Post-hoc Nonparametric Analysis of Forced Vital Capacity in the COMET Trial Demonstrates Superiority of Avalglucosidase Alfa vs Alglucosidase Alfa.

In the COMET trial of patients with late-onset Pompe disease, greater improvement in upright forced vital capacity (FVC) % predicted was observed with avalglucosidase alfa (AVA) vs alglucosidase alfa (ALGLU) (estimated treatment difference: 2.43%). The pre-specified mixed model repeated measures (MMRM) analysis demonstrated non-inferiority of AVA (P = 0.0074) and narrowly missed superiority (P = 0.063; 95% CI: -0.13-4.99). We report superiority of AVA in two post-hoc analyses that account for an extreme outlier participant with low FVC and severe chronic obstructive pulmonary disease at baseline: MMRM excluding the outlier (P = 0.013) and non-parametric analysis of all data with repeated measures analysis of covariance (P = 0.019).

Methylation hotspots evidenced by deep sequencing in patients with facioscapulohumeral dystrophy and mosaicism.

OBJECTIVE: To investigate the distribution of cytosine-guanine dinucleotide (CpG) sites with a variable level of DNA methylation of the D4Z4 macrosatellite element in patients with facioscapulohumeral dystrophy (FSHD). METHODS: By adapting bisulfite modification to deep sequencing, we performed a comprehensive analysis of D4Z4 methylation across D4Z4 repeats and adjacent 4qA sequence in DNA from patients with FSHD1, FSHD2, or mosaicism and controls. RESULTS: Using hierarchical clustering, we identified clusters with different levels of methylation and separated, thereby the different groups of samples (controls, FSHD1, and FSHD2) based on their respective level of methylation. We further show that deep sequencing-based methylation analysis discriminates mosaic cases for which methylation changes have never been evaluated previously. CONCLUSIONS: Altogether, our approach offers a new high throughput tool for estimation of the D4Z4 methylation level in the different subcategories of patients having FSHD. This methodology allows for a comprehensive and discriminative analysis of different regions along the macrosatellite repeat and identification of focal regions or CpG sites differentially methylated in patients with FSHD1 and FSHD2 but also complex cases such as those presenting mosaicism.

Segregation between SMCHD1 mutation, D4Z4 hypomethylation and Facio-Scapulo-Humeral Dystrophy: a case report.

BACKGROUND: The main form of Facio-Scapulo-Humeral muscular Dystrophy is linked to copy number reduction of the 4q D4Z4 macrosatellite (FSHD1). In 5 % of cases, FSHD phenotype appears in the absence of D4Z4 reduction (FSHD2). In 70-80 % of these patients, variants of the SMCHD1 gene segregate with 4qA haplotypes and D4Z4 hypomethylation. CASE PRESENTATION: We report a family presenting with neuromuscular symptoms reminiscent of FSHD but without D4Z4 copy reduction. We characterized the 4q35 region using molecular combing, searched for mutation in the SMCHD1 gene and determined D4Z4 methylation level by sodium bisulfite sequencing. We further investigated the impact of the SMCHD1 mutation at the protein level and on the NMD-dependent degradation of transcript. In muscle, we observe moderate but significant reduction in D4Z4 methylation, not correlated with DUX4-fl expression. Exome sequencing revealed a heterozygous insertion of 7 bp in exon 37 of the SMCHD1 gene producing a loss of frame with premature stop codon 4 amino acids after the insertion (c.4614-4615insTATAATA). Both wild-type and mutated transcripts are detected. CONCLUSION: The truncated protein is absent and the full-length protein level is similar in patients and controls indicating that in this family, FSHD is not associated with SMCHD1 haploinsufficiency.