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

The molecular diagnosis of type 1 facioscapulohumeral muscular dystrophy (FSHD1) relies on the detection of a shortened D4Z4 array at the 4q35 locus. Until recently, the diagnosis of FSHD2 relied solely on the absence of a shortened D4Z4 allele in clinically affected patients. It is now established that most FSHD2 cases carry a heterozygous variant in the SMCHD1 gene. A decrease in D4Z4 DNA methylation is observed in both FSHD1 and FSHD2 patients. To refine the molecular diagnosis of FSHD2, we performed a molecular diagnosis of SMCHD1 in 54 patients with a clinical diagnosis of FSHD. All patients carry a D4Z4 array of more than 10 D4Z4 units, or a cis-duplication of the locus. Forty-eight of them carry a variant in SMCHD1 and six other cases are hemizygous for the 18p32 locus encompassing SMCHD1. Genetic and epigenetic analyses were considered to assess the pathogenicity of new SMCHD1 variants and of variants previously classified as likely pathogenic. In comparison to the healthy population and FSHD1 patients, we defined a threshold of 40% of methylation at the D4Z4 DR1 site as associated with SMCHD1 variants or SMCHD1 hemizygosity. We also showed that the number of D4Z4 on the shortest 4q allele ranges from 11 up to 35 units in these same patients. Using variant interpretation and protein structure prediction tools, we also highlight the difficulty in interpreting the impact of pathogenic variants on SMCHD1 function. Our study further emphasizes the intriguing relationship between D4Z4 methylation, SMCHD1 variants with SMCHD1 protein structure-function in FSHD.