Caratterizzazione molecolare del gene SMCHD1 in pazienti affetti da FSHD: applicazione clinica della medicina traslazionale

Introduction: Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder which is typically transmitted by an autosomal dominant pattern, although reduced penetrance and sporadic cases, caused by de novo mutations, are often observed. FSHD may be caused by a contraction of a repetitive element, located on chromosome 4 (4q35). The D4Z4 is normally hypermethylated and the genes located on this locus are silenced. In case of FSHD, the D4Z4 region is characterized by 1–10 repeats and results in the region being hypomethylated. However, 5% of FSHD cases do not carry the short allele of D4Z4 region. To date, two forms of FSHD (FSHD1 and FSHD2) are known. FSHD2 is usually observed in patients without the D4Z4 fragment contraction and carrying variants in SMCHD1 gene (18p11.32). SMCHD1 (18p11.32) is a gene coding for a chromatin remodeller involved in the epigenetic silencing of different targets, including genes located in D4Z4 locus (4q35). SMCHD1 loss of function mutations can hamper its function, leading to the aberrant expression of D4Z4 genes associated with FSHD. Aim of the study: This study aimed to characterize a group of 58 patients with a clinical suspect of FSHD, by molecular analysis of SMCHD1 sequence, using both traditional and innovative genetic sequencing techniques in order to investigate pathogenic mechanisms and translate the results into the clinical diagnostic routine. Materials and Methods: 58 patients with a clinical suspect of FSHD, previously analysed for D4Z4 region, were enrolled and were analyzed by NGS methodology and direct sequencing. In particular, Ion Torrent PGM sequencing was used for this study. The detected variants were investigated by bioinformatics tools in order to assess their frequences among the European population, predict their impact on the protein structure and function and thus evaluate the potential pathogenic effect on FSHD. Moreover, these variants were classified according to the ACMG-AMP standards and guidelines. The variant c.5150_5151delAA was further investigated analysing its segregation within a family and performing epigenetic analysis of the methylation pattern at the D4Z4 locus. Results: Molecular analysis of SMCHD1 sequence revealed the presence of exonic, intronic and 3’UTR variants. Among them, four novel exonic variants (c.183_184insGT; c.2130_2131insC; c.3469G>T; c.5150_5151delAA) were predicted to be likely pathogenic and pathogenic for FSHD, causing a substantial alteration of protein structure and function. These variants can cause the creation of PTCs or the alteration of splicing mechanisms, probably leading to the induction of NMD process or to the production of a truncated protein lacking functional domains. Moreover, seven exonic variants were found to be benign (rs2430853, rs635132, rs2276092, rs633422, rs12327477, rs2304859, rs483547), one likely benign (rs117771893) and four variants were found to be of uncertain significance (c.853G>C, c.2748G>T, c.4310A>C, c.5870A>G). One novel intronic variant was found to be pathogenic, since it was predicted to alterate a canonical splice donor site, leading to the impairment of correct splicing. In addition, four variants (c.7394A>C; c.7597G>A; c.*1397A>G; c.*1889G>C) in 3’UTR were predicted to affect the binding of different miRNAs. The expression levels of SMCHD1 mRNA and protein were assessed in silico in muscleskeletal samples, reporting a moderate RPKM value of 3.7 (+-1) for its mRNA and not detected protein levels. The investigation of the c.5150_5151delAA segregation revealed its presence in the mother and in the maternal uncle of the index subject. Furthermore, the analysis of D4Z4 fragment resulted to be 8 RU in the proband, 26 RU in the mother and 25 RU in the maternal uncle. Epigenetic analysis of CpG6 methylation regions showed significant hypomethylation in the affected patient (54%) and in the mother (56%), in contrast to the father (88%) and the uncle (81%) carrying higher methylation levels. The analysis of DR1 methylation levels reported hypomethylation for the proband (19%), the mother (11%), and the uncle (16%). Discussions: Molecular analysis of SMCHD1 allowed to detect novel potential pathogenic variants in patients with a clinical suspect of FSHD. In particular, those variants may modify the structure and function of SMCHD1 protein, leading thereby to the disruption of its physiological activity and, ultimately, contributing to FSHD etiopathogenesis. Variants found to be of uncertain significance will be further investigated to assess their potential effect on SMCHD1 and the common benign and likely-benign variants may be analysed in case-control studies in order to establish risk profiles on FSHD. Moreover, the 3’UTR variants unveiled a possible impact of miRNA-dependent regulation on FSHDrelated pathways: evidence from bioinformatics tools supports a possible effect for the post-transcriptional regulation on SMCHD1 levels and may be exploited for the modulation of SMCHD1 in innovative therapies. The variant c.5150_5151delAA was further investigated analysing its segregation within a family and performing epigenetic analysis of the methylation pattern at the D4Z4 locus, establishing its pathogenicity and the crucial utility of an integrated approach for an efficacious and complete diagnosis of FSHD. Future Perspectives and Conclusions: Variants within SMCHD1 will be further investigated to characterize their effect on the pathology. Moreover, since SMCHD1 is known to be involved in the 3D genome organization, the occurrence of modifications in the chromatin conformations in FSHD primary cells compared to controls will be assessed. miRNAs binding to SMCHD1 will be studied in order to assess the relevance of the creation or the disruption of binding sites in its 3’UTR; moreover, the global expression of miRNAs will be assessed to find differential expressions between FSHD samples and controls. In conclusion, the molecular characterization of SMCHD1 suggests the existence of potential pathogenic mechanisms that need to be studied: the dissection of the etiopathogenesis of FSHD will provide new therapeutical strategies and will improve the effectiveness of diagnostic protocols. As demonstrated for the c.5150_5151delAA, the integration of traditional and innovative genetic and epigenetic approaches will determine a more complete and effective diagnosis in the context of FSHD.