of D4Z4 repeats the two 10q and two 4q alleles, r=0.4504, pvalue=0.0006).\u003c/p\u003e","description":"","filename":"GeXXrardDelourmeetal.Figures2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881525/v1/94a1d2677661f0d7be8c9b7f.jpg"},{"id":50938008,"identity":"df697219-01a3-45c4-9f9e-3a1a52c6dee2","added_by":"auto","created_at":"2024-02-09 21:22:54","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":904214,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrediction of the deleterious impact of missense variant on SMCHD1 function.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Schematic representation of the SMCHD1 protein. The positions of the different domains are indicated from the N to the C terminus. UBL, Ubiquitin like domain (amino acids 25 to 109); GHKL-ATPase domain (110-395) including the Strap (110-120); BAH, Bromo adjacent homology Domain (or transducer domain (TD), 396-578); SMC hinge domain (1616-1963). The position of the different missense variants is indicated. is indicated. \u003cstrong\u003eB-E.\u003c/strong\u003e Cartoon and surface representation of the crystal structure (PDB 6MW7) of the dimeric N-terminal ATPase module (25-578) of SMCHD1. \u003cstrong\u003eB.\u003c/strong\u003e The ubiquitin-like fold (UBL), the Strap, the GHKL ATPase and the transducer domain (BAH domain) are indicated. FSHD2 variants mapping at the dimer interface (p.T111M, p.G128C, p.T527M), in the catalytic domain (p.Q193P) and in the transducer domain (p.P440L, p.R479Q) are indicated in red, green and yellow respectively. \u003cstrong\u003eC.\u003c/strong\u003e Major stretches of amino-acid residues contributing to the homodimer interface are represented in sphere style. Position of interface variants within these stretches is indicated between parentheses. \u003cstrong\u003eD.\u003c/strong\u003e Zoom on the strap region. The strap (red) forms a β-strand displayed here in stick style. Dashed lines indicate the numerous H-bond formed between the strap residues (including T111) and the adjacent protomer. \u003cstrong\u003eE\u003c/strong\u003e. Zoom on SMCHD1 catalytic site, illustrating the possible contribution of Q193 to ATP stabilization. \u003cstrong\u003eF\u003c/strong\u003e. Cartoon and surface representation of the structure of the linker region (571-1615) of human SMCHD1. Structure prediction was performed using AlphaFold2 via ColabFold. Position of the p.R585H, p.Y774C and p.G1834V variants is indicated. \u003cstrong\u003eG-H.\u003c/strong\u003e Cartoon and surface representation of the structure of the SMC hinge region of human SMCHD1, including the contiguous N-terminal and C-terminal coiled-coils (1617-1958). Structure prediction was performed using AlphaFold2 via ColabFold. Of note, SAXS data \u003ca href=\"#_ENREF_46\" title=\"Chen, 2016 #8783\"\u003e\u003csup\u003e46\u003c/sup\u003e\u003c/a\u003e suggests that N-terminal helices intertwine to form an intermolecular coiled-coil and the C-terminal helices similarly, but in opposite directions. D1631V maps within the N-terminal coiled-coil. D1833V maps within the pore of the SMC hinge homodimer; G1864E maps within a conserved SMC motif GX\u003csub\u003e6\u003c/sub\u003eGX\u003csub\u003e2\u003c/sub\u003eGG (red surface within red dashes). \u003cstrong\u003eH\u003c/strong\u003e. Zoom on the conserved SMC motif GX\u003csub\u003e6\u003c/sub\u003eGX\u003csub\u003e2\u003c/sub\u003eGG (red) illustrating its contribution to both the homodimer interface and to a positively charged cluster, involved in DNA binding.\u003c/p\u003e","description":"","filename":"GeXXrardDelourmeetal.Figures3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881525/v1/d932181c3a00d1ce9bdead49.jpg"},{"id":50938013,"identity":"788a0d26-5062-4a2b-bce9-fd9c31dc8689","added_by":"auto","created_at":"2024-02-09 21:22:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":353454,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorkflow for FSHD2 diagnosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the clinical diagnosis of FSHD clinical features, FSHD molecular diagnosis provides the size of each D4Z4 array (4q and 10q alleles) together with associated A or B haplotypes. A molecular diagnosis of FSHD1 is concluded in the presence of a short D4Z4 array (\u0026lt; 10 D4Z4 units) carried on a 4qA haplotype. In the absence of a short D4Z4 array but a clinical diagnosis evocative of FSHD in symptomatic patients, the D4Z4 DNA methylation level is analyzed by Sodium Bisulfite Sequencing and a whole exome sequencing is performed. The pathogenicity of \u003cem\u003eSMCHD1\u003c/em\u003e variant is determined using prediction tools and validated when the D4Z4 methylation level is lower than 40%.\u003c/p\u003e","description":"","filename":"GeXXrardDelourmeetal.Figures4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881525/v1/b602d5a73b374011a1db4645.jpg"},{"id":72448145,"identity":"1e8e94a2-c024-486a-ab11-4300dc069247","added_by":"auto","created_at":"2024-12-27 08:05:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2521901,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3881525/v1/bae0675e-b100-4b26-876a-5a8458368a3a.pdf"},{"id":50938241,"identity":"5e24c749-147e-4d04-aaf6-1fb941bd6d9a","added_by":"auto","created_at":"2024-02-09 21:30:54","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":308102,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881525/v1/5fd01447590dd52f87854a24.jpg"},{"id":50938242,"identity":"f1087afc-712a-4f08-ae0b-aa80cc0240a0","added_by":"auto","created_at":"2024-02-09 21:30:54","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":33020,"visible":true,"origin":"","legend":"","description":"","filename":"GeXXrardetal.Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3881525/v1/2847080895c2b69028a2043b.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"SMCHD1 genetic variants in type 2 FacioScapuloHumeral dystrophy and challenges in predicting pathogenicity and disease penetrance.