Facioscapulohumeral muscular dystrophy (FSHD) is characterized by incomplete penetrance and intra-familial clinical variability. The disease has been associated with the genetic and epigenetic features of the D4Z4 repetitive elements at 4q35. Recently, D4Z4 hypomethylation has been proposed as a reliable marker in the FSHD diagnosis. We exploited the Italian Registry for FSHD, in which FSHD families are classified using the Clinical Comprehensive Evaluation Form (CCEF). A total of 122 index cases showing a classical FSHD phenotype (CCEF, category A) and 110 relatives were selected to test with the receiver operating characteristic (ROC) curve, the diagnostic and predictive value of D4Z4 methylation. Moreover, we performed DNA methylation analysis in selected large families with reduced penetrance characterized by the co-presence of subjects carriers of one D4Z4 reduced allele with no signs of disease or presenting the classic FSHD clinical phenotype. We observed a wide variability in the D4Z4 methylation levels among index cases revealing no association with clinical manifestation or disease severity. By extending the analysis to family members, we revealed the low predictive value of D4Z4 methylation in detecting the affected condition. In view of the variability in D4Z4 methylation profiles observed in our large cohort, we conclude that D4Z4 methylation does not mirror the clinical expression of FSHD. We recommend that measurement of this epigenetic mark must be interpreted with caution in clinical practice.

OBJECTIVES: Facioscapulohumeral muscular dystrophy type 1 (FSHD1) has been genetically linked to reduced numbers (≤8) of D4Z4 repeats at 4q35. Particularly severe FSHD cases, characterised by an infantile onset and presence of additional extra-muscular features, have been associated with the shortest D4Z4 reduced alleles with 1-3 repeats (1-3 DRA). We searched for signs of perinatal onset and evaluated disease outcome through the systematic collection of clinical and anamnestic records of de novo and familial index cases and their relatives, carrying 1-3 DRA. SETTING: Italy. PARTICIPANTS: 66 index cases and 33 relatives carrying 1-3 DRA. OUTCOMES: The clinical examination was performed using the standardised FSHD evaluation form with validated inter-rater reliability. To investigate the earliest signs of disease, we designed the Infantile Anamnestic Questionnaire (IAQ). Comparison of age at onset was performed using the non-parametric Wilcoxon rank-sum or Kruskal-Wallis test. Comparison of the FSHD score was performed using a general linear model and Wald test. Kaplan-Meier survival analysis was used to estimate the age-specific cumulative motor impairment risk. RESULTS: No patients had perinatal onset. Among index cases, 36 (54.5%) showed the first signs by 10 years of age. The large majority of patients with early disease onset (26 out of 36, 72.2%) were de novo; whereas the majority of patients with disease onset after 10 years of age were familial (16, 53.3%). Comparison of the disease severity outcome between index cases with age at onset before and over 10 years of age, failed to detect statistical significance (Wald test p value=0.064). Of 61 index cases, only 17 (27.9%) presented extra-muscular conditions. Relatives carrying 1-3 DRA showed a large clinical variability ranging from healthy subjects, to patients with severe motor impairment. CONCLUSIONS: The size of the D4Z4 allele is not always predictive of severe clinical outcome. The high degree of clinical variability suggests that additional factors contribute to the phenotype complexity.