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWith a prevalence of 1:8 000\u0026ndash;1:12 000, FacioScapuloHumeral Dystrophy (FSHD) is one of the most common muscular dystrophies \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The clinical symptoms involve asymmetric weakness followed by atrophy of specific facial, shoulder and upper arm muscles with progression to the lower extremities \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The most prevalent form of FSHD (FSHD1, 95% of cases) is associated to the monoallelic contraction of the D4Z4 macrosatellite repeat array on chromosome 4q. At this 4q35 locus, individuals suffering of FSHD carry between 1 and 10 copies of the repetitive DNA element while this number is higher than 11 units in healthy individuals. In approximately 5% of individuals displaying the typical clinical FSHD features, referred to as type 2 (FSHD2), the number of D4Z4 is similar to healthy individuals. Approximately 80% of these patients carry a heterozygous variant in the \u003cem\u003eSMCHD1\u003c/em\u003e gene encoding the Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 protein \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In rare cases, heterozygous variants in \u003cem\u003eDNMT3B\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, homozygous \u003cem\u003eLRIF\u003c/em\u003e1 variants \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, or hemizygosity of the p18 locus encompassing \u003cem\u003eSMCHD1\u003c/em\u003e \u003csup\u003e7\u0026ndash;9\u003c/sup\u003e were reported.\u003c/p\u003e \u003cp\u003eAs most of repetitive DNA elements \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, D4Z4 is enriched in CpG dinucleotides with \u0026gt;\u0026thinsp;65% of them methylated in healthy individuals \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e but hypomethylated in FSHD patients \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Besides FSHD, \u003cem\u003eSMCHD1\u003c/em\u003e hemizygosity or sequence variants are associated with at least three other syndromes with very distinct phenotypical features, such as 18p deletion syndrome \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, Bosma Arhinia and Microphtalmia Syndrome (BAMS) \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e or Isolated Hypogonadotrophic Hypogonadism (IHH) with Combined Pituitary Hormone Deficiency (CHPD) and Septo-Optic Dysplasia (SOD) \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eReduced D4Z4 DNA methylation in blood DNA is consistently associated with \u003cem\u003eSMCHD1\u003c/em\u003e variants in FSHD as well as in the other diseases \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Thus, this epigenetic mark appears as a good standard for validating the pathogenicity of \u003cem\u003eSMCHD1\u003c/em\u003e variants, in particular for variants of unknown significance (VUS) \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, or to aid in the diagnosis of a patient showing neuromuscular symptoms evocative of FSHD in the absence of a short D4Z4 array.\u003c/p\u003e \u003cp\u003eWe explored here a cohort of 54 patients clinically diagnosed with FSHD and carrying a non-pathogenic 4qA allele. Among these 54 cases, 48 are carriers of a variant in \u003cem\u003eSMCHD1\u003c/em\u003e and 6 of a deletion of the 18p32.1 locus encompassing the gene. In all patients, we analyzed D4Z4 methylation level by sodium bisulfite sequencing (BSS). In \u003cem\u003eSMCHD1\u003c/em\u003e variant carriers, we defined that the D4Z4 methylation level is below a threshold of 40% with the shortest D4Z4 allele ranging from 10 to 35 units. Combining the identification of \u003cem\u003eSMCHD1\u003c/em\u003e variants by whole exome sequencing (WES), DNA methylation analysis with information of patient\u0026rsquo;s clinical phenotype led us to define a threshold of FSHD2-associated methylation level, together with the classification of several VUS and propose a workflow in the diagnosis process of FSHD2 now applicable in molecular diagnosis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification of new and recurrent\u003c/b\u003e \u003cb\u003eSMCHD1\u003c/b\u003e \u003cb\u003evariants in 48 type 2 FSHD patients.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing Molecular Combing (MC), WES and BSS, we molecularly explored 48 patients presenting with clinical symptoms specific to FSHD. These patients were classified as FSHD2 in the absence of a short D4Z4 allele (Sup table 1). Seven patients out of 48 are carriers of a \u003cem\u003ecis\u003c/em\u003e-duplication of the D4Z4 array at the 4q35 locus \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Interpretation of variants was done following the American College of Medical Genetics (ACMG) guidelines \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e (Sup Table\u0026nbsp;1). Thirty-one were not reported before and 10 are listed in the LOVD database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://databases.lovd.nl/shared/genes/SMCHD1\u003c/span\u003e\u003cspan address=\"https://databases.lovd.nl/shared/genes/SMCHD1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong previously identified variants, we identified three unrelated cases (17073; 24045; 26871) carrying a missense variant in the region encoding the ATPase domain of the protein and reported as pathogenic (c.1580C\u0026thinsp;\u0026gt;\u0026thinsp;T; p.T527M). Three other cases were reported \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The first one is a female carrying a 50RU 4qA-type allele (methylation level of 24% at the \u003cem\u003eFse\u003c/em\u003eI site as determined by Southern blotting (SB)). The two other cases are the affected father (9RU-4qA; \u003cem\u003eFse\u003c/em\u003eI site, 18%) and affected son (9RU-4qA; \u003cem\u003eFse\u003c/em\u003eI site, 11%). Given the different genetic features between patients, we concluded of six independent cases. This recurrent variant associated with D4Z4 hypomethylation (11\u0026ndash;24% at the \u003cem\u003eFse\u003c/em\u003eI site, 8\u0026ndash;27% by BSS) confirming its pathogenicity.\u003c/p\u003e \u003cp\u003eAnother recurrent variant at position c.2088_2138del was previously reported for a father and daughter carrying a 28 D4Z4 4qA allele and 1 female patient carrying a 16 D4Z4 4qA allele (\u003cem\u003eFse\u003c/em\u003eI site, 10%). As all three patients were diagnosed in France, it is likely that the female patient described in our cohort was reported previously (carrier of a 16RU) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. It is interesting to note that the level of methylation defined at different positions by BSS or SB \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e yield similar results (8% versus 11%, respectively).\u003c/p\u003e \u003cp\u003eBesides our #11440 patient who carries a c.2338\u0026thinsp;+\u0026thinsp;4A\u0026thinsp;\u0026gt;\u0026thinsp;G insertion reported as likely pathogenic, three other cases with an insertion at the same position were also found \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe report one patient carrying a c.3048\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;C splicing variant likely associated with exon skipping. The exact same variant was not reported before but two other variants near the same position are listed in LOVD. We also report here three unrelated individuals carrying insertion, deletion or substitution at position c.3274. These variants classified as likely pathogenic occur in a highly repetitive region and cause deletion of Lysine 1092 together with the creation of a cryptic splice site possibly causing the skipping of exon 25 and disruption of the open reading frame, leading to haploinsufficiency. Eight identical variants were also reported at position c.3274 \u003csup\u003e4,24\u0026ndash;27\u003c/sup\u003e together with eight individuals carrying a variant at position c.3276 \u003csup\u003e24,27\u0026ndash;30\u003c/sup\u003e. Among them, two were identified in large scale WES of patients phenotypically diagnosed as suffering of Limb Girdle Muscular Dystrophy (LGMD) \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. All variants are associated with D4Z4 hypomethylation and 4qA alleles of different sizes (19 to 47 RU) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe also report two patients in which multiple \u003cem\u003eSMCHD1\u003c/em\u003e variants were detected. The first one (18267) carries three variants. The first variant (c.1436G\u0026thinsp;\u0026gt;\u0026thinsp;A), predicted as pathogenic as it may alter the ATPase domain was found in another patient carrying 23 RUs and 2% of methylation at the most proximal \u003cem\u003eFse\u003c/em\u003eI site \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The second variant (c.3801\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;C), likely associated with a frameshift and premature stop was also reported in another patient (14 RU; 10% at \u003cem\u003eFse\u003c/em\u003eI) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The third variant was not described before. The second patient (25897; 21 RU, 16.7% of DR1 methylation) carries a c.5281C\u0026thinsp;\u0026gt;\u0026thinsp;T variant in the region encoding the Hinge domain, never reported before and predicted as a VUS. The other variant, predicted as likely pathogenic (c.5705del) is also located in the region encoding the Hinge domain and was not reported either.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacterization of 6 cases carrying a 18p deletion.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDeletion of the 18p locus is associated with a multisystemic syndrome with a variable phenotype depending on the breakpoint and size of the deleted region. This syndrome associated with deletion of 1 to 101 genes in the short arm of chromosome 18 involves cognitive impairment, congenital heart disease, small stature and minor facial dysmorphism \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Based on the prevalence of 4qA alleles in the population, approximately 12% of patients carrying a deletion encompassing \u003cem\u003eSMCHD1\u003c/em\u003e might be at risk for FSHD \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. We analyzed here 6 patients carrying a heterozygous deletion of a variable size of the 18p32 locus encompassing \u003cem\u003eSMCHD1\u003c/em\u003e and referred to our center for a suspicion of FSHD (Sup Table\u0026nbsp;2). All of them carry a type A allele with a number of D4Z4 units ranging from 10 to 25 units, on their shortest 4q chromosome.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMapping of D4Z4 unit counts in\u003c/b\u003e \u003cb\u003eSMCHD1\u003c/b\u003e \u003cb\u003evariant-carriers.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to determine the distribution of the shortest D4Z4 4qA array in patients carrying a \u003cem\u003eSMCHD1\u003c/em\u003e variant, we plotted the number of D4Z4 units determined by MC for 40 \u003cem\u003eSMCHD1\u003c/em\u003e variants carriers (Fig.\u0026nbsp;1A). We excluded the eight cases carrying a \u003cem\u003ecis\u003c/em\u003e-duplication and the six patients carrying a deletion of the 18p locus. The number of D4Z4 units ranges from 10 to 35 RUs with a mean size of 16 RUs. 27/40 patients (67.5%) carry less than 16 RUs, 5/40 patients (12.5%) carry 16\u0026ndash;20 RUs and 8/40 patients (20%) carry more than 20 units considered as the threshold for which \u003cem\u003eSMCHD1\u003c/em\u003e variants are associated with FSHD \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;1A).\u003c/p\u003e \u003cp\u003eFor patients carrying an 18p deletion, the mean size of the array is around 15 D4Z4 with 1/6 patients carrying more than 20 D4Z4 units (Sup Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eAs a threshold of 20 D4Z4 units has been proposed as associated with the clinical signs of the disease in FSHD2 patients carrying a pathogenic \u003cem\u003eSMCHD1\u003c/em\u003e variant\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, we reanalyzed the data reported in the LOVD database to determine the proportion of patients below or above this limit for the 4 listed categories of variants: benign, VUS, likely pathogenic or pathogenic (Fig.\u0026nbsp;1B). For pathogenic variants (174), we calculated a mean threshold of 16 D4Z4 units with the majority of patients carrying a shorter D4Z4 array. We noticed that 31/174 (17.8%) individuals carrying a pathogenic variant carry more than 20 D4Z4 units on their shortest 4q chromosome (Fig.\u0026nbsp;1B). This proportion is in the same range as the one determined in our cohort (17.8% versus 20%, respectively). We therefore consider that the limit of 20 repeated units of D4Z4 as permissive for FSHD2 cannot be strictly applied for the molecular diagnosis of FSHD when a variant in \u003cem\u003eSMCHD1\u003c/em\u003e is identified and when this variant is associated with D4Z4 hypomethylation and more importantly a clinical phenotype.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of DNA methylation profile for validation of\u003c/b\u003e \u003cb\u003eSMCHD1\u003c/b\u003e \u003cb\u003evariants pathogenicity.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe next evaluated the impact of the different \u003cem\u003eSMCHD1\u003c/em\u003e variants on D4Z4 DNA methylation by BSS of the DR1 site \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The mean methylation level was 19.6%, with a 5\u0026ndash;40% range for individuals carrying a variant in \u003cem\u003eSMCHD1\u003c/em\u003e or a deletion of the 18p locus (Fig.\u0026nbsp;2A) regardless of the type of variant (Supplementary Fig.\u0026nbsp;1B). By analyzing the distribution of the methylation percentage, we noticed one outlier (15667, 52%) who carries a deletion in exon 2 likely leading to a frameshift and premature stop codon. This patient also carries a \u003cem\u003ecis\u003c/em\u003e-duplication of the D4Z4 array that might be pathogenic\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAltogether, the level of methylation slightly correlates with the number of D4Z4 units of the shortest A-type 4q35 allele (Fig.\u0026nbsp;2B) but more strongly correlates with the total number of D4Z4 units on 4q ends (Fig.\u0026nbsp;2C) or total number of D4Z4 units of the two 4q and the two 10q alleles (Fig.\u0026nbsp;2D). This is consistent with a role for SMCHD1 in regulating D4Z4 methylation, regardless of its chromosomal position.\u003c/p\u003e \u003cp\u003eBased on these results, we now consider a threshold of 40% of DR1 methylation as associated with a pathogenic \u003cem\u003eSMCHD1\u003c/em\u003e variant or hemizygosity of the 18p locus. Moreover, if the mean D4Z4 number in FSHD2 patients is of 16, approximately half of patients carries a longer D4Z4 array strongly arguing against a strict threshold in D4Z4 number for FSHD2 \u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImpact of missense variants on SMCHD1 protein conformation and function.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe report here 12 different missense variants leading to amino-acid substitution affecting the different domains of the protein (Fig.\u0026nbsp;3A). However, their impact on the function of the protein remains unknown. A majority of FSHD2 variants map to the N-terminal region, which harbors an ubiquitin-like (UBL) fold (amino acid (AA) residues 25\u0026ndash;109), required for stabilizing the ATPase dimer, the GHKL-ATPase catalytic domain (AA 110\u0026ndash;395) and a transducer domain (TD, AA 396\u0026ndash;577) (Fig.\u0026nbsp;3A). The crystal structure of the GHKL-ATPase and SMC hinge domains were solved, with identification of amino acids of critical function for ATPase activity, SMCHD1 binding to chromatin or homodimerization \u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Among the missense variants reported here, two variants in the ATPase domain associated with D4Z4 hypomethylation abrogate (p.Q193P \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e) or diminish (p.T527M \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e) the ATPase activity.\u003c/p\u003e \u003cp\u003eBased on the known structure of the various domains or AlphaFold2-based predictions of the structure, we further analyzed the potential impact of all missense variants on SMCHD1 function.\u003c/p\u003e \u003cp\u003eThe p.T111M variant is located at the limit between the UBL fold required for stabilization of the homodimer and chromatin localization and the ATPase domain \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;3B-D). This variant maps to a β-strand within the so-called \u0026ldquo;straps\u0026rdquo; that connect the UBL to the GHKL-ATPase cores and are swapped between monomers (Fig.\u0026nbsp;3B-C) \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. By extending across to the neighboring molecule in the dimer, the strap provides an additional β-strand, in anti-parallel configuration, to the β-sheet formed by the adjacent protomer, providing extensive hydrogen bonds between the two monomers (Fig.\u0026nbsp;3D). Thr111 forms hydrogen bonds with Asn289 in the ATPase domain of the adjacent monomer (Fig.\u0026nbsp;3D). Variants mapping within the strap, including p.T111M or the previously described p.A110T, are likely to disturb this critical interface, with a potential impact on SMCHD1 dimerization and binding to DNA. The p.A110T variant associated with D4Z4 hypomethylation (13%) was reported as likely pathogenic in a patient with mild symptoms, facial sparing and late onset \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The patient reported here (p. T111M, 24996) has a milder decrease in D4Z4 methylation (31.4%) associated with an atypical phenotype (no scapular involvement) and a late onset (at age 58) suggesting that variants located in this part of the protein might be associated with a mild phenotype.\u003c/p\u003e \u003cp\u003eThe p.G128C variant, predicted as pathogenic is also associated with a marked D4Z4 hypomethylation (13.7%). It maps to the long segment (AA 122\u0026ndash;134), next to the strap, that interacts with segment (AA 520\u0026ndash;529) in the switch loop of the TD of the adjacent monomer, prolonging the interface between the two protomers (Fig.\u0026nbsp;3B,C). This variant is susceptible to disturb this interface. The p.T527M variant at same interface might similarly affect protomers interaction (Fig.\u0026nbsp;3B,C). In addition, the fact that Thr527 also maps to Motif V of the GHKL-ATPase which functions by positioning the \u0026ldquo;switch\u0026rdquo; lysine, Lys525, at the ATP binding site for a hydrogen bond with the γ-phosphate \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, could explain why p.T527M displays a decreased ATPase activity \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The p.T527M variant reported here in three patients was also reported before. This variant was first described in a patient with a severe clinical phenotype diagnosed with both FSHD1 and FSHD2 while his mother, carrying only the p.T527M \u003cem\u003eSMCHD1\u003c/em\u003e variant had a low clinical score \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The three patients of our cohort carry a D4Z4 allele of \u0026gt;\u0026thinsp;11 units associated with a decreased D4Z4 methylation (7 to 15%). All three patients display a typical FSHD phenotype with onset during early adulthood suggesting that variants in the interface of the ATPase dimer might be associated to clinical features of variable severity but are likely causative of the disease.\u003c/p\u003e \u003cp\u003eThe GHKL-ATPase catalytic domain consists of a Bergerat ATP-binding fold defined by motif I-V and a flexible ATP lid. ATP bound to the active site is stabilized by interaction with residues from each of these motifs, whereas ATP lid closing is required for hydrolysis. Gln193 is located within the α-helix between motif II and this ATP lid. It is involved in water-mediated hydrogen bond-based interactions with ATP (Fig.\u0026nbsp;3B, E). The p.Q193P variant identified in a patient with a typical and severe FSHD phenotype might thus interfere directly or indirectly with SMCHD1 catalytic activity. In agreement, this variant associated with a marked hypomethylation (14.7%) but with an increased D4Z4 chromatin binding is devoid of ATPase activity suggesting that abrogation of the ATPase activity might inhibit the release of the homodimer from DNA \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Mutation of the adjacent Leu194 in an SMCHD1 ATPase construct similarly impedes ATPase activity and diminishes homodimerization \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe identified two variants in the TD, whose effect on SMCHD1 structure is less obvious. The outcome of the p.P440L variant, predicted as likely pathogenic but never described before is unclear. It is located next to a highly anionic region, flanking a cavity formed between the TD (Fig.\u0026nbsp;3B) that might be important to accommodate potential positively charged clients. The variant, which might perturb the positioning of the disordered Asp/Glu loop and the binding to a potential polypeptide client is associated with hypomethylation in a patient with a typical phenotype and onset at the age of 25 and showing 16% of methylation, confirming its probable pathogenicity.\u003c/p\u003e \u003cp\u003eThe p.R479Q predicted as pathogenic was identified in a severely affected patient. Several other previously reported variants at Arg479 are associated with hypomethylation \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. According to the crystal structure, Arg479 is localized within a β-hairpin at the interface with the ATPase domain Motif I, Arg479 forming a hydrogen bond with Asp150, between the key catalytic residues Glu147 and Asn151. As mutation of the adjacent Gly478 leads to a defective ATPase activity and dimerization \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, it would thus be interesting to also evaluate how this variant impacts the ATPase activity.\u003c/p\u003e \u003cp\u003eVery little is known on the function of the middle region (AA 578\u0026ndash;1615) that occupies more than half of the protein size in SMCHD1 function as it does not contain any recognizable domains. We identified three missense variants mapped to this region associated with D4Z4 hypomethylation. In the monomer, AlphaFold2 prediction indicates that the linker region is organized as a succession of nine modules, formed mostly by β-sheets, connected by flexible loops (Fig.\u0026nbsp;3F). This type of structure is a unique feature among the SMC proteins, which are entirely α-helical in the corresponding region. The organization of this region within the SMCHD1 dimer and its function are unknown. However, negative stain electron microscopy of full-length SMCHD1 indicates that, within the dimer, the protomers are aligned head-to-head, to form an extended rod that connect the SMC hinge to the GHKL-type ATPase domain \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. A recent model proposed that these central linker regions open up upon SMC hinge DNA binding, allowing DNA to reel through the dimer \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, it is not possible to clearly predict how these variants might impact this function. The p.R585H variant maps into a loop within the first module contributes to stabilize the relative orientation of the β-sheets, via numerous hydrogen bonds with proximal and distant amino-acid residues. This variant was described in a mother and daughter carrying 11 D4Z4 units. The daughter, clinically diagnosed at the age of 14 with myopathic features displays a D4Z4 methylation level of 31%. Her mother, clinically diagnosed with a typical FSHD at the age of 30 displays a methylation level of 19% suggesting that the variant is pathogenic. Similarly, Tyr774 connects two distinct β-sheets within the second module via multiple interactions, including hydrogen bonds, which are likely to be disturbed within the p.Y774C variant. At the opposite ends of the linker, the p.G1384V variant also maps to a long flexible loop. In the absence of SMCHD1 dimer structure, we cannot exclude that these variants also disturb the interaction between SMCHD1 protomers and/or interaction with other proteins.\u003c/p\u003e \u003cp\u003eWe report two novel FSHD variants of the core SMC hinge. This domain is an obligate homodimer that forms the principal interface for SMCHD1 dimer and can interact with chromatin (Fig.\u0026nbsp;3G). A recent crystal structure resolution permitted the characterization of the homodimer interface and identified two positively charged clusters critically required for DNA binding \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. A number of FSHD2-associated variants were associated with a reduced thermal stability of the protein or reduced stability of the SMCHD1 dimer \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. We identified two missense variants located at the dimer interface (D1833V and G1864E) that likely affect the function of the SMC hinge in different ways. Asp1833 maps to the lumen of the hinge domain dimer donut (Fig.\u0026nbsp;3G). Its possible impact on SMCHD1 function is unknown, since it is apparently not directly involved in dimer formation, nor nucleic acid binding. The D1833V is the only FSHD2 variant that maps within the homodimer pore, whose contribution to SMCHD1 function is unknown. Other FSHD2 hinge variants map either to the dimer interface or to charged clusters at the surface of the molecule\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. This variant was identified in a patient carrying a 11RU, with a low methylation level but presenting with a mild form and a late onset at the age of 65.\u003c/p\u003e \u003cp\u003eThe novel p.G1864E variant maps to the first position of a triglycine motif conserved in SMC hinge domains (Fig.\u0026nbsp;3G-H). Residues in SMCHD1 motif GX\u003csub\u003e6\u003c/sub\u003eGX\u003csub\u003e2\u003c/sub\u003eG contribute both to the dimer interface and to a patch of positively charged residues called cluster 3 (Fig.\u0026nbsp;3G-H), involved in DNA binding \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. It was observed that mutation of the FSHD2-related Arg1866, Gly1871 or Phe1873 within the conserved motif reduced thermal stability of the hinge dimer \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Moreover, mutation of the same Arg1866, but also charged residues Arg1868 and Lys1872, exhibited a strong reduction in the hinge domain affinity for nucleic acids \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, supporting the two critical functions of this conserved triglycine motif. Thus, like the recurrent p.R1866G/p.R1866Q variants, substitution of Gly1864 by a bulkier and negatively charged Glu in the p.G1864E variant, is likely to compromise SMCHD1 motif GX\u003csub\u003e6\u003c/sub\u003eGX\u003csub\u003e2\u003c/sub\u003eG function in both dimer formation and nuclei acid binding.\u003c/p\u003e \u003cp\u003eFinally, the p.D1631V variant does not map to the hinge \u003cem\u003eper se\u003c/em\u003e, but to the N-terminal coiled-coil region preceding the SMC hinge. Contrary to what occurs in other SMC proteins, the short coiled-coils flanking the hinge domain extend in opposite directions to form intermolecular coiled-coils contributing to SMCHD1 homodimerization \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Other variants in this region were identified in LGMD patients \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In our cohort, this variant was identified in a patient carrying a \u003cem\u003ecis\u003c/em\u003e-duplication and a level of methylation around 30%. As this is the first FSHD2 variant mapping to the coiled-coil regions, it will be of interest to investigate whether it affects SMCHD1 function and whether it is associated with the disease.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePathogenicity prediction of \u003cem\u003eSMCHD1\u003c/em\u003e variants by available algorithms remains limited and numerous variants are classified as VUS or likely pathogenic according to the ACMG classification \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. With the goal of refining the diagnosis of patients affected with FSHD, we report here a cohort of 48 patients initially categorized as FSHD2 and carrying a variant in \u003cem\u003eSMCHD1\u003c/em\u003e together with 6 cases with hemizygosity of the 18p32 locus, encompassing \u003cem\u003eSMCHD1\u003c/em\u003e. By combining genetic and epigenetic analyses in light of information on the clinical phenotype, we confirm the pathogenicity of 12 novel variants and of 2 previously reported ones.\u003c/p\u003e \u003cp\u003eUsing BSS, we observed a significant decrease in D4Z4 DNA methylation for all reported patients with a methylation level lower than 40% at the DR1 site \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In our cohort, the number of D4Z4 units on the shortest 4qA allele ranges from 10 to up to 35 repeats suggesting a variable range in size for the shortest 4qA allele among FSHD2 patients. We further concluded that the threshold of 16\u0026ndash;20 units proposed for the classification of FSHD2 patients \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e cannot be strictly applied in the molecular diagnosis of FSHD2. We showed here that for all patients combined, the methylation of the DR1 site is below 40%, a threshold that we now consistently apply this threshold in the validation of variants pathogenicity for the molecular diagnosis of FSHD2, regardless of the number of D4Z4 units. We did not find clear evidence that amino acid substitutions are associated with a more marked D4Z4 hypomethylation compared to frameshift variants.\u003c/p\u003e \u003cp\u003eHeterozygous germline \u003cem\u003eSMCHD1\u003c/em\u003e mutations are associated with at least three distinct rare human genetic diseases, FSHD2 \u003csup\u003e4,39,40\u003c/sup\u003e, BAMS \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and observed in rare cases of Isolated IHH/CHPD/SOD \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In addition, hemizygosity of the 18p32 locus encompassing \u003cem\u003eSMCHD1\u003c/em\u003e is associated with the 18p deletion syndrome \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e but also FSHD \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In BAMS, \u003cem\u003eSMCHD1\u003c/em\u003e variants span the GHKL-type ATPase domain and the region immediately C-terminal to it \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In this syndrome, several variants are missense mutations that cause a gain of function of SMCHD1 ATPase activity \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In FSHD2, missense, splice and truncating mutations dispersed across the whole coding region lead to a loss-of-function or haploinsufficiency \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In our cohort, the 12 different missense variants causing amino-acid substitution are predicted to affect the functional domains of the protein, either by altering the function of the catalytic domain or formation of the homodimer.\u003c/p\u003e \u003cp\u003eThe pleiotropic impact of \u003cem\u003eSMCHD1\u003c/em\u003e variants or haploinsufficiency remains a key question to understand how variants in the same gene might lead to at least four different syndromes, \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e in which D4Z4 hypomethylation but also \u003cem\u003eDUX4\u003c/em\u003e expression are consistently observed \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In FSHD2, \u003cem\u003eSMCHD1\u003c/em\u003e variants were previously located around the ATP binding site or on a loop adjacent to the ATP binding pocket \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. On the other hand, the majority of BAMS variants were positioned at the dimer interface and proposed to alter the dimerization properties \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. We report three variants that may also affect the formation of the SMCHD1 homodimer (T111, G128, Q193) suggesting that altered SMCHD1 dimerization is not be the main cause of the marked phenotypical differences between FSHD and BAMS.\u003c/p\u003e \u003cp\u003eUnderstanding the consequences of \u003cem\u003eSMCHD1\u003c/em\u003e variants at the protein level thus offer an extraordinary platform for investigating DNA methylation dynamics at D4Z4 and for understanding the impact of the different \u003cem\u003eSMCHD1\u003c/em\u003e variants on chromatin structure or manipulation of the epigenetic machinery to treat FSHD.\u003c/p\u003e \u003cp\u003eIt is worth noting that multiple \u003cem\u003eSMCHD1\u003c/em\u003e variants listed in the LOVD database were also reported in large cohorts of patients clinically diagnosed LGMD \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. This further highlights the importance of integrating all molecular data with clinical features with the goal of improving the diagnosis rate and FSHD patients\u0026rsquo; classification.\u003c/p\u003e \u003cp\u003eOverall, determining the pathogenicity remains a challenge for many of them, in particular in the diagnosis of FSHD as it requires other features. Hence our diagnostics workflow for FSHD2 (Fig.\u0026nbsp;4) involves sizing of all 4 D4Z4 regions (4q and 10q alleles), identification of associated A or B haplotypes and D4Z4 methylation analysis together with \u003cem\u003eSMCHD1\u003c/em\u003e variant screening. Importantly, this workflow also considers the importance of having access to information on the clinical features of patients provided by prescribers to refine the classification of variants in particular in case of borderline alleles (8\u0026ndash;10 units) or complex rearrangements \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In this scenario, pre-natal and pre-symptomatic testing and genetic counselling in FSHD2 genetic pedigrees remain a challenge to evaluate the recurrence risk in developing the disease as clinical information is crucial in the interpretation of molecular findings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval statement.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll individuals have provided written informed consent for the use of DNA sample for medical research and the study was done in accordance with the Declaration of Helsinki.\u0026nbsp;Samples were provided by the Center for biological Resources (Department of Medical Genetics, La Timone Children\u0026rsquo;s hospital) with the AC 2011-1312 and N\u0026deg;IE-2013-710 accreditation numbers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are indebted and thank all patients for participating in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by \u0026ldquo;Association Fran\u0026ccedil;aise contre les Myopathies\u0026rdquo; (AFM\u0026nbsp;T\u0026eacute;l\u0026eacute;thon; TRIM-RD; MoThARD grants)\u0026nbsp;and Agence Nationale pour la Recherche, ANR-21-CE45-0001-01. The project leading to this publication has received funding from the Excellence Initiative of Aix-Marseille University-A*Midex, a French \u0026ldquo;investissement d\u0026rsquo;avenir programme\u0026rdquo; AMX-19-IET-007. CL and MD are the recipient of a fellowship from the French Ministry of Higher Education and Research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflict of interest declared.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLG and CC performed Molecular Combing for the diagnosis of patients.\u003c/p\u003e\n\u003cp\u003eMD, BG, PP, NE, CL and JPT conducted the DNA methylation experiments and analyzed the data.\u003c/p\u003e\n\u003cp\u003eAB performed the protein prediction analyses and edited the manuscript.\u003c/p\u003e\n\u003cp\u003eRB, KB, CM, CT and KN provided patients\u0026rsquo; samples and clinical data, analyzed the data and edited the manuscript.\u003c/p\u003e\n\u003cp\u003eGB, AB, PC, FC, ADLC, ED, TE, MF, NH, LK, PL, CL, AM, VM, JN, AP, GS, MS, TS, JS, CT, CT, CV, ESC, SA provided patients\u0026rsquo; samples and clinical expertise.\u003c/p\u003e\n\u003cp\u003eFM designed and supervised the study, obtained funding, analyzed the data, wrote and edited the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMostacciuolo ML, Pastorello E, Vazza G \u003cem\u003eet al\u003c/em\u003e: Facioscapulohumeral muscular dystrophy: epidemiological and molecular study in a north-east Italian population sample. 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Biomedicines 2021; 9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohassel P, Chang N, Inoue K \u003cem\u003eet al\u003c/em\u003e: Cross-sectional Neuromuscular Phenotyping Study of Patients With Arhinia With SMCHD1 Variants. Neurology 2022; 98: e1384-e1396.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen K, Broucqsault N, Chaix C \u003cem\u003eet al\u003c/em\u003e: Deciphering the complexity of the 4q and 10q subtelomeres by molecular combing in healthy individuals and patients with facioscapulohumeral dystrophy. J Med Genet 2019.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelourme M, Charlene C, Gerard L \u003cem\u003eet al\u003c/em\u003e: Complex 4q35 and 10q26 Rearrangements: A Challenge for Molecular Diagnosis of Patients With Facioscapulohumeral Dystrophy. Neurol Genet 2023; 9: e200076.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen K, Dobson RC, Lucet IS \u003cem\u003eet al\u003c/em\u003e: The epigenetic regulator Smchd1 contains a functional GHKL-type ATPase domain. Biochem J 2016; 473: 1733\u0026ndash;1744.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"FacioScapuloHumeral Dystrophy, DNA methylation, D4Z4, SMCHD1, Diagnosis, pathogenicity","lastPublishedDoi":"10.21203/rs.3.rs-3881525/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3881525/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe molecular diagnosis of type 1 FacioScapuloHumeral Dystrophy (FSHD1) relies on the detection of a shortened D4Z4 array at the 4q35 locus while until recently, the diagnosis of FSHD2 relied on the absence of a shortened D4Z4 allele in clinically affected patients. The vast majority of FSHD2 patients carry a heterozygous variant in the \u003cem\u003eSMCHD1\u003c/em\u003e gene. In addition, a decreased in D4Z4 DNA methylation is consistently associated with FSHD1 and FSHD2. In molecular genetic diagnostics, predicting the pathogenicity of \u003cem\u003eSMCHD\u003c/em\u003e1 variants remains challenging, as many are classified as variants of unknown significance or likely pathogenic. To refine the diagnosis of FSHD2, define 4q-associated molecular features and validate the pathogenicity of \u003cem\u003eSMCHD1\u003c/em\u003e variants, we explored a cohort of 54 FSHD2 patients carrying a variant in \u003cem\u003eSMCHD1\u003c/em\u003e or hemizygosity of the 18p32 locus encompassing the gene. Genetic and epigenetic analyses together with a clinical description of patients were combined to confirm the pathogenicity of new \u003cem\u003eSMCHD1\u003c/em\u003e variants and previously reported ones initially classified as likely pathogenic. We defined a threshold of 40% of methylation at the D4Z4 DR1 site as associated with \u003cem\u003eSMCHD1\u003c/em\u003e pathogenic variants. We also showed that the number of D4Z4 units on the shortest 4qA allele ranges from 11 up to 35 units in patients clinically affected with FSHD2. Using prediction tools, our study further highlighted the difficulty in interpretating the impact of pathogenic variants on the severity of the disease. Our study further emphasizes the complex relationship between D4Z4 methylation, \u003cem\u003eSMCHD1\u003c/em\u003e variants, and disease penetrance in FSHD.\u003c/p\u003e","manuscriptTitle":"SMCHD1 genetic variants in type 2 FacioScapuloHumeral dystrophy and challenges in predicting pathogenicity and disease penetrance.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-09 21:22:49","doi":"10.21203/rs.3.rs-3881525/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-04-11T18:51:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-04-10T16:19:40+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-03-22T19:45:04+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-03-11T08:28:56+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-03-07T12:46:18+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-03-03T10:22:09+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-02-18T08:18:02+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-02-07T17:33:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-29T10:34:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Human Genetics","date":"2024-01-26T09:20:26+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2024-01-22T19:42:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-20T12:25:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f4bfef26-3720-4f74-a82e-11f592b3d61d","owner":[],"postedDate":"February 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28424801,"name":"Health sciences/Diseases/Neurological disorders/Neuromuscular disease"},{"id":28424802,"name":"Biological sciences/Genetics/Epigenetics/DNA methylation"}],"tags":[],"updatedAt":"2024-12-27T08:05:38+00:00","versionOfRecord":{"articleIdentity":"rs-3881525","link":"https://doi.org/10.1038/s41431-024-01781-x","journal":{"identity":"european-journal-of-human-genetics","isVorOnly":false,"title":"European Journal of Human Genetics"},"publishedOn":"2024-12-26 05:00:00","publishedOnDateReadable":"December 26th, 2024"},"versionCreatedAt":"2024-02-09 21:22:49","video":"","vorDoi":"10.1038/s41431-024-01781-x","vorDoiUrl":"https://doi.org/10.1038/s41431-024-01781-x","workflowStages":[]},"version":"v1","identity":"rs-3881525","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3881525","identity":"rs-3881525","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